slc3a2  (Cell Signaling Technology Inc)


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    Structured Review

    Cell Signaling Technology Inc slc3a2
    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 <t>(SLC3A2)</t> AR, or actin. Molecular weight markers are indicated in kilodaltons.
    Slc3a2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis"

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    Journal: eLife

    doi: 10.7554/eLife.81083

    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.
    Figure Legend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Techniques Used: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

    ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .
    Figure Legend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Techniques Used: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

    ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Techniques Used: Two Tailed Test, Standard Deviation, Western Blot

    ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Techniques Used: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

    LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.
    Figure Legend Snippet: LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Techniques Used: Transduction, CRISPR, Cell Culture, Amplification

    ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

    ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.
    Figure Legend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Techniques Used: Activation Assay, Expressing

    ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Variant Assay, Molecular Weight, Transduction, Plasmid Preparation

    ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.
    Figure Legend Snippet: ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Techniques Used: Staining, Two Tailed Test, Standard Deviation, Injection

    ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .
    Figure Legend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Techniques Used: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

    ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Standard Deviation, Western Blot, Variant Assay, Molecular Weight

    ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.
    Figure Legend Snippet: ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Techniques Used: Standard Deviation, Western Blot, Molecular Weight

    Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.
    Figure Legend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).
    Figure Legend Snippet: ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Techniques Used: Microarray, Immunohistochemistry, Two Tailed Test, Staining

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    Cell Signaling Technology Inc slc3a2
    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 <t>(SLC3A2)</t> AR, or actin. Molecular weight markers are indicated in kilodaltons.
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    1) Product Images from "GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis"

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    Journal: eLife

    doi: 10.7554/eLife.81083

    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.
    Figure Legend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Techniques Used: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

    ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .
    Figure Legend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Techniques Used: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

    ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Techniques Used: Two Tailed Test, Standard Deviation, Western Blot

    ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Techniques Used: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

    LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.
    Figure Legend Snippet: LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Techniques Used: Transduction, CRISPR, Cell Culture, Amplification

    ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

    ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.
    Figure Legend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Techniques Used: Activation Assay, Expressing

    ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Variant Assay, Molecular Weight, Transduction, Plasmid Preparation

    ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.
    Figure Legend Snippet: ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Techniques Used: Staining, Two Tailed Test, Standard Deviation, Injection

    ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .
    Figure Legend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Techniques Used: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

    ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Standard Deviation, Western Blot, Variant Assay, Molecular Weight

    ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.
    Figure Legend Snippet: ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Techniques Used: Standard Deviation, Western Blot, Molecular Weight

    Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.
    Figure Legend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).
    Figure Legend Snippet: ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Techniques Used: Microarray, Immunohistochemistry, Two Tailed Test, Staining

    slc3a2 4f2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc slc3a2 4f2
    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), <t>4F2</t> <t>(SLC3A2)</t> AR, or actin. Molecular weight markers are indicated in kilodaltons.
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    1) Product Images from "GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis"

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    Journal: eLife

    doi: 10.7554/eLife.81083

    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.
    Figure Legend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Techniques Used: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

    ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .
    Figure Legend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Techniques Used: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

    ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Techniques Used: Two Tailed Test, Standard Deviation, Western Blot

    ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Techniques Used: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

    LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.
    Figure Legend Snippet: LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Techniques Used: Transduction, CRISPR, Cell Culture, Amplification

    ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

    ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Techniques Used: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.
    Figure Legend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Techniques Used: Activation Assay, Expressing

    ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Variant Assay, Molecular Weight, Transduction, Plasmid Preparation

    ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.
    Figure Legend Snippet: ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Techniques Used: Staining, Two Tailed Test, Standard Deviation, Injection

    ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .
    Figure Legend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Techniques Used: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

    ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
    Figure Legend Snippet: ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Techniques Used: Standard Deviation, Western Blot, Variant Assay, Molecular Weight

    ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.
    Figure Legend Snippet: ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Techniques Used: Standard Deviation, Western Blot, Molecular Weight

    Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.
    Figure Legend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Techniques Used: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).
    Figure Legend Snippet: ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Techniques Used: Microarray, Immunohistochemistry, Two Tailed Test, Staining

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    Cell Signaling Technology Inc slc3a2 4f2
    (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), <t>SLC3A2</t> <t>(4F2),</t> SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.
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    1) Product Images from "GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis"

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    Journal: bioRxiv

    doi: 10.1101/2022.06.17.496598

    (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), SLC3A2 (4F2), SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), SLC3A2 (4F2), SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    (A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold-change for the sgRNAs for each gene is shown on the x axis. Significantly depleted genes ( p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC -genes in red are dependent on GCN2 for expression. (B) Plot of -Log10 (p-value) for depleted genes identified in CRISPR screen for LNCaP vs. 22Rv1 cells. Significantly depleted genes ( p ≥ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC-genes in red are GCN2-dependent. (C) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hours, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2 (4F2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. (D) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+ His), or media depleted of histidine (- His) for 24 hours. Lysates were analyzed by Immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2, or actin. (E) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hours or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. (F) SLC3A2 (4F2) was knocked down in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA control. Cell growth was measured in replicate wells (N = 5) for up to 6 days and are plotted relative to day 0 (mean ± SD). (G) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hours. Lysate were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, SLC3A2, or actin. (H) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells (N = 5) and is plotted relative to day 0 (mean ± SD). Statistical analysis is shown in Supplementary File 1 . (I) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 (N = 4), 4F2 (SLC3A2, N = 4), or scramble control (N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons Error bars indicate SD; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
    Figure Legend Snippet: (A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold-change for the sgRNAs for each gene is shown on the x axis. Significantly depleted genes ( p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC -genes in red are dependent on GCN2 for expression. (B) Plot of -Log10 (p-value) for depleted genes identified in CRISPR screen for LNCaP vs. 22Rv1 cells. Significantly depleted genes ( p ≥ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC-genes in red are GCN2-dependent. (C) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hours, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2 (4F2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. (D) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+ His), or media depleted of histidine (- His) for 24 hours. Lysates were analyzed by Immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2, or actin. (E) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hours or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. (F) SLC3A2 (4F2) was knocked down in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA control. Cell growth was measured in replicate wells (N = 5) for up to 6 days and are plotted relative to day 0 (mean ± SD). (G) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hours. Lysate were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, SLC3A2, or actin. (H) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells (N = 5) and is plotted relative to day 0 (mean ± SD). Statistical analysis is shown in Supplementary File 1 . (I) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 (N = 4), 4F2 (SLC3A2, N = 4), or scramble control (N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons Error bars indicate SD; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Techniques Used: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Transfection, Stable Transfection, Plasmid Preparation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including SLC3A2 (4F2) to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.
    Figure Legend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including SLC3A2 (4F2) to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Techniques Used: Activation Assay, Expressing

    (A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the materials and methods section. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± SEM (N = 4). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; *, p ≤ 0.05; ****, p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; **, p ≤ 0.01. (B) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tail t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05; ***, p ≤ 0.001. (C) Tumor growth of PC-3 WT and PC- 3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM (N = 5); ***, p ≤ 0.001; ****, p ≤ 0.0001. On the right bar graphs, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 5); *, p ≤ 0.01. (D) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of SLC7A5 (LAT1), SLC7A11 (xCT), and SLC3A2 (4F2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); **, p ≤ 0.01 . (E) 22Rv1 WT (N = 4), 22Rv1 GCN2 KO (clone 7, N = 5) and 22Rv1 ATF4 KO (N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. (F) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05. (G) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for figure , with or without supplementation of EAA in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT (N = 4) and 22Rv1 KO (N = 5) are the same tumor growth curves shown in 6E . 22Rv1 WT + EAA (N = 5), 22Rv1 GCN2 KO + EAA (N = 5) (H) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (I) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced SLC3A2 (4F2) lentivirus (WT + SLC3A2 and GCN2 KO + SLC3A2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM (N = 5).
    Figure Legend Snippet: (A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the materials and methods section. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± SEM (N = 4). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; *, p ≤ 0.05; ****, p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; **, p ≤ 0.01. (B) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tail t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05; ***, p ≤ 0.001. (C) Tumor growth of PC-3 WT and PC- 3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM (N = 5); ***, p ≤ 0.001; ****, p ≤ 0.0001. On the right bar graphs, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 5); *, p ≤ 0.01. (D) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of SLC7A5 (LAT1), SLC7A11 (xCT), and SLC3A2 (4F2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); **, p ≤ 0.01 . (E) 22Rv1 WT (N = 4), 22Rv1 GCN2 KO (clone 7, N = 5) and 22Rv1 ATF4 KO (N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. (F) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05. (G) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for figure , with or without supplementation of EAA in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT (N = 4) and 22Rv1 KO (N = 5) are the same tumor growth curves shown in 6E . 22Rv1 WT + EAA (N = 5), 22Rv1 GCN2 KO + EAA (N = 5) (H) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (I) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced SLC3A2 (4F2) lentivirus (WT + SLC3A2 and GCN2 KO + SLC3A2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM (N = 5).

    Techniques Used: Injection, Two Tailed Test, Western Blot, Variant Assay, Molecular Weight, Plasmid Preparation

    Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N=4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors (N = 5) (D). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days per week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; *, p ≤ 0.05. (E) Protein lysates were prepared from 22Rv1 tumors treated with Vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05. (F) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (G) Pearson correlation between p-GCN2-T899 and SLC3A2 (4F2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal (N = 10), hyperplasia (N = 20) and malignant (N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and SLC3A2 (4F2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and SLC3A2 (4F2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). (H) Correlation of expression of SLC3A2 (4F2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the materials and methods.
    Figure Legend Snippet: Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N=4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors (N = 5) (D). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days per week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; *, p ≤ 0.05. (E) Protein lysates were prepared from 22Rv1 tumors treated with Vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05. (F) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (G) Pearson correlation between p-GCN2-T899 and SLC3A2 (4F2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal (N = 10), hyperplasia (N = 20) and malignant (N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and SLC3A2 (4F2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and SLC3A2 (4F2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). (H) Correlation of expression of SLC3A2 (4F2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the materials and methods.

    Techniques Used: Injection, Two Tailed Test, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

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    (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), <t>SLC3A2</t> (4F2), SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.
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    1) Product Images from "GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis"

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    Journal: bioRxiv

    doi: 10.1101/2022.06.17.496598

    (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), SLC3A2 (4F2), SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.
    Figure Legend Snippet: (A) Volcano plot illustrating log2 fold change with adjusted p -value (−log10) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hours. Several amino acid transporters reduced by GCN2iB treatment are highlighted. (B) Enrichment plots from Gene Set Enrichment Analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hours versus vehicle control. (C) Heat map displaying differential gene expression of SLC genes comparing LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hours. SLC genes with log2 fold change ≤ -0.5 and p ≤ 0.05 are shown. (D) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hours and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, SLC7A11 (xCT), SLC3A2 (4F2), SLC7A1 (CAT1), SLC1A4 (ASCT1), SLC1A5 (ASCT2), or actin. Molecular weight markers are indicated in kilodaltons. (E) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hours. Lysates were prepared and analyzed by immunoblot for the indicated proteins. (F) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hours. (G) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hours. Statistical significance was determined using an unpaired two-tailed t -test (N = 4), * p < 0.05, ** p < 0.01, *** p < 0.001.

    Techniques Used: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    (A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold-change for the sgRNAs for each gene is shown on the x axis. Significantly depleted genes ( p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC -genes in red are dependent on GCN2 for expression. (B) Plot of -Log10 (p-value) for depleted genes identified in CRISPR screen for LNCaP vs. 22Rv1 cells. Significantly depleted genes ( p ≥ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC-genes in red are GCN2-dependent. (C) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hours, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2 (4F2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. (D) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+ His), or media depleted of histidine (- His) for 24 hours. Lysates were analyzed by Immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2, or actin. (E) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hours or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. (F) SLC3A2 (4F2) was knocked down in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA control. Cell growth was measured in replicate wells (N = 5) for up to 6 days and are plotted relative to day 0 (mean ± SD). (G) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hours. Lysate were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, SLC3A2, or actin. (H) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells (N = 5) and is plotted relative to day 0 (mean ± SD). Statistical analysis is shown in Supplementary File 1 . (I) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 (N = 4), 4F2 (SLC3A2, N = 4), or scramble control (N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons Error bars indicate SD; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.
    Figure Legend Snippet: (A) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold-change for the sgRNAs for each gene is shown on the x axis. Significantly depleted genes ( p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC -genes in red are dependent on GCN2 for expression. (B) Plot of -Log10 (p-value) for depleted genes identified in CRISPR screen for LNCaP vs. 22Rv1 cells. Significantly depleted genes ( p ≥ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC-genes in red are GCN2-dependent. (C) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hours, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2 (4F2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. (D) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+ His), or media depleted of histidine (- His) for 24 hours. Lysates were analyzed by Immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, SLC3A2, or actin. (E) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hours or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. (F) SLC3A2 (4F2) was knocked down in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA control. Cell growth was measured in replicate wells (N = 5) for up to 6 days and are plotted relative to day 0 (mean ± SD). (G) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hours. Lysate were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, SLC3A2, or actin. (H) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells (N = 5) and is plotted relative to day 0 (mean ± SD). Statistical analysis is shown in Supplementary File 1 . (I) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 (N = 4), 4F2 (SLC3A2, N = 4), or scramble control (N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons Error bars indicate SD; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001.

    Techniques Used: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Transfection, Stable Transfection, Plasmid Preparation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including SLC3A2 (4F2) to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.
    Figure Legend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including SLC3A2 (4F2) to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Techniques Used: Activation Assay, Expressing

    (A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the materials and methods section. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± SEM (N = 4). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; *, p ≤ 0.05; ****, p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; **, p ≤ 0.01. (B) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tail t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05; ***, p ≤ 0.001. (C) Tumor growth of PC-3 WT and PC- 3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM (N = 5); ***, p ≤ 0.001; ****, p ≤ 0.0001. On the right bar graphs, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 5); *, p ≤ 0.01. (D) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of SLC7A5 (LAT1), SLC7A11 (xCT), and SLC3A2 (4F2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); **, p ≤ 0.01 . (E) 22Rv1 WT (N = 4), 22Rv1 GCN2 KO (clone 7, N = 5) and 22Rv1 ATF4 KO (N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. (F) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05. (G) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for figure , with or without supplementation of EAA in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT (N = 4) and 22Rv1 KO (N = 5) are the same tumor growth curves shown in 6E . 22Rv1 WT + EAA (N = 5), 22Rv1 GCN2 KO + EAA (N = 5) (H) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (I) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced SLC3A2 (4F2) lentivirus (WT + SLC3A2 and GCN2 KO + SLC3A2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM (N = 5).
    Figure Legend Snippet: (A) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the materials and methods section. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± SEM (N = 4). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; *, p ≤ 0.05; ****, p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; **, p ≤ 0.01. (B) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tail t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05; ***, p ≤ 0.001. (C) Tumor growth of PC-3 WT and PC- 3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM (N = 5); ***, p ≤ 0.001; ****, p ≤ 0.0001. On the right bar graphs, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 5); *, p ≤ 0.01. (D) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of SLC7A5 (LAT1), SLC7A11 (xCT), and SLC3A2 (4F2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); **, p ≤ 0.01 . (E) 22Rv1 WT (N = 4), 22Rv1 GCN2 KO (clone 7, N = 5) and 22Rv1 ATF4 KO (N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. (F) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05. (G) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for figure , with or without supplementation of EAA in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT (N = 4) and 22Rv1 KO (N = 5) are the same tumor growth curves shown in 6E . 22Rv1 WT + EAA (N = 5), 22Rv1 GCN2 KO + EAA (N = 5) (H) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (I) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced SLC3A2 (4F2) lentivirus (WT + SLC3A2 and GCN2 KO + SLC3A2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM (N = 5).

    Techniques Used: Injection, Two Tailed Test, Western Blot, Variant Assay, Molecular Weight, Plasmid Preparation

    Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N=4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors (N = 5) (D). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days per week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; *, p ≤ 0.05. (E) Protein lysates were prepared from 22Rv1 tumors treated with Vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05. (F) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (G) Pearson correlation between p-GCN2-T899 and SLC3A2 (4F2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal (N = 10), hyperplasia (N = 20) and malignant (N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and SLC3A2 (4F2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and SLC3A2 (4F2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). (H) Correlation of expression of SLC3A2 (4F2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the materials and methods.
    Figure Legend Snippet: Male NSG mice were injected subcutaneously with LNCaP (N = 5) (A) or 22Rv1 (N=4) (B) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 (N = 5) (C). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors (N = 5) (D). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days per week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD; *, p ≤ 0.05. (E) Protein lysates were prepared from 22Rv1 tumors treated with Vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, SLC7A5 (LAT1), SLC7A11 (xCT), SLC3A2 (4F2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); *, p ≤ 0.05. (F) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD (N = 4); #, p ≤ 0.1; *, p ≤ 0.05; **, p ≤ 0.01. (G) Pearson correlation between p-GCN2-T899 and SLC3A2 (4F2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal (N = 10), hyperplasia (N = 20) and malignant (N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and SLC3A2 (4F2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and SLC3A2 (4F2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). (H) Correlation of expression of SLC3A2 (4F2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the materials and methods.

    Techniques Used: Injection, Two Tailed Test, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    4f2hc  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc 4f2hc
    The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: <t>4F2hc,</t> <t>4F2</t> <t>heavy</t> <t>chain</t> of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.
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    1) Product Images from "Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study"

    Article Title: Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

    Journal: Environmental Health Perspectives

    doi: 10.1289/EHP9505

    The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: 4F2hc, 4F2 heavy chain of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.
    Figure Legend Snippet: The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: 4F2hc, 4F2 heavy chain of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.

    Techniques Used: Cell Culture, Western Blot, Inhibition, Expressing, Small Interfering RNA

    anti 4f2hc  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti 4f2hc
    Anti 4f2hc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    slc3a2 4f2hc cd98  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc slc3a2 4f2hc cd98
    FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and <t>SLC3A2</t> were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.
    Slc3a2 4f2hc Cd98, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "GPX4 Plays a Crucial Role in Fuzheng Kang’ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis"

    Article Title: GPX4 Plays a Crucial Role in Fuzheng Kang’ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2022.851680

    FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and SLC3A2 were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.
    Figure Legend Snippet: FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and SLC3A2 were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.

    Techniques Used: Expressing, Western Blot, GSSG Assay

    Validation of FZKA-induced NSCLC cell ferroptosis in vivo . (A) , Mice tumor photograph and tumor weight was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (B) , Tumor volume in each group was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (C) , Western blot analyses of GPX4, SLC7A11 and SLC3A2 expression from tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (D) , Immunohistochemistry was carried out to measure the expression of GPX4, SLC7A11 and SLC3A2 in mice tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (E) , The diagram showing FZKA induced NSCLC cell ferroptosis through system xc − /GSH/GPX4 axis, and, importantly, GPX4 is the crucial molecular in the process. Finally, inhibition of GPX4 by FZKA leads to NSCLC cell ferroptosis.
    Figure Legend Snippet: Validation of FZKA-induced NSCLC cell ferroptosis in vivo . (A) , Mice tumor photograph and tumor weight was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (B) , Tumor volume in each group was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (C) , Western blot analyses of GPX4, SLC7A11 and SLC3A2 expression from tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (D) , Immunohistochemistry was carried out to measure the expression of GPX4, SLC7A11 and SLC3A2 in mice tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (E) , The diagram showing FZKA induced NSCLC cell ferroptosis through system xc − /GSH/GPX4 axis, and, importantly, GPX4 is the crucial molecular in the process. Finally, inhibition of GPX4 by FZKA leads to NSCLC cell ferroptosis.

    Techniques Used: In Vivo, Western Blot, Expressing, Immunohistochemistry, Inhibition

    anti cd98hc  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cd98hc
    Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, <t>CD98hc</t> and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown
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    1) Product Images from "Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer"

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-022-02330-4

    Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, CD98hc and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown
    Figure Legend Snippet: Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, CD98hc and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown

    Techniques Used: Microarray, Expressing, Western Blot, Quantitation Assay, Immunoprecipitation

    Expression of CD98hc in normal and TNBC tumoral tissue. A Immunohistochemical stainings of CD98hc showing tumor tissue scored as high (+++), intermediate (++) and low (+), and normal tissue with negative (-) staining. Magnification: 40X. B Immunohistochemical staining of CD98hc in a sample of a patient, showing staining of CD98hc only in tumor tissue and not in normal tissue. Magnification: 20 X. C Box plot showing SLC3A2 gene expression levels in normal and tumor tissue of breast cancer patients. Data were obtained from the TNMplot database. D Box plot showing SLC3A2 gene expression levels in different breast cancer subtypes from patients with breast cancer. The data were obtained using the Xena online tool
    Figure Legend Snippet: Expression of CD98hc in normal and TNBC tumoral tissue. A Immunohistochemical stainings of CD98hc showing tumor tissue scored as high (+++), intermediate (++) and low (+), and normal tissue with negative (-) staining. Magnification: 40X. B Immunohistochemical staining of CD98hc in a sample of a patient, showing staining of CD98hc only in tumor tissue and not in normal tissue. Magnification: 20 X. C Box plot showing SLC3A2 gene expression levels in normal and tumor tissue of breast cancer patients. Data were obtained from the TNMplot database. D Box plot showing SLC3A2 gene expression levels in different breast cancer subtypes from patients with breast cancer. The data were obtained using the Xena online tool

    Techniques Used: Expressing, Immunohistochemical staining, Negative Staining, Staining

    Internalization of an antibody against CD98hc. A Subcellular localization of CD98hc in MDA-MB231 and HCC3153 cells was analyzed by immunofluorescence. Scale bar = 25 μm. B Cell surface immunoprecipitation of CD98hc. The different TNBC cell lines treated or not with 10 nM of anti-CD98hc for 2 hours at 4°C were lysed and cell extracts precipitated with protein A-sepharose. CD98hc in those immunoprecipitates was analyzed by Western. C Protease protection experiments of CD98hc. Cells were treated with proteinase K, lysed and subjected to immunoprecipitation and Western analysis with the anti-CD98hc antibody. D FACS analyses of CD98hc cell surface expression. MDA-MB231 cells were incubated with 10 nM of anti-CD98hc for 20 minutes at 37°C. Cells were detached, incubated with an anti-mouse antibody conjugated to FITC, and fluorescence intensity was measured with a BD FACSAria TM III cytometer. The yellow histogram corresponds to signals from cells incubated with the secondary antibody alone, whereas the pink histogram represents the fluorescence due to the expression of CD98hc. E MDA-MB231 cells were seeded on coverslips and treated with 10 nM of anti-CD98hc for the times indicated. Scale bar = 25 μm. The images at the bottom of this section correspond to magnifications of a cell present in the images obtained at 24 hours. The white arrows indicate colocalization of CD98hc and LAMP1. Scale bar = 7.5 μm
    Figure Legend Snippet: Internalization of an antibody against CD98hc. A Subcellular localization of CD98hc in MDA-MB231 and HCC3153 cells was analyzed by immunofluorescence. Scale bar = 25 μm. B Cell surface immunoprecipitation of CD98hc. The different TNBC cell lines treated or not with 10 nM of anti-CD98hc for 2 hours at 4°C were lysed and cell extracts precipitated with protein A-sepharose. CD98hc in those immunoprecipitates was analyzed by Western. C Protease protection experiments of CD98hc. Cells were treated with proteinase K, lysed and subjected to immunoprecipitation and Western analysis with the anti-CD98hc antibody. D FACS analyses of CD98hc cell surface expression. MDA-MB231 cells were incubated with 10 nM of anti-CD98hc for 20 minutes at 37°C. Cells were detached, incubated with an anti-mouse antibody conjugated to FITC, and fluorescence intensity was measured with a BD FACSAria TM III cytometer. The yellow histogram corresponds to signals from cells incubated with the secondary antibody alone, whereas the pink histogram represents the fluorescence due to the expression of CD98hc. E MDA-MB231 cells were seeded on coverslips and treated with 10 nM of anti-CD98hc for the times indicated. Scale bar = 25 μm. The images at the bottom of this section correspond to magnifications of a cell present in the images obtained at 24 hours. The white arrows indicate colocalization of CD98hc and LAMP1. Scale bar = 7.5 μm

    Techniques Used: Immunofluorescence, Immunoprecipitation, Western Blot, Expressing, Incubation, Fluorescence, Cytometry

    Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T ( D ) and MDA-MB231 ( E ) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times
    Figure Legend Snippet: Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T ( D ) and MDA-MB231 ( E ) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times

    Techniques Used: Activity Assay, Western Blot, SDS Page, Staining, Knock-Out, CRISPR, Clone Assay, Expressing

    The anti-CD98hc-DM1 antibody provokes cell cycle arrest in mitosis and mitotic catastrophe. A Effect of anti-CD98-DM1 (10 nM, 24 hours) on the morphology of MDA-MB231 and HS578T cells grown as monolayers. The images were taken at 10X magnification. B Quantitative analyses of the action of anti-CD98hc-DM1 on the distribution of the different cell cycle phases in MDA-MB231 and HS578T cell lines. C MDA-MB231 and HS578T cells were treated with anti-CD98hc-DM1 (10 nM) and lysed at the indicated times. Analyses of the amounts of the different proteins studied were performed by Western blotting. GAPDH was used as loading control. D Effect of anti-CD98hc-DM1 on spindle assembly and organization. MDA-MB231 cells seeded on coverslips were treated with CD98hc-DM1 (10 nM) for 24 hours, fixed and stained. Scale bars are indicated. E Detection of giant multinucleated cells after anti-CD98hc-DM1 treatment. MDA-MB231 cells were treated with 10 nM anti-CD98hc-DM1 for 48 hours, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 7.5 μm. F Quantitation of abnormal mitoses, normal mitoses and interphase cells from the experiment shown in ( D ). G Bar graph representation of the percentage of viable (Annexin V-negative/PI-negative) and non-viable MDA-MB231 cells at 48 and 72 hours of treatment with 10 nM anti-CD98hc-DM1. H Effect of anti-CD98hc-DM1 on the levels of several apoptosis-related proteins. MDA-MB231 and HS578T cells were treated with 10 nM anti-CD98hc-DM1, lysed at 0, 1, 2, or 3 days and the indicated proteins analyzed by Western
    Figure Legend Snippet: The anti-CD98hc-DM1 antibody provokes cell cycle arrest in mitosis and mitotic catastrophe. A Effect of anti-CD98-DM1 (10 nM, 24 hours) on the morphology of MDA-MB231 and HS578T cells grown as monolayers. The images were taken at 10X magnification. B Quantitative analyses of the action of anti-CD98hc-DM1 on the distribution of the different cell cycle phases in MDA-MB231 and HS578T cell lines. C MDA-MB231 and HS578T cells were treated with anti-CD98hc-DM1 (10 nM) and lysed at the indicated times. Analyses of the amounts of the different proteins studied were performed by Western blotting. GAPDH was used as loading control. D Effect of anti-CD98hc-DM1 on spindle assembly and organization. MDA-MB231 cells seeded on coverslips were treated with CD98hc-DM1 (10 nM) for 24 hours, fixed and stained. Scale bars are indicated. E Detection of giant multinucleated cells after anti-CD98hc-DM1 treatment. MDA-MB231 cells were treated with 10 nM anti-CD98hc-DM1 for 48 hours, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 7.5 μm. F Quantitation of abnormal mitoses, normal mitoses and interphase cells from the experiment shown in ( D ). G Bar graph representation of the percentage of viable (Annexin V-negative/PI-negative) and non-viable MDA-MB231 cells at 48 and 72 hours of treatment with 10 nM anti-CD98hc-DM1. H Effect of anti-CD98hc-DM1 on the levels of several apoptosis-related proteins. MDA-MB231 and HS578T cells were treated with 10 nM anti-CD98hc-DM1, lysed at 0, 1, 2, or 3 days and the indicated proteins analyzed by Western

    Techniques Used: Western Blot, Staining, Quantitation Assay

    The anti-CD98hc-DM1 has antitumoral activity in vivo and potentiates the action of standard of care drugs. A Analysis of the “in vivo” effect of anti-CD98hc-DM1 on tumor growth in nude mice implanted with MDA-MB231 cells. Arrows indicate days of administration of anti-CD98hc-DM1. Data are plotted as mean tumor volumes ± SEM. P values were calculated using Student t test (two-sided). B Effect of CD98hc-DM1 on the weight of mice. Data are plotted as mean ± SD of six mice/group. C Analyses of DM1, pH3 and pH2AX levels in tumors from mice. Seventeen days after the last treatment, tumors were resected and immediately frozen in liquid nitrogen. Expression of DM1, pH3 and pH2AX was analyzed by Western. β-actin was used as a loading control. D Quantitation of the levels of DM1, pH3 and pH2AX of the experiment performed in ( C ). The graphs represent the mean ± S.D of the different proteins (arbitrary units) of control and treated (anti-CD98hc-DM1) groups. Student’s t test was used to analyze differences among groups. ( E and F ) MDA-MB231 cells were treated with CD98hc-DM1 alone or in combination with Docetaxel ( E ) or Vinorelbine ( F ) for four days at the indicated doses (nM)
    Figure Legend Snippet: The anti-CD98hc-DM1 has antitumoral activity in vivo and potentiates the action of standard of care drugs. A Analysis of the “in vivo” effect of anti-CD98hc-DM1 on tumor growth in nude mice implanted with MDA-MB231 cells. Arrows indicate days of administration of anti-CD98hc-DM1. Data are plotted as mean tumor volumes ± SEM. P values were calculated using Student t test (two-sided). B Effect of CD98hc-DM1 on the weight of mice. Data are plotted as mean ± SD of six mice/group. C Analyses of DM1, pH3 and pH2AX levels in tumors from mice. Seventeen days after the last treatment, tumors were resected and immediately frozen in liquid nitrogen. Expression of DM1, pH3 and pH2AX was analyzed by Western. β-actin was used as a loading control. D Quantitation of the levels of DM1, pH3 and pH2AX of the experiment performed in ( C ). The graphs represent the mean ± S.D of the different proteins (arbitrary units) of control and treated (anti-CD98hc-DM1) groups. Student’s t test was used to analyze differences among groups. ( E and F ) MDA-MB231 cells were treated with CD98hc-DM1 alone or in combination with Docetaxel ( E ) or Vinorelbine ( F ) for four days at the indicated doses (nM)

    Techniques Used: Activity Assay, In Vivo, Expressing, Western Blot, Quantitation Assay

    anti slc3a2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti slc3a2 antibody
    (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) <t>SLC3A2</t> expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.
    Anti Slc3a2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "TYRO3 induces anti–PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis"

    Article Title: TYRO3 induces anti–PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI139434

    (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) SLC3A2 expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.
    Figure Legend Snippet: (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) SLC3A2 expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.

    Techniques Used: Expressing, Luciferase, Reporter Assay, Plasmid Preparation

    cd98hc  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc cd98hc
    ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse <t>LAT1/CD98hc</t> expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.
    Cd98hc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target"

    Article Title: Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27981

    ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse LAT1/CD98hc expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.
    Figure Legend Snippet: ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse LAT1/CD98hc expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, Negative Control, SDS Page

    ( A ) Anchorage-dependent growth in Matrigel. After the cell culture for 7 days, the number of colonies larger than 50 μm (Lower left) and colonies with pseudopods (Lower right) was counted. The white arrow indicates a colony with pseudopod (Upper). ( B ) Anchorage-independent growth in 3D culture. Cells (2.5 × 10 2 ) in 6-well plates were cultured for 14 days in soft agar, stained with Giemsa solution, and the number of colonies was analyzed by Image J. Statistical analysis was carried out using one-way ANOVA. ( C ) Tumor formation by NIH/3T3 cell lines overexpressing LAT1. Tumor volumes were measured every two days. ( D ) FCM using dispersed cell suspension from in vitro cultured and in vivo (tumor-derived) naLAT1/3T3 was carried out. Ab1, anti-human LAT1 mAb; HR35, anti-human CD98hc mAb.
    Figure Legend Snippet: ( A ) Anchorage-dependent growth in Matrigel. After the cell culture for 7 days, the number of colonies larger than 50 μm (Lower left) and colonies with pseudopods (Lower right) was counted. The white arrow indicates a colony with pseudopod (Upper). ( B ) Anchorage-independent growth in 3D culture. Cells (2.5 × 10 2 ) in 6-well plates were cultured for 14 days in soft agar, stained with Giemsa solution, and the number of colonies was analyzed by Image J. Statistical analysis was carried out using one-way ANOVA. ( C ) Tumor formation by NIH/3T3 cell lines overexpressing LAT1. Tumor volumes were measured every two days. ( D ) FCM using dispersed cell suspension from in vitro cultured and in vivo (tumor-derived) naLAT1/3T3 was carried out. Ab1, anti-human LAT1 mAb; HR35, anti-human CD98hc mAb.

    Techniques Used: Cell Culture, Staining, In Vitro, In Vivo, Derivative Assay

    TCGA data ( A – C ) were obtained from the UCSC Xena browser. (A) Box-whisker plot of the expression of CD98hc (right) /LAT1 (left) in normal and tumor colorectal tissues. P -values were calculated by the Student’s t test. (B) Box-whisker plot of the expression of LAT1 in non-metastatic and metastatic ccRCC. The presence of metastasis was determined based on pathological TMN classification. P -values were calculated by the Student’s t test. (C) Kaplan-Meier survival analysis with the log rank test of ccRCC and bladder cancer patient. The correlation between mRNA expression (top 25% and lower 25%) and survival was evaluated. ( D ) Effects of CD98hc-KO on the expression of LAT1. CD98hc in HE293F and SW1116 cells was disrupted by the CRISPR/Cas9-based method, and the expression of CD98hc and LAT1 was analyzed by FCM. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using various human cell lines. Lysates were subjected to SDS-PAGE under reducing or non-reducing conditions, and to WB with anti-LAT1 rabbit pAb.
    Figure Legend Snippet: TCGA data ( A – C ) were obtained from the UCSC Xena browser. (A) Box-whisker plot of the expression of CD98hc (right) /LAT1 (left) in normal and tumor colorectal tissues. P -values were calculated by the Student’s t test. (B) Box-whisker plot of the expression of LAT1 in non-metastatic and metastatic ccRCC. The presence of metastasis was determined based on pathological TMN classification. P -values were calculated by the Student’s t test. (C) Kaplan-Meier survival analysis with the log rank test of ccRCC and bladder cancer patient. The correlation between mRNA expression (top 25% and lower 25%) and survival was evaluated. ( D ) Effects of CD98hc-KO on the expression of LAT1. CD98hc in HE293F and SW1116 cells was disrupted by the CRISPR/Cas9-based method, and the expression of CD98hc and LAT1 was analyzed by FCM. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using various human cell lines. Lysates were subjected to SDS-PAGE under reducing or non-reducing conditions, and to WB with anti-LAT1 rabbit pAb.

    Techniques Used: Whisker Assay, Expressing, CRISPR, SDS Page

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    Cell Signaling Technology Inc slc3a2
    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 <t>(SLC3A2)</t> AR, or actin. Molecular weight markers are indicated in kilodaltons.
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    Cell Signaling Technology Inc slc3a2 4f2
    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), <t>4F2</t> <t>(SLC3A2)</t> AR, or actin. Molecular weight markers are indicated in kilodaltons.
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    Cell Signaling Technology Inc 4f2hc
    The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: <t>4F2hc,</t> <t>4F2</t> <t>heavy</t> <t>chain</t> of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.
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    Cell Signaling Technology Inc anti 4f2hc
    The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: <t>4F2hc,</t> <t>4F2</t> <t>heavy</t> <t>chain</t> of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.
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    Cell Signaling Technology Inc slc3a2 4f2hc cd98
    FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and <t>SLC3A2</t> were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.
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    Cell Signaling Technology Inc anti cd98hc
    Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, <t>CD98hc</t> and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown
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    Cell Signaling Technology Inc anti slc3a2 antibody
    (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) <t>SLC3A2</t> expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.
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    Cell Signaling Technology Inc cd98hc
    ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse <t>LAT1/CD98hc</t> expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.
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    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

    ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

    ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Two Tailed Test, Standard Deviation, Western Blot

    ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

    LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Transduction, CRISPR, Cell Culture, Amplification

    ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

    ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Activation Assay, Expressing

    ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Injection, Two Tailed Test, Standard Deviation, Western Blot, Variant Assay, Molecular Weight, Transduction, Plasmid Preparation

    ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Staining, Two Tailed Test, Standard Deviation, Injection

    ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

    ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Standard Deviation, Western Blot, Variant Assay, Molecular Weight

    ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Standard Deviation, Western Blot, Molecular Weight

    Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Article Snippet: Tissue blocking was performed with 5% Horse Serum (Vector Laboratories Cat. #PK-7200) in PBS and incubated with primary antibodies: Ki-67 (1:200, Abcam Cat. #ab16667, RRID: AB_302459 ), p-GCN2-T899 (1:100, Abcam Cat. #ab75836, RRID: AB_1310260 ), and SLC3A2 (4F2hc/CD98) (1:500, Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ).

    Techniques: Microarray, Immunohistochemistry, Two Tailed Test, Staining

    ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

    ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

    ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Volcano plot illustrating log 2 fold change in gene transcript levels with adjusted p value (−log 10 ) comparing LNCaP cells treated with GCN2iB (2 µM) versus vehicle control (DMSO) for 24 hr. Several amino acid transporters reduced by GCN2iB treatment are highlighted. ( B ) Plots from gene set enrichment analysis (GSEA) of gene expression in LNCaP cells treated with GCN2iB (2 µM) for 24 hr versus vehicle control. ( C ) Heat map displaying significantly downregulated SLC genes as indicated in panel A . The heat map compares gene transcript levels from LNCaP cells treated with vehicle (DMSO), or GCN2iB (2 µM) for 6 or 24 hr. Four biological replicates were measured for each treatment group. Transcript levels (normalized read counts) are shown relative to the average of the vehicle control samples for each gene. ( D ) Lysates were prepared from LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr and immunoblot analysis were carried out using antibodies that recognize ATF4, ASNS, xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), or actin. Molecular weight markers are indicated in kilodaltons. ( E ) 22Rv1 WT cells, 22Rv1 GCN2 KO cells, and 22Rv1 GCN2 KO complemented with GCN2 cells were cultured for 24 hr. Lysates were prepared and analyzed by immunoblot for the indicated proteins. ( F ) Amino acid uptake measurements in LNCaP and 22Rv1 cells treated with vehicle (DMSO) or GCN2iB (2 µM) for 24 hr. ( G ) Amino acid uptake measurements for 22Rv1 WT or 22Rv1 GCN2 KO cells cultured for 24 hr. Statistical significance was determined using an unpaired two-tailed t -test ( N = 4); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Expressing, Western Blot, Molecular Weight, Cell Culture, Two Tailed Test

    ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) LNCaP cells were treated with 2 µM GCN2iB or vehicle control (DMSO) for 6 or 24 hr, protein lysates were prepared, and immunoblotted for the indicated proteins. The bar graphs show the relative levels of the indicated proteins normalized to actin. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. ( B ) Immunoblot analysis of PC-3 WT, PC-3 GCN2 KO (clone C-2), and PC-3 GCN2 KO (clone C-3) lysates using antibodies that recognize GCN2, ATF4, ASNS, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), ASNS, or actin.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Two Tailed Test, Standard Deviation, Western Blot

    ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

    LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: LNCaP and 22Rv1 cells were transduced with a SLC KO CRISPR/Cas9 library targeting 394 human SLC genes and pseudogenes. Transduced cells were cultured for 3–4 weeks and single guide RNA (sgRNA) sequences were amplified by PCR and sequenced to determine the composition of the sgRNA library compared to Control (day 0). Scatterplot of sgRNA counts (log10, normalized) for control ( N = 3) versus LNCaP ( N = 3) and control ( N = 3) versus 22Rv1 ( N = 3). Non-targeting sgRNAs (red), significant depleted sgRNAs (blue) (p ≤ 0.05), and 4F2 (SLC3A2) sgRNAs (orange) are indicated.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Transduction, CRISPR, Cell Culture, Amplification

    ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) 4F2 (SLC3A2) and ATF4 mRNA were measured by qRT-PCR as described in the Materials and methods in LNCaP cells treated with 2 µM GCN2iB for 6 or 24 hr or vehicle control (DMSO), ( B ) cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (− His) for 24 hr, or ( C ) treated with 100 nM halofuginone (HF) for 2 or 6 hr or untreated (DMSO control). Error bars indicate standard deviation (SD) ( N = 3). An unpaired two-tailed t -test was used to determine statistical significance; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Quantitative RT-PCR, Cell Culture, Standard Deviation, Two Tailed Test

    ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

    Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: Model depicting the role of GCN2 in regulating SLC amino acid transporters. Enhanced translation and altered metabolism driven by oncogenes deplete amino acid pools resulting in accumulation of uncharged tRNAs, leading to activation of GCN2. Active GCN2 results in increased expression of SLC amino acid transporters, including 4F2 (SLC3A2), to increase uptake of amino acids. Loss of GCN2 function disrupts amino acid homeostasis decreasing proliferation of prostate cancer cells.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Activation Assay, Expressing

    ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) WT or GCN2 KO 22Rv1 (clone 7) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volume (TV) was measured on indicated days and is plotted as average TV ± standard error of the mean (SEM) ( N = 4). Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison; *p ≤ 0.05; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint and statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); **p ≤ 0.01. ( B ) Protein lysates were prepared from WT and GCN2 KO 22Rv1 tumors and analyzed by immunoblot to measure total GCN2, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the SLC proteins normalized to actin are shown in the bar graph (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05; ***p ≤ 0.001. ( C ) Tumor growth of PC-3 WT and PC-3 GCN2 KO (clone 3) cells was analyzed in a mouse xenograft study as in A. Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison. Error bars indicate SEM ( N = 5); ***p ≤ 0.001; ****p ≤ 0.0001. On the right bar graph, the final tumor weight was measured at endpoint. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.01. ( D ) Protein lysates were prepared from the PC-3 WT and PC-3 GCN2 KO tumors and analyzed by immunoblot for the indicated proteins. (Right panels) Quantification of protein levels of LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2) normalized to actin are shown in the bar graphs. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); **p ≤ 0.01. ( E ) 22Rv1 WT ( N = 4), 22Rv1 GCN2 KO (clone 7, N = 5), and 22Rv1 ATF4 KO ( N = 5) were evaluated in the mouse xenograft model. Tumor volumes were measured on the indicated days. Error bars indicated SEM. ( F ) Amino acid measurements of 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); # p ≤ 0.1, *p ≤ 0.05. ( G ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were analyzed in a xenograft model as described for ( A ), with or without supplementation of essential amino acid (EAA) in the drinking water. Tumor volume was measured on indicated days. 22Rv1 WT ( N = 4) and 22Rv1 KO ( N = 5) are the same tumor growth curves shown in ( E ). 22Rv1 WT + EAA ( N = 5), 22Rv1 GCN2 KO + EAA ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001. ( H ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2, and 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids present in EAA supplemented water. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicated SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( I ) Tumor growth curves for 22Rv1 WT or 22Rv1 GCN2 KO (clone 11) transduced with 4F2 (SLC3A2) lentivirus (WT + 4F2 and GCN2 KO + 4F2) or empty vector (WT + EV and GCN2 KO + EV). Tumor volumes were measured on indicated days. Error bars indicate SEM ( N = 5). Statistical significance was determined using a two-way ANOVA with Sidak’s multiple comparison; ****p ≤ 0.0001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Injection, Two Tailed Test, Standard Deviation, Western Blot, Variant Assay, Molecular Weight, Transduction, Plasmid Preparation

    ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of 22Rv1 WT and 22Rv1 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 4); **p ≤ 0.01, ****p ≤ 0.0001. ( B ) PC-3 WT, and PC-3 GCN2 KO clone 2 (C-2), or PC-3 GCN2 KO clone 3 (C-3) cells were injected subcutaneously into the dorsal flank of mice as described in the Materials and methods. Tumor volumes were measured on the indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM) ( N = 5); *p ≤ 0.05; ***p ≤ 0.001; ****p ≤ 0.0001. Bar graph indicates final tumor weight measured at endpoint. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 5); **p ≤ 0.01. ( C ) Representative images for Ki-67, p-GCN2-T899, or 4F2 (SLC3A2) staining of PC-3 WT and PC-3 GCN2 KO tumors are shown. Bar graph shows quantification for percent positive nuclear staining (Ki67) or Histoscore (p-GCN2-T899 and 4F2). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 5); *p ≤ 0.05; **p ≤ 0.01.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Staining, Two Tailed Test, Standard Deviation, Injection

    ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

    ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Body weight measurements from mice bearing 22Rv1 WT or 22Rv1 GCN2 KO tumors with or without EAA supplementation in the drinking water as described in . Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison, **p ≤ 0.01. ( B ) Amino acid measurements for 22Rv1 WT, 22Rv1 GCN2 KO, or 22Rv1 GCN2 KO + EAA tumors. Bar graphs show only amino acids not present in EAA supplemented water. Error bars indicate standard deviation (SD) ( N = 4). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1; *p ≤ 0.05. ( C ) Protein lysates were prepared from tumors described in and analyzed by immunoblot to measure total GCN2, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASNS, androgen receptor (AR), AR splice variant 7 (ARv7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. The levels of the indicated proteins normalized to actin are shown in the bar graphs. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 4); ns, p > 0.05; *p ≤ 0.05, **,p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Standard Deviation, Western Blot, Variant Assay, Molecular Weight

    ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) Amino acid measurements from 22Rv1 WT + EV, 22Rv1 GCN2 KO + EV, and 22Rv1 GCN2 KO + 4F2 (SLC3A2) tumors. Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); # p ≤ 0.1, **p ≤ 0.01. ( B ) Lysates were prepared from these tumors in A were analyzed by immunoblot to measure total GCN2, 4F2 (SLC3A2), or actin. A long and short exposure for the 4F2 (SLC3A2) immunoblot is shown. Molecular weight markers are indicated in kilodaltons for each immunoblot panel.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Standard Deviation, Western Blot, Molecular Weight

    Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: Male NSG mice were injected subcutaneously with LNCaP ( N = 5) ( A ) or 22Rv1 ( N = 4) ( B ) cells, or alternatively implanted with tumor fragments from an androgen-sensitive tumor TM00298 ( N = 5) ( C ). Male castrated NSG mice were implanted with tumor fragments from LuCaP-35 CR tumors ( N = 5) ( D ). Mice were treated with vehicle or 30 mg/kg GCN2iB twice daily for 5 days/week and tumor volumes were measured on indicated days. Statistical significance was determined using a two-way analysis of variance (ANOVA) with Sidak’s multiple comparison. Error bars indicate standard error of the mean (SEM); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Final tumor weight was measured at endpoint and is represented in bar graphs (right panels). Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD); *p ≤ 0.05. ( E ) Protein lysates were prepared from 22Rv1 tumors treated with vehicle or GCN2iB and analyzed by immunoblot for phosphorylated GCN2-T899, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), and 4F2 (SLC3A2), and actin. The levels of the SLC proteins normalized to actin are shown. Phosphorylated GCN2-T899 was normalized to total GCN2. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); *p ≤ 0.05. ( F ) Amino acid measurements of 22Rv1 tumors treated with vehicle or GCN2iB. Bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate SD ( N = 4); # p ≤ 0.1; *p ≤ 0.05; **p ≤ 0.01. ( G ) Pearson correlation between p-GCN2-T899 and 4F2 (SLC3A2) histoscores calculated from IHC staining from a prostate tumor microarray (Biomax PR807c) containing normal ( N = 10), hyperplasia ( N = 20), and malignant ( N = 50) for all tissues (combined) or Gleason scores 4 and 5. The center lines depict linear regression (95% confidence intervals). Not all samples were analyzed due to damaged/quality of tissue samples. Levels of p-GCN2-T899 and 4F2 (SLC3A2) were measured by IHC staining and QuPath was used to determine the histoscore. Two representative cases are shown for high (Case 1) and low (Case 2) p-GCN2-T899 and 4F2 (SLC3A2) staining. Scale bar indicates 200 µm (main image) and 20 µm (insert). ( H ) Correlation of expression of 4F2 (SLC3A2) and a GCN2-dependent gene signature in prostate adenocarcinoma (PRAD, N = 551) from the Cancer Genome Atlas (TCGA). The GCN2-dependepent gene signature was derived from RNA-seq data as described in the Materials and methods.

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Injection, Two Tailed Test, Standard Deviation, Western Blot, Immunohistochemistry, Microarray, Staining, Expressing, Derivative Assay, RNA Sequencing Assay

    ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Journal: eLife

    Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

    doi: 10.7554/eLife.81083

    Figure Lengend Snippet: ( A ) The protein levels of 4F2 (SLC3A2) were measured in a prostate tumor microarray (Biomax PR807c) using immunohistochemistry (IHC). Staining for 4F2 (SLC3A2) from normal prostate tissue ( N = 10) and malignant prostate cancer tissue ( N = 50) was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing 4F2 (SLC3A2) staining of normal and malignant prostate tissues are shown. Scale bars: 200 µm (main image) and 20 µm (insert). ( B ) H&E and IHC staining using p-GCN2-T899 or 4F2 (SLC3A2) antibody in prostate needle biopsy specimens from patients with high grade prostate cancer with Gleason scores 5 + 4 = 9 (top) or 4 + 5 = 9 (bottom).

    Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

    Techniques: Microarray, Immunohistochemistry, Two Tailed Test, Staining

    The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: 4F2hc, 4F2 heavy chain of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.

    Journal: Environmental Health Perspectives

    Article Title: Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study

    doi: 10.1289/EHP9505

    Figure Lengend Snippet: The levels of PQ + transporters in cultured astrocytes with or without DRP1 knockdown exposed to control or PQ + . (A,B) Representative images of Western blots showing three major monovalent cation transporters in GL261 astrocytes with or without DRP1 inhibition by Drp1 siRNA or mdivi-1. Scrambled siRNA was used as control for Drp1 siRNA exposed to control or PQ + . (C–H) Quantitative analysis of the relative expression of the transporters shown in A and B. The group of PQ + (–) scramble siRNA were normalized to 1. n = 5 independent experiments. Data are shown as mean ± SEM . Two-way ANOVA followed by the Bonferroni multiple comparison test. ***, p < 0.001 . The numeric data are shown in Excel Table S6. In two-way ANOVAs, “*” was used to present the statistical difference between groups both treated by PBS or PQ 2 + . Note: 4F2hc, 4F2 heavy chain of L-type amino acid transporter 1; ANOVA, analysis of variance; ASCT2, alanine serine cysteine transporter 2; DRP1, dynamic related protein-1; mdivi-1, mitochondrial division inhibitor-1; PBS, phosphate-buffered saline; PQ, paraquat; Scramble, empty AAV for control; SEM, standard error of the mean; siRNA, small interfering RNA; SLC1A4, solute carrier family 1 member 4.

    Article Snippet: The membranes were blocked with 5% wt/vol bovine serum albumin (BSA) for 1 h and incubated in primary antibodies (1:1,000 vol/vol) of DRP1 (Cell Signaling Technology; Cat. No. 8570), ASCT2 (Cell Signaling Technology; Cat. No. 8057), SLC1A4 (Cell Signaling Technology; Cat. No. 8442), 4F2hc (Cell Signaling Technology; Cat. No. 47213), β -actin (Cell Signaling Pathway; Cat. No. 13E5), and OCT3 (Affinity; Cat. No. AF5358) separately at 4°C overnight.

    Techniques: Cell Culture, Western Blot, Inhibition, Expressing, Small Interfering RNA

    FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and SLC3A2 were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.

    Journal: Frontiers in Pharmacology

    Article Title: GPX4 Plays a Crucial Role in Fuzheng Kang’ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis

    doi: 10.3389/fphar.2022.851680

    Figure Lengend Snippet: FZKA decreased the ratio of GSH/GSSG and expression of system xc − . (A) , The protein expression levels of SLC7A11 and SLC3A2 were detected by Western blot. (B) , the levels of GSH and GSSG were measured by GSH and GSSG Assay kit. Each point represents the mean ± SEM, n = 3. * p < 0.05.

    Article Snippet: The antibodies were obtained from the following sources: GPX4 (ab125066) and GAPDH (ab9485) were purchased from Abcam (Cambridge, United Kingdom); SLC3A2 (4F2hc/CD98) (47213S), SLC7A11 (12691S), horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (7074S), were from Cell Signalling Technology (Danvers, MA); SLC7A11 (bs-6883R) for immunohistochemistry was obtained from Bioss Biological Technology Co. Ltd. (Beijing, China).

    Techniques: Expressing, Western Blot, GSSG Assay

    Validation of FZKA-induced NSCLC cell ferroptosis in vivo . (A) , Mice tumor photograph and tumor weight was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (B) , Tumor volume in each group was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (C) , Western blot analyses of GPX4, SLC7A11 and SLC3A2 expression from tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (D) , Immunohistochemistry was carried out to measure the expression of GPX4, SLC7A11 and SLC3A2 in mice tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (E) , The diagram showing FZKA induced NSCLC cell ferroptosis through system xc − /GSH/GPX4 axis, and, importantly, GPX4 is the crucial molecular in the process. Finally, inhibition of GPX4 by FZKA leads to NSCLC cell ferroptosis.

    Journal: Frontiers in Pharmacology

    Article Title: GPX4 Plays a Crucial Role in Fuzheng Kang’ai Decoction-Induced Non-Small Cell Lung Cancer Cell Ferroptosis

    doi: 10.3389/fphar.2022.851680

    Figure Lengend Snippet: Validation of FZKA-induced NSCLC cell ferroptosis in vivo . (A) , Mice tumor photograph and tumor weight was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (B) , Tumor volume in each group was showed. Data represents Mean ± SEM, n = 7. * p < 0.05. (C) , Western blot analyses of GPX4, SLC7A11 and SLC3A2 expression from tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (D) , Immunohistochemistry was carried out to measure the expression of GPX4, SLC7A11 and SLC3A2 in mice tumor tissues. Data represents Mean ± SEM, n = 7. * p < 0.05. (E) , The diagram showing FZKA induced NSCLC cell ferroptosis through system xc − /GSH/GPX4 axis, and, importantly, GPX4 is the crucial molecular in the process. Finally, inhibition of GPX4 by FZKA leads to NSCLC cell ferroptosis.

    Article Snippet: The antibodies were obtained from the following sources: GPX4 (ab125066) and GAPDH (ab9485) were purchased from Abcam (Cambridge, United Kingdom); SLC3A2 (4F2hc/CD98) (47213S), SLC7A11 (12691S), horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (7074S), were from Cell Signalling Technology (Danvers, MA); SLC7A11 (bs-6883R) for immunohistochemistry was obtained from Bioss Biological Technology Co. Ltd. (Beijing, China).

    Techniques: In Vivo, Western Blot, Expressing, Immunohistochemistry, Inhibition

    Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, CD98hc and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: Identification of surface proteins differentially expressed in TNBC. A Schematic representation of the genomic and proteomic approaches. For the genomic approach, microarray data from normal and tumoral triple negative breast cancer of patients obtained by us (SUH) or deposited in databases (HBS and TBC) were used. The number of up-regulated cell surface proteins found using each dataset is shown. The proteomic approach was based on cell surface biotinylation and plasma membrane enrichment to detect surface proteins from MDA-MB231, BT549 and HS578T cells. Upon Orbitrap identification, plasma membrane proteins were selected using the Surfaceome database. B List of possible protein targets (score ≥5) ranked from highest to lowest score. The scoring criteria (one point per analysis) are described in the main text of this paper. The maximum score of 9, would be given to a protein identified in the three cell lines in the two proteomic methods and also identified in the three gene expression arrays. C Levels of expression of LAT1, CD98hc and GLUT1 in a panel of TNBC cell lines. Cell extracts of different TNBC cell lines were used to identified LAT1, CD98hc and GLUT1 by Western blot. Calnexin was used as a loading control. D Quantitation of expression of CD98hc and LAT1 of the experiment shown in ( C ). The graph represents the expression values of CD98hc and LAT1 for each cell line. Quantitation of CD98hc and LAT1 was made as described in the experimental procedures section. Pearson’s correlation coefficient and the p value are shown. E Co-immunoprecipitation studies of CD98hc and LAT1. One mg of HCC3153 extracts were immunoprecipitated with the anti-CD98hc antibody and the immunocomplexes were analyzed by Western with the anti-LAT1 antibody. Mouse IgG was used as a control. F Expression of CD98hc and LAT1 in tumoral samples of patients with TNBC. The tumours were homogenized and lysed. CD98hc and LAT1 were analyzed by Western blot. β-actin was used as a loading control. G Quantitation of expression of CD98hc and LAT1 of the experiment performed in ( F ). The graph represents the expression values of CD98hc and LAT1 for each tumor sample. Pearson’s correlation coefficient and the p value are shown

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Microarray, Expressing, Western Blot, Quantitation Assay, Immunoprecipitation

    Expression of CD98hc in normal and TNBC tumoral tissue. A Immunohistochemical stainings of CD98hc showing tumor tissue scored as high (+++), intermediate (++) and low (+), and normal tissue with negative (-) staining. Magnification: 40X. B Immunohistochemical staining of CD98hc in a sample of a patient, showing staining of CD98hc only in tumor tissue and not in normal tissue. Magnification: 20 X. C Box plot showing SLC3A2 gene expression levels in normal and tumor tissue of breast cancer patients. Data were obtained from the TNMplot database. D Box plot showing SLC3A2 gene expression levels in different breast cancer subtypes from patients with breast cancer. The data were obtained using the Xena online tool

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: Expression of CD98hc in normal and TNBC tumoral tissue. A Immunohistochemical stainings of CD98hc showing tumor tissue scored as high (+++), intermediate (++) and low (+), and normal tissue with negative (-) staining. Magnification: 40X. B Immunohistochemical staining of CD98hc in a sample of a patient, showing staining of CD98hc only in tumor tissue and not in normal tissue. Magnification: 20 X. C Box plot showing SLC3A2 gene expression levels in normal and tumor tissue of breast cancer patients. Data were obtained from the TNMplot database. D Box plot showing SLC3A2 gene expression levels in different breast cancer subtypes from patients with breast cancer. The data were obtained using the Xena online tool

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Expressing, Immunohistochemical staining, Negative Staining, Staining

    Internalization of an antibody against CD98hc. A Subcellular localization of CD98hc in MDA-MB231 and HCC3153 cells was analyzed by immunofluorescence. Scale bar = 25 μm. B Cell surface immunoprecipitation of CD98hc. The different TNBC cell lines treated or not with 10 nM of anti-CD98hc for 2 hours at 4°C were lysed and cell extracts precipitated with protein A-sepharose. CD98hc in those immunoprecipitates was analyzed by Western. C Protease protection experiments of CD98hc. Cells were treated with proteinase K, lysed and subjected to immunoprecipitation and Western analysis with the anti-CD98hc antibody. D FACS analyses of CD98hc cell surface expression. MDA-MB231 cells were incubated with 10 nM of anti-CD98hc for 20 minutes at 37°C. Cells were detached, incubated with an anti-mouse antibody conjugated to FITC, and fluorescence intensity was measured with a BD FACSAria TM III cytometer. The yellow histogram corresponds to signals from cells incubated with the secondary antibody alone, whereas the pink histogram represents the fluorescence due to the expression of CD98hc. E MDA-MB231 cells were seeded on coverslips and treated with 10 nM of anti-CD98hc for the times indicated. Scale bar = 25 μm. The images at the bottom of this section correspond to magnifications of a cell present in the images obtained at 24 hours. The white arrows indicate colocalization of CD98hc and LAMP1. Scale bar = 7.5 μm

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: Internalization of an antibody against CD98hc. A Subcellular localization of CD98hc in MDA-MB231 and HCC3153 cells was analyzed by immunofluorescence. Scale bar = 25 μm. B Cell surface immunoprecipitation of CD98hc. The different TNBC cell lines treated or not with 10 nM of anti-CD98hc for 2 hours at 4°C were lysed and cell extracts precipitated with protein A-sepharose. CD98hc in those immunoprecipitates was analyzed by Western. C Protease protection experiments of CD98hc. Cells were treated with proteinase K, lysed and subjected to immunoprecipitation and Western analysis with the anti-CD98hc antibody. D FACS analyses of CD98hc cell surface expression. MDA-MB231 cells were incubated with 10 nM of anti-CD98hc for 20 minutes at 37°C. Cells were detached, incubated with an anti-mouse antibody conjugated to FITC, and fluorescence intensity was measured with a BD FACSAria TM III cytometer. The yellow histogram corresponds to signals from cells incubated with the secondary antibody alone, whereas the pink histogram represents the fluorescence due to the expression of CD98hc. E MDA-MB231 cells were seeded on coverslips and treated with 10 nM of anti-CD98hc for the times indicated. Scale bar = 25 μm. The images at the bottom of this section correspond to magnifications of a cell present in the images obtained at 24 hours. The white arrows indicate colocalization of CD98hc and LAMP1. Scale bar = 7.5 μm

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Immunofluorescence, Immunoprecipitation, Western Blot, Expressing, Incubation, Fluorescence, Cytometry

    Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T ( D ) and MDA-MB231 ( E ) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: Generation and anti-proliferative activity of an antibody-drug conjugate targeting CD98hc. A Preparation of the antibody-drug conjugate targeting CD98hc. The coupling of DM1 to the anti-CD98hc antibody was analyzed by Western, by using an anti-DM1 antibody. Twenty nanograms of this ADC (αCD98hc-DM1), the nude anti-CD98hc (αCD98hc), trastuzumab or T-DM1 were loaded in 12% SDS-PAGE gels and analyzed for total protein (stain-free blot, lower image) and DM1 reactivity (upper panel). Trastuzumab and T-DM1 were used as a negative and positive controls. B Effect of anti-CD98hc-DM1 in a panel of TNBC cell lines. Cells were treated with anti-CD98hc and anti-CD98hc-DM1 10 nM for four days. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of triplicates of an experiment repeated twice. C Dose-response analyses of the effect of anti-CD98hc-DM1 on four TNBC cell lines. Cells were treated with the ADC for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. Results are shown as the mean ± SD of quadruplicates of an experiment repeated two times. D and E HS578T ( D ) and MDA-MB231 ( E ) cells were treated with anti-CD98hc, anti-CD98hc-DM1 or DM1 for four days at the indicated doses. The data are plotted as the percentage of MTT metabolization with respect to control. F Knockout of CD98hc in MDA-MB231 cells by CRISPR/Cas9. Parental MDA-MB231 cells and two different clones knocked out for CD98hc were lysed. The levels of expression of CD98hc and LAT1 were analyzed by Western blot. Calnexin was used as a loading control. G Dose-response analyses of the effect of anti-CD98hc-DM1 on parental and CD98hc CRISPR #B3, #G3 MDA-MB231 cells. Cells were treated with anti-CD98hc-DM1 for four days. Results are shown as the mean ± SD of quadruplicates of an experiment repeated three times

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Activity Assay, Western Blot, SDS Page, Staining, Knock-Out, CRISPR, Clone Assay, Expressing

    The anti-CD98hc-DM1 antibody provokes cell cycle arrest in mitosis and mitotic catastrophe. A Effect of anti-CD98-DM1 (10 nM, 24 hours) on the morphology of MDA-MB231 and HS578T cells grown as monolayers. The images were taken at 10X magnification. B Quantitative analyses of the action of anti-CD98hc-DM1 on the distribution of the different cell cycle phases in MDA-MB231 and HS578T cell lines. C MDA-MB231 and HS578T cells were treated with anti-CD98hc-DM1 (10 nM) and lysed at the indicated times. Analyses of the amounts of the different proteins studied were performed by Western blotting. GAPDH was used as loading control. D Effect of anti-CD98hc-DM1 on spindle assembly and organization. MDA-MB231 cells seeded on coverslips were treated with CD98hc-DM1 (10 nM) for 24 hours, fixed and stained. Scale bars are indicated. E Detection of giant multinucleated cells after anti-CD98hc-DM1 treatment. MDA-MB231 cells were treated with 10 nM anti-CD98hc-DM1 for 48 hours, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 7.5 μm. F Quantitation of abnormal mitoses, normal mitoses and interphase cells from the experiment shown in ( D ). G Bar graph representation of the percentage of viable (Annexin V-negative/PI-negative) and non-viable MDA-MB231 cells at 48 and 72 hours of treatment with 10 nM anti-CD98hc-DM1. H Effect of anti-CD98hc-DM1 on the levels of several apoptosis-related proteins. MDA-MB231 and HS578T cells were treated with 10 nM anti-CD98hc-DM1, lysed at 0, 1, 2, or 3 days and the indicated proteins analyzed by Western

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: The anti-CD98hc-DM1 antibody provokes cell cycle arrest in mitosis and mitotic catastrophe. A Effect of anti-CD98-DM1 (10 nM, 24 hours) on the morphology of MDA-MB231 and HS578T cells grown as monolayers. The images were taken at 10X magnification. B Quantitative analyses of the action of anti-CD98hc-DM1 on the distribution of the different cell cycle phases in MDA-MB231 and HS578T cell lines. C MDA-MB231 and HS578T cells were treated with anti-CD98hc-DM1 (10 nM) and lysed at the indicated times. Analyses of the amounts of the different proteins studied were performed by Western blotting. GAPDH was used as loading control. D Effect of anti-CD98hc-DM1 on spindle assembly and organization. MDA-MB231 cells seeded on coverslips were treated with CD98hc-DM1 (10 nM) for 24 hours, fixed and stained. Scale bars are indicated. E Detection of giant multinucleated cells after anti-CD98hc-DM1 treatment. MDA-MB231 cells were treated with 10 nM anti-CD98hc-DM1 for 48 hours, fixed and stained for nucleoporin p62 (red) and DNA (blue). Scale bar = 7.5 μm. F Quantitation of abnormal mitoses, normal mitoses and interphase cells from the experiment shown in ( D ). G Bar graph representation of the percentage of viable (Annexin V-negative/PI-negative) and non-viable MDA-MB231 cells at 48 and 72 hours of treatment with 10 nM anti-CD98hc-DM1. H Effect of anti-CD98hc-DM1 on the levels of several apoptosis-related proteins. MDA-MB231 and HS578T cells were treated with 10 nM anti-CD98hc-DM1, lysed at 0, 1, 2, or 3 days and the indicated proteins analyzed by Western

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Western Blot, Staining, Quantitation Assay

    The anti-CD98hc-DM1 has antitumoral activity in vivo and potentiates the action of standard of care drugs. A Analysis of the “in vivo” effect of anti-CD98hc-DM1 on tumor growth in nude mice implanted with MDA-MB231 cells. Arrows indicate days of administration of anti-CD98hc-DM1. Data are plotted as mean tumor volumes ± SEM. P values were calculated using Student t test (two-sided). B Effect of CD98hc-DM1 on the weight of mice. Data are plotted as mean ± SD of six mice/group. C Analyses of DM1, pH3 and pH2AX levels in tumors from mice. Seventeen days after the last treatment, tumors were resected and immediately frozen in liquid nitrogen. Expression of DM1, pH3 and pH2AX was analyzed by Western. β-actin was used as a loading control. D Quantitation of the levels of DM1, pH3 and pH2AX of the experiment performed in ( C ). The graphs represent the mean ± S.D of the different proteins (arbitrary units) of control and treated (anti-CD98hc-DM1) groups. Student’s t test was used to analyze differences among groups. ( E and F ) MDA-MB231 cells were treated with CD98hc-DM1 alone or in combination with Docetaxel ( E ) or Vinorelbine ( F ) for four days at the indicated doses (nM)

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Surfaceome analyses uncover CD98hc as an antibody drug-conjugate target in triple negative breast cancer

    doi: 10.1186/s13046-022-02330-4

    Figure Lengend Snippet: The anti-CD98hc-DM1 has antitumoral activity in vivo and potentiates the action of standard of care drugs. A Analysis of the “in vivo” effect of anti-CD98hc-DM1 on tumor growth in nude mice implanted with MDA-MB231 cells. Arrows indicate days of administration of anti-CD98hc-DM1. Data are plotted as mean tumor volumes ± SEM. P values were calculated using Student t test (two-sided). B Effect of CD98hc-DM1 on the weight of mice. Data are plotted as mean ± SD of six mice/group. C Analyses of DM1, pH3 and pH2AX levels in tumors from mice. Seventeen days after the last treatment, tumors were resected and immediately frozen in liquid nitrogen. Expression of DM1, pH3 and pH2AX was analyzed by Western. β-actin was used as a loading control. D Quantitation of the levels of DM1, pH3 and pH2AX of the experiment performed in ( C ). The graphs represent the mean ± S.D of the different proteins (arbitrary units) of control and treated (anti-CD98hc-DM1) groups. Student’s t test was used to analyze differences among groups. ( E and F ) MDA-MB231 cells were treated with CD98hc-DM1 alone or in combination with Docetaxel ( E ) or Vinorelbine ( F ) for four days at the indicated doses (nM)

    Article Snippet: CD98hc expression was analyzed using the anti-CD98hc (Cell Signaling Technology, #47213) dilution 1:500, 20 minutes of incubation.

    Techniques: Activity Assay, In Vivo, Expressing, Western Blot, Quantitation Assay

    (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) SLC3A2 expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.

    Journal: The Journal of Clinical Investigation

    Article Title: TYRO3 induces anti–PD-1/PD-L1 therapy resistance by limiting innate immunity and tumoral ferroptosis

    doi: 10.1172/JCI139434

    Figure Lengend Snippet: (A) Volcano plot of 4T1-P and Tyro3-OE differentially expressed genes. adj, adjusted. (B) SLC3A2 expression in patients with melanoma with high (n = 52) and low (n = 19) TYRO3 expression levels who received anti–PD-1 therapy. ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (C) Relative lipid ROS in CD45− tumor cells. 4T1-P plus IgG versus 4T1-P plus anti–PD-1, *P = 0.037; Tyro3-OE plus IgG versus Tyro3-OE plus anti–PD-1, NS P = 0.92; and 4T1-P plus anti–PD-1 versus Tyro3-OE plus anti–PD-1, ***P = 0.0004, by 2-way ANOVA. (D) MFI of IFN-γ expression in CD8+ T cells from anti–PD-1–treated 4T1-P and Tyro3-OE tumors. NS P = 0.626, by 2-tailed, unpaired Student’s t test. (E) Percentage of 7-AAD+ cells in 4T1-P and Tyro3-OE cells treated with 2 μM erastin and/or 5 μM Fer-1 for 48 hours (n = 3). £P = 0.013, by 2-tailed, unpaired Student’s t test. (F) Relative lipid ROS in 4T1-P and Tyro3-OE cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). **P = 0.0013, by 2-tailed, unpaired Student’s t test. (G) Percentage of 7-AAD+ cells in 4T1-R and Tyro3−/− cells treated with 2 μM erastin and/or 5 μM Fer-1 for 24 hours (n = 3). ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (H) Relative lipid ROS in 4T1-R and Tyro3−/− cells treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ****P < 0.0001, †††P = 0.000124, and ††P = 0.00125, by 2-tailed, unpaired Student’s t test. (I) A dual-luciferase reporter assay was performed by cotransfecting ARE-reporter-luciferase and pRL-TK with a TYRO3-OE plasmid, and cells were primed with 2 μM MK2206 for 24 hours (n = 3). ##P = 0.002 and NS P = 0.115, by 2-tailed, unpaired Student’s t test. (J) Relative lipid ROS in 4T1-P and Tyro3-OE cells primed with 2 μM MK2206 for 24 hours, and then treated with 10 μM erastin for 8 hours (n = 3). §P = 0.02, §§P = 0.003, NS P = 0.052, and NS P = 0.79, by 2-tailed, unpaired Student’s t test. (K) Relative lipid ROS in 4T1 cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). ¶P = 0.013 and ****P < 0.0001, by 2-tailed, unpaired Student’s t test. (L) Relative lipid ROS in 4T1 Tyro3−/− cells primed with or without 200 nM Pros1 for 24 hours and then treated with 10 μM erastin and/or 10 μM Fer-1 for 8 hours (n = 3). NS P = 0.059, NS P = 0.53, and NS P = 0.58, by 2-tailed, unpaired Student’s t test. Data are presented as the mean ± SD.

    Article Snippet: Paraffin-embedded tissue array slides containing melanoma sections (ME804b, US Biomax) were pretreated using a Melanin Bleach Kit (24883-1, Polysciences) and stained with anti-SLC3A2 antibody (1:100; 47213S, Cell Signaling Technology) and anti-TYRO3 (1:100; OM223993, Omnimabs) as described above.

    Techniques: Expressing, Luciferase, Reporter Assay, Plasmid Preparation

    ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse LAT1/CD98hc expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.

    Journal: Oncotarget

    Article Title: Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target

    doi: 10.18632/oncotarget.27981

    Figure Lengend Snippet: ( A ) Schematic illustration of the cell surface of NIH/3T3 cell lines expressing human native or mutant LAT1. ( B ) RT-qPCR analysis of human and mouse LAT1/CD98hc expression in NIH/3T3 cell lines. ( C ) Association of exogenously expressed human naLAT1, but not muLAT1, with endogenous mouse CD98hc. Cell lysates from NIH/3T3 cell lines were subjected to IP with anti-mouse CD98hc mAb and to WB with anti-human LAT1 rabbit pAb. ( D ) Protein expression of human LAT1/CD98hc in whole lysates or membrane fraction was analyzed by WB. Blots of hCD98hc were used as the negative control. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using NIH/3T3 cell lines. Lysates from NIH/3T3 cell lines were subjected to SDS-PAGE in under reducing or non-reducing conditions, and to WB with anti-LAT1 pAb.

    Article Snippet: Rabbit pAbs against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, #9101), p44/42 MAPK (ERK1/2, #9102), LAT1 (#5347), CD98hc (#47213), HER1/EGFR (#4267), and GAPDH (#2118) (Cell signaling technology, Danvers, MA, USA) were also used.

    Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Negative Control, SDS Page

    ( A ) Anchorage-dependent growth in Matrigel. After the cell culture for 7 days, the number of colonies larger than 50 μm (Lower left) and colonies with pseudopods (Lower right) was counted. The white arrow indicates a colony with pseudopod (Upper). ( B ) Anchorage-independent growth in 3D culture. Cells (2.5 × 10 2 ) in 6-well plates were cultured for 14 days in soft agar, stained with Giemsa solution, and the number of colonies was analyzed by Image J. Statistical analysis was carried out using one-way ANOVA. ( C ) Tumor formation by NIH/3T3 cell lines overexpressing LAT1. Tumor volumes were measured every two days. ( D ) FCM using dispersed cell suspension from in vitro cultured and in vivo (tumor-derived) naLAT1/3T3 was carried out. Ab1, anti-human LAT1 mAb; HR35, anti-human CD98hc mAb.

    Journal: Oncotarget

    Article Title: Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target

    doi: 10.18632/oncotarget.27981

    Figure Lengend Snippet: ( A ) Anchorage-dependent growth in Matrigel. After the cell culture for 7 days, the number of colonies larger than 50 μm (Lower left) and colonies with pseudopods (Lower right) was counted. The white arrow indicates a colony with pseudopod (Upper). ( B ) Anchorage-independent growth in 3D culture. Cells (2.5 × 10 2 ) in 6-well plates were cultured for 14 days in soft agar, stained with Giemsa solution, and the number of colonies was analyzed by Image J. Statistical analysis was carried out using one-way ANOVA. ( C ) Tumor formation by NIH/3T3 cell lines overexpressing LAT1. Tumor volumes were measured every two days. ( D ) FCM using dispersed cell suspension from in vitro cultured and in vivo (tumor-derived) naLAT1/3T3 was carried out. Ab1, anti-human LAT1 mAb; HR35, anti-human CD98hc mAb.

    Article Snippet: Rabbit pAbs against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, #9101), p44/42 MAPK (ERK1/2, #9102), LAT1 (#5347), CD98hc (#47213), HER1/EGFR (#4267), and GAPDH (#2118) (Cell signaling technology, Danvers, MA, USA) were also used.

    Techniques: Cell Culture, Staining, In Vitro, In Vivo, Derivative Assay

    TCGA data ( A – C ) were obtained from the UCSC Xena browser. (A) Box-whisker plot of the expression of CD98hc (right) /LAT1 (left) in normal and tumor colorectal tissues. P -values were calculated by the Student’s t test. (B) Box-whisker plot of the expression of LAT1 in non-metastatic and metastatic ccRCC. The presence of metastasis was determined based on pathological TMN classification. P -values were calculated by the Student’s t test. (C) Kaplan-Meier survival analysis with the log rank test of ccRCC and bladder cancer patient. The correlation between mRNA expression (top 25% and lower 25%) and survival was evaluated. ( D ) Effects of CD98hc-KO on the expression of LAT1. CD98hc in HE293F and SW1116 cells was disrupted by the CRISPR/Cas9-based method, and the expression of CD98hc and LAT1 was analyzed by FCM. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using various human cell lines. Lysates were subjected to SDS-PAGE under reducing or non-reducing conditions, and to WB with anti-LAT1 rabbit pAb.

    Journal: Oncotarget

    Article Title: Oncogenic transformation of NIH/3T3 cells by the overexpression of L-type amino acid transporter 1, a promising anti-cancer target

    doi: 10.18632/oncotarget.27981

    Figure Lengend Snippet: TCGA data ( A – C ) were obtained from the UCSC Xena browser. (A) Box-whisker plot of the expression of CD98hc (right) /LAT1 (left) in normal and tumor colorectal tissues. P -values were calculated by the Student’s t test. (B) Box-whisker plot of the expression of LAT1 in non-metastatic and metastatic ccRCC. The presence of metastasis was determined based on pathological TMN classification. P -values were calculated by the Student’s t test. (C) Kaplan-Meier survival analysis with the log rank test of ccRCC and bladder cancer patient. The correlation between mRNA expression (top 25% and lower 25%) and survival was evaluated. ( D ) Effects of CD98hc-KO on the expression of LAT1. CD98hc in HE293F and SW1116 cells was disrupted by the CRISPR/Cas9-based method, and the expression of CD98hc and LAT1 was analyzed by FCM. ( E ) WB analysis of LAT1 protein under reducing or non-reducing conditions using various human cell lines. Lysates were subjected to SDS-PAGE under reducing or non-reducing conditions, and to WB with anti-LAT1 rabbit pAb.

    Article Snippet: Rabbit pAbs against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204, #9101), p44/42 MAPK (ERK1/2, #9102), LAT1 (#5347), CD98hc (#47213), HER1/EGFR (#4267), and GAPDH (#2118) (Cell signaling technology, Danvers, MA, USA) were also used.

    Techniques: Whisker Assay, Expressing, CRISPR, SDS Page