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Cell Signaling Technology Inc anti gapdh
HGF stimulation is abrogated by <t>IQSEC1</t> depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and <t>GAPDH</t> (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.
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1) Product Images from "Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis"

Article Title: Spatial restriction of phosphoinositide metabolism is a molecular switch to promote metastasis

Journal: bioRxiv

doi: 10.1101/851410

HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.
Figure Legend Snippet: HGF stimulation is abrogated by IQSEC1 depletion. (A) Western blot of PC3 cells expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 30 minutes using anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, ARF5, ARF6 and GAPDH (shown for Akt blot) antibodies. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. Values, mean ± s.d. n=3 independent experiments. (C) Phase contrast images of PC3 acini expressing Scr or IQSEC1 KD4 shRNA stimulated with HGF for 96 hours. Scale bars, 100μm. Cartoon, depicts acini phenotype representative of each condition. (D) Quantitation of PC3 acini shown in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 4 replicates/condition, 1,400-4,025 acini/condition. (E) Western blots of PC3 cells expressing Scr or IQSEC1 KD4 shRNA treated with cycloheximide (CHX) for various times using anti-IQSEC1, Met and GAPDH (shown for Met blot) antibodies. Quantitation of Met expression levels normalised to time 0 is shown. Values, mean ± s.e. n=3. p-values (one-way ANOVA): *p≤0.05, ***p≤0.001 and ****p≤0.0001.

Techniques Used: Western Blot, Expressing, shRNA, Quantitation Assay

IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.
Figure Legend Snippet: IQSEC1 isoforms differentially regulate collective invasion. (A) Schema, domain structure of IQSEC1 variants (v) 1-4. All variants possess an IQ motif, SEC7 and PH domains. v1 and v2 contain N-terminal extensions. A nuclear localization signal (NLS) is present in v1-3, lost in v4. v2/v3 share a common C-terminus. Common domains shown in grey, unique domains indicated by colour; blue; v1, pink; v2 and v3, green; v4. (B) Western blot in 2D and 3D using anti-IQSEC1 antibody. GAPDH is shown for IQSEC1 blot. Relative expression of all IQSEC1 bands normalised to 2D RWPE1 is shown. n=6 independent experiments. Values, mean ± s.d. p-values (Student’s t-test), *p ≤0.05; n.s. not significant. (C) Cartoon, isoform specific IQSEC1 functions were identified by comparing the growth and invasion of acini expressing different GFP-IQSEC1 variants upon depletion of endogenous IQSEC1 (shRNA KD4). (D) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4, and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, GFP and GAPDH antibodies. All antibodies used on same membrane. GAPDH is shown for IQSEC1 blot. Different exposures of IQSEC1 (long, short) are presented to demonstrate expression of all variants. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (E) Phase images of acini from cells described in D. GFP positive acini are outlined in yellow. Scale bars, 100μm. (F) Quantitation of images shown in E. Heatmap shows area and compactness measurements as Z-score-normalised to control (+-+-) values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower panels). n=3, 3 replicates/condition, 300-650 acini per condition. (G) PC3 acini described in D were stained for F-actin and nuclei (black and magenta respectively in upper panels) after 4 days. Green arrowheads, protrusions. Scale bars, 20μm. Localisation of GFP-IQSEC1 can be appreciated from FIRE pseudo coloured Look Up Table (FIRE LUT) (middle panels). Magnified images of boxed regions are inset. Cartoon, localization of GFP-IQSEC1 variants in PC3 acini (lower panel). (H) Western blot of PC3 cells expressing GFP or GFP-IQSEC1 v1-v4 using anti-IQSEC1 (specific for v2) and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. Different exposures of GFP (long, short) are presented showing GFP-IQSEC1 proteins. Upper and lower parts of same GFP blot are shown to demonstrate expression of GFP-IQSEC1 variants and GFP control respectively. (I) Schema, summary of the effect of IQSEC1 KD and expression of GFP-IQSEC1 v2 on growth and protrusive ability of PC3 acini. (J) Endogenous IQSEC1 v2 co-stained with F-actin (green and red respectively in merge). Magnified images of boxed region are shown. Red arrowhead indicates localisation at protrusion tip.

Techniques Used: Western Blot, Expressing, shRNA, Quantitation Assay, Staining

IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.
Figure Legend Snippet: IQSEC1 regulates growth and invasion in multiple prostate cancer cell lines. (A) Heatmap shows mRNA expression levels, mined from CCLE, of the Met-PI3K-Akt pathway across different prostate cancer cell lines. (B) Western blot of prostate cancer cell lines using anti-IQSEC1, phospho-Y1234/1234 Met, total Met, LRP1, ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. (C) Schema, effect of NAV-2729 on ARF GTPase cycle. (D) Phase images of acini (+ and – HGF) at different time points are shown. Acini were also treated with NAV-2729 for 96 hours. Scale bars, 100μm. (E) Schema, summarizes the effect of each treatment described in D on acini growth and invasion. (F-I) Quantitation of 22Rv1 (F) , LNCaP (G) , DU145 (H) and DU145 + HGF (I) acini formation in the absence or presence of NAV-2729. 22Rv1 and DU145 acini were also expressing LRP1-GFP and IQSEC1-GFP v2 respectively. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2, replicates/condition, 400-1600 acini/condition.

Techniques Used: Expressing, Western Blot, Quantitation Assay

IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.
Figure Legend Snippet: IQSEC1 interacts with multiple transmembrane proteins. (A) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-IQSEC1, LRP1, Met, SORL1, RICTOR, Sin1, ARFGAP1, 14-3-3ζ/Δ, PPC6, GFP and GAPDH (shown for GFP blot) antibodies. (B) Western blot of PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (C) Western blot of Epi and EMT14 PC3 subclones using anti-IQSEC1, ARF5, ARF6, ARFGAP1, LRP1, phospho-Y1234/1235 Met, Met, SORL1, RICTOR and GAPDH (shown for IQSEC1 blot) antibodies. (D) Met immunoprecipitation was carried out on PC3 cells expressing Scr or IQSEC1 KD4 shRNA. Western blot was then performed on lysates (upper panels) and IPs (lower panels) using anti-LRP1, Met, IQSEC1 and GAPDH (shown for IQSEC1 blot) antibodies. Quantitation of LRP1/Met interaction normalised to Scr is shown. Values, mean ± s.d. n=3 independent experiments. p values (one-way ANOVA): n.s. not significant. (E-F) Western blot analysis of PC3 cells expressing Scr or (E) LRP1 and (F) Met shRNA. GAPDH is shown for Akt blots. (G) Quantitation of shape classification of PC3 cells expressing shRNAs for IQSEC1 binding partners as log2 fold change of each phenotype over control (left heatmap). p-values (one-way ANOVA): greyscale values as indicated (right heatmap). n=2, 4 replicates per condition, minimum 14,000 cells imaged/condition. (H) Quantitation of PC3 acini expressing GFP or LRP1-GFP. Heatmaps for area and compactness measurements shown as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 3 replicates, minimum 150 acini/condition. (I) Western blot of PC3 cells expressing SORL1 shRNA. GAPDH is shown for Akt blots. (J) Representative phase contrast images of PC3 acini expressing Scr (also shown in Figure 4H ), SORL1_ 2 and RICTOR_2 shRNA taken after 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum 700 acini/condition. (L-N) Western blots of PC3 cells expressing (L) RICTOR or (M) SIN1 or (N) ARFGAP1 shRNA. GAPDH is shown for Akt blots. (O) Representative phase contrast images of PC3 acini expressing Scr, ARFGAP1_1 and ARFGAP1_2 shRNA taken after 96 hours. Scale bars, 100μm. (P) Quantitation of PC3 acini shown in O. n=2, 4 replicates, minimum 1,100 acini/condition. (Q) Cartoon, depicts representative acini phenotype upon depletion of ARFGAP1 and ARF GTPase cycle.

Techniques Used: Immunoprecipitation, Expressing, Western Blot, shRNA, Quantitation Assay, Binding Assay

IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.
Figure Legend Snippet: IQSEC1 activates ARF5/6 in distinct locations within protrusions. (A) Western blot of PC3 cells expressing Scr, ARF5 or ARF6 shRNA, alone or together, using anti ARF5, ARF6 and GAPDH antibodies. GAPDH is shown for ARF6 blot. ARF intensity normalised to Scr is quantified. n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA). (B) Phase images of PC3 acini described in A. Yellow outlines indicate shRNA (mVenus) positive acini. Scale bars, 100μm. (C) Quantitation of images shown in B. Quantitation is shown for area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=3, 5 replicates/condition, 2880 - 3188 acini quantified/condition. Cartoon, acini phenotype representative of each condition. (D) Western blot of PC3 cells stably co-overexpressing (OX) mNeonGreen (mNG) and TagRFP-T (RFP) (Control) or ARF5-mNG and ARF6-RFP (ARF5/6), and either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, ARF5, ARF6 and GAPDH antibodies were used. Both endogenous and exogenous (OX) ARFs were detected. GAPDH is shown for ARF5 blot. (E) Phase images of PC3 acini described in D. Scale bars, 100μm. (F) Quantitation of images shown in E. n=2, 4 replicates/condition, 1,254 – 1,567 acini/condition. (G) PC3 cells co-overexpressing ARF5-mNG and ARF6-RFP (ARF5/6 OX) were stained for IQSEC1 v2 and merged image of spindle shaped cell shown (left panel). Magnified images of boxed region are shown. White arrowheads indicate areas of colocalization. Scale bars, 20μm. (H) PC3 acini expressing either ARF5-mNG or ARF6-mNG were stained for IQSEC1. Localisation of these proteins is shown using FIRE LUT. Yellow and white arrowheads, colocalization in juxtanuclear region and protrusive tips, respectively. White arrows, lack of colocalization. Scale bars, 5μm. (I) Schema, GTPase cycle of ARF5 and ARF6, site of action of the IQSEC1-inhibiting molecules NAV-2729, the pan ARFGAP-inhibitor QS11, and GTP-loaded ARF detection by a GGA1-NGAT probe. (J) PC3 acini expressing either ARF5-mNG or ARF6-mNG and GGA1-NGAT-RFP were fixed and FIRE LUT of maximum projections (upper panels) and a single Z-slice of boxed regions (lower panels) shown. White and green arrowheads, colocalization in protrusions, white arrows, colocalization in cell body. Scale bars, 10μm. (K-L) PC3 cells expressing (K) ARF5-NG and GGA1-NGAT or (L) ARF6-NG and GGA1-NGAT were stably transfected with either Scr or IQSEC1 KD4 shRNA. Cells were treated with NAV-2729 or QS11 for 24 hours prior to fixation. Quantitation of % overlap of ARF and ARF-GTP probe per cell is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 3 replicates/condition with a minimum of 500 cells/condition. p-values (one-way ANOVA): *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001. (M) Schema, different localization of IQSEC1 and active ARFs in protrusions is shown.

Techniques Used: Western Blot, Expressing, shRNA, Quantitation Assay, Stable Transfection, Staining, Transfection

IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
Figure Legend Snippet: IQSEC1-LRP1 complex regulates Met endocytic trafficking. (A) Western blot of PC3 cells co-expressing mNG and RFP (Control) or ARF5-mNG and ARF6-RFP (ARF5/6) with either Scr or IQSEC1 KD4 shRNA. Anti-IQSEC1, phospho-Y1234/1235 Met, Met, phospho-S473 Akt, Akt, and GAPDH (sample control) antibodies were used. (B) Quantitation of phospho/total Met and phospho/total Akt expression is presented as signal intensity relative to control. n=2 independent experiments. Values, mean ± s.d. (C) Schema, effect of IQSEC1 on Met trafficking. (D) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were incubated with a Met-647 fluorescent antibody at 4°C (Surface) prior to stimulation with HGF for either 10 or 30 minutes (Internalisation). PC3 cells were also treated with chloroquine to allow accumulation of surface-derived Met (black) for 1 hour at 17°C prior to stimulation with HGF for either 10 or 30 minutes (Recycling). Cells were stained with F-actin (green outlines) and Hoechst (nuclei in magenta). Magnified images of boxed regions are inset. Scale bars 20µm. (E) Intensity of Met antibody in each cell was quantified (Z-score normalised); shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (F) Intensity of Met antibody in membrane, cytoplasmic and juxtanuclear regions was quantified. Line graphs show relative region intensities compared to the intensity of the whole cell (Z-score normalised). n=2, 4 replicates/condition, minimum 500 cells per condition. p values: (Welsh’s t-test). (G) Schema, sub-cellular re-localisation of active Met upon HGF treatment. (H) PC3 cells expressing Scr, IQSEC1 KD4, LRP1 or SORL1 shRNA were stimulated with HGF for 30 minutes. Cells were stained for phospho-Met (black), F-actin (green outlines) and Hoechst/nuclei (magenta). Magnified images of boxed regions are shown (lower panels). Cartoon, sub-cellular localization of active Met under different conditions. (I-J) Quantitation of spot intensity (Z-score normalised) is shown for images in H in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, minimum 500 cells per condition. p-values (one-way ANOVA). (K) Schema, regulation of Met internalisation, but not recycling, by IQSEC1 and LRP1. All p values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

Techniques Used: Western Blot, Expressing, shRNA, Quantitation Assay, Incubation, Derivative Assay, Staining

ARF5 and ARF6 co-operate to regulate both growth and invasion. (A) Quantitation of shape classification of PC3 cells expressing Scr, ARF5, ARF6 or AR5/6 shRNA is shown. Heatmaps show log2 fold change of each phenotype over control (left heatmaps). p-values (one-way ANOVA): greyscale values as indicated (right heatmaps). n=2, 4 replicates. Minimum of 4,000 cells/condition analysed in total. (B) Schema, comparison of activation of WT ARF GTP and fast-cycling ARF mutants. (C) Western blot of PC3 cells expressing mNG, ARF5-mNG, ARF5-mNG fast cycling (TA), ARF6-mNG or ARF6-mNG fast cycling (TN) using anti-ARF5, ARF6 and GAPDH (shown for ARF5 blot) antibodies. (D) Quantitation of shape classification of PC3 cells described in C is shown. n=2, 4 replicates. (E) Quantitation of PC3 acini described in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2 with 4 replicates per condition. Minimum of 900 acini/condition analysed in total. Cartoon, depicts acini phenotype representative of each condition. (F) Quantitation of shape classification of PC3 cells expressing mNG, ARF5-mNG and ARF6-RFP or ARF5-mNG TA and ARF6-RFP TN is shown. n=3, 3 replicates. (G) Quantitation of PC3 acini. n=2, 4 replicates. Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of shape classification of PC3 cells expressing control or ARF5/ARF6 and Scr or IQSEC1 KD4 shRNA. n=2, 4 replicates. (I) PC3 cells expressing Scr or IQSEC1 shRNA were serum starved overnight and HGF was then added for 30 minutes. GGA3 pulldown was carried out and western blot analysis performed using anti-IQSEC1, ARF5, ARF6, and GAPDH (shown for ARF6 blot) antibodies. Quantitation of ARF5 and ARF6 GTP loading normalised to control is shown (right panels). n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA): no significance. (J) Schema, modulation of ARF activity using SecinH3, NAV-2729 or QS11. (K) In vitro fluorescent polarisation assay was carried out to study exchange of fluorescent nucleotides (MANT) on ARF5. IQSEC1 v2 SEC7-PH WT and GEF dead mutant IQSEC1 v2 SEC7-PH GEF* were used to test the role of IQSEC1 in activation of ARF5. ARFs, nucleotides, GEFs and inhibitors were added at time points indicated. Changes in polarisation over time are shown. (L) Western blot of PC3 cells treated for 24 hours with NAV-2729, SecinH3 or QS11 using anti-IQSEC1, ARF5, ARF6 and GAPDH (shown for IQSEC1) antibodies. (M) Representative phase contrast images of PC3 acini treated with NAV-2729, SecinH3 or QS11 after 96 hours. Scale bars, 100μm. (N) Quantitation of PC3 acini shown in M. n=1, 4 replicates. (O) Cartoon, depicts representative acini phenotype upon treatment with inhibitors.
Figure Legend Snippet: ARF5 and ARF6 co-operate to regulate both growth and invasion. (A) Quantitation of shape classification of PC3 cells expressing Scr, ARF5, ARF6 or AR5/6 shRNA is shown. Heatmaps show log2 fold change of each phenotype over control (left heatmaps). p-values (one-way ANOVA): greyscale values as indicated (right heatmaps). n=2, 4 replicates. Minimum of 4,000 cells/condition analysed in total. (B) Schema, comparison of activation of WT ARF GTP and fast-cycling ARF mutants. (C) Western blot of PC3 cells expressing mNG, ARF5-mNG, ARF5-mNG fast cycling (TA), ARF6-mNG or ARF6-mNG fast cycling (TN) using anti-ARF5, ARF6 and GAPDH (shown for ARF5 blot) antibodies. (D) Quantitation of shape classification of PC3 cells described in C is shown. n=2, 4 replicates. (E) Quantitation of PC3 acini described in C. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=2 with 4 replicates per condition. Minimum of 900 acini/condition analysed in total. Cartoon, depicts acini phenotype representative of each condition. (F) Quantitation of shape classification of PC3 cells expressing mNG, ARF5-mNG and ARF6-RFP or ARF5-mNG TA and ARF6-RFP TN is shown. n=3, 3 replicates. (G) Quantitation of PC3 acini. n=2, 4 replicates. Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of shape classification of PC3 cells expressing control or ARF5/ARF6 and Scr or IQSEC1 KD4 shRNA. n=2, 4 replicates. (I) PC3 cells expressing Scr or IQSEC1 shRNA were serum starved overnight and HGF was then added for 30 minutes. GGA3 pulldown was carried out and western blot analysis performed using anti-IQSEC1, ARF5, ARF6, and GAPDH (shown for ARF6 blot) antibodies. Quantitation of ARF5 and ARF6 GTP loading normalised to control is shown (right panels). n=3 independent experiments. Values, mean ± s.d. p-values (one-way ANOVA): no significance. (J) Schema, modulation of ARF activity using SecinH3, NAV-2729 or QS11. (K) In vitro fluorescent polarisation assay was carried out to study exchange of fluorescent nucleotides (MANT) on ARF5. IQSEC1 v2 SEC7-PH WT and GEF dead mutant IQSEC1 v2 SEC7-PH GEF* were used to test the role of IQSEC1 in activation of ARF5. ARFs, nucleotides, GEFs and inhibitors were added at time points indicated. Changes in polarisation over time are shown. (L) Western blot of PC3 cells treated for 24 hours with NAV-2729, SecinH3 or QS11 using anti-IQSEC1, ARF5, ARF6 and GAPDH (shown for IQSEC1) antibodies. (M) Representative phase contrast images of PC3 acini treated with NAV-2729, SecinH3 or QS11 after 96 hours. Scale bars, 100μm. (N) Quantitation of PC3 acini shown in M. n=1, 4 replicates. (O) Cartoon, depicts representative acini phenotype upon treatment with inhibitors.

Techniques Used: Quantitation Assay, Expressing, shRNA, Activation Assay, Western Blot, Activity Assay, In Vitro, Mutagenesis

IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.
Figure Legend Snippet: IQSEC1 is a scaffold for Met signalling. (A) Schematic, IQSEC1 chimeras and variants display distinct phenotypes in 3D culture. GFP-trap immunoprecipitation was performed on PC3 cells expressing these proteins followed by tryptic digestion “on-beads” and LC/MS/MS analysis. IQSEC1 domain-specific interactions were identified and sorted by mRNA expression compared in paired PC3 subclones. (B) STRING network analysis of IQSEC1 binding partners identified by MS visualized using Cytoscape. Most IQSEC1 binding partners could be clustered into 8 protein complexes (colour coded). (C) Schema, indicates the protein complex each binding partner is associated with. Heatmap shows the fold change of IQSEC1 interactors from panel B binding to different IQSEC1 domains over GFP control. Values are −log 2 (FC), very high = −7 to −5, high = −5 to −2.5, medium −2.5 to −1, low = −1 to −0.05, no binding > 0.05. p-values are indicated in grey heatmap. The specific IQSEC1 domain to which each interactor binds is also depicted. Interactions were sorted according to the fold change in mRNA of non-invasive PC3 subclones (GS689.Li, EMT) compared to invasive subclones (PC3E, Epi) (RNAseq). (D) GFP-trap immunoprecipitation was performed on PC3 cells expressing GFP, GFP-IQSEC1 v2 or v4. Western blot analysis was then carried out using anti-Met, LRP1, GFP, IQSEC1 and GAPDH antibodies. GAPDH is shown for LRP1 blot. Quantitation of LRP1/IQSEC1 and Met/IQSEC1 interactions for each GFP-trap are shown. n=3 and n=4, respectively. Values, mean ± s.d. p values (one-way ANOVA): **p≤0.01 and n.s. not significant. (E) Cartoon, depicts domain specific binding of IQSEC1 interactors. (F) PC3 acini expressing ARF5-mNG or ARF6-mNG were stained for Met and LRP1. FIRE LUT images are displayed with magnified images of boxed regions shown in lower panels. White arrowheads indicate localisation in protrusion tips. Scale bars, 5µm (upper) and 10µm (lower). (G) Schema, colocalization of IQSEC1-ARF interactors in protrusive tips. (H) Phase images of PC3 acini expressing Scr, LRP1_1, Met_2, SORL1_1, RICTOR_1 or SIN1_2 shRNA after 96 hours. Scale bars, 100μm. (I) Quantitation of images shown in H. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates/condition, 700-2,400 acini/condition. Cartoon, depicts acini phenotype representative of each condition.

Techniques Used: Immunoprecipitation, Expressing, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Western Blot, Quantitation Assay, Staining, shRNA

Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
Figure Legend Snippet: Upregulation of IQSEC1 is associated with tumorigenesis. (A) Schema, prostate cell lines forming non-invasive or invasive 3D acini in extracellular matrix (ECM). (B) Cartoon, phenotype of typical RWPE-1 and RWPE-2 acini. Confocal (F-actin (red) and nuclei (blue)) and brightfield images show RWPE-1 and RWPE-2 acini at 120 hours. Arrowheads, protrusions. Scale bar, 20μm. (C) Schema, PC3 acini form (grow) and invade (protrusions) through ECM over time. Phase contrast images of PC3 acini where higher magnification of boxed region at different time points is shown. Arrowheads, protrusions. Scale bar, 100μm. (D) Cartoon, ARF GTPase cycle. (E) Heatmap representation of mRNA expression. Data are normalized to RWPE-1 and presented as the log2-transformed fold change compared to the average of all values. Bar graphs summarise fold changes of ARF and IQSEC mRNA levels. n=3 technical replicates. Values, mean ± s.d. p-values (Student’s t-test). (F) Schema, elevated activation of ARF GTPases in PC3 cells by GEFs such as IQSEC1. (G) Graph generated using RNAseq data from the Cancer Cell Line Encyclopedia (CCLE) comparing IQSEC1 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (H) Western blot analysis of androgen receptor (AR) proficient or deficient prostate cell lines using anti-ARF1, ARF6, pan-IQSEC1 isoform and GAPDH (as sample control) antibodies. (I) Quantitation of IQSEC1 expression levels in either 52 normal or 487 tumour samples mined from The Cancer Genome Atlas (TCGA). Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA). (J) IQSEC1 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Heatmap shows clustering of expression values. p values (Logrank). All p-values: *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

Techniques Used: Expressing, Transformation Assay, Activation Assay, Generated, Western Blot, Quantitation Assay

IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.
Figure Legend Snippet: IQSEC1-ARF signalling controls phosphoinositide generation in invasive protrusion tips. (A) Schema, signalling pathways in protrusive tips of PC3 acini. (B) PC3 acini expressing mNeonGreen tagged PH-PLCδ or PH-Grp1 were fixed after 3 days. PC3 acini were also stained with phospho-S473 Akt antibody. FIRE LUT used to show localisation and intensity of mNeonGreen or pAkt. Magnified images of boxed regions are also shown. White arrowheads, localisation at protrusive tips. Scale bars, 10 µm. Cartoon, spatial PIP production in protrusive tips. (C) Quantitation of cortical enrichment of PI(4,5)P 2 (upper panel) or PIP 3 per cell in the presence or absence of IQSEC1 is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition, > 500 cells/condition. p-values (one-way ANOVA): ****p≤0.0001. (D) Cartoon, PIPK targeting in presence or absence of IQSEC1. (E) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control), Myr-FLAG-PIP5Kα, Myr-FLAG-PIP5Kβ or Myr-FLAG-PI3Kβ and either Scr or IQSEC1 shRNA (KD4). Anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies were used. GAPDH is shown for IQSEC1 blot. (F) Western blot of PC3 cells expressing Myr-FLAG-Cre (Control) or Myr-Akt1 and either Scr or IQSEC1 KD4 shRNA using anti-IQSEC1, phospho-S473 Akt, total Akt and GAPDH antibodies. GAPDH is shown for IQSEC1 blot. (G) Phase images of PC3 acini described in E are shown. Scr acini were also treated with NAV-2729 (IQSEC1 inhibitor). Scale bars, 100μm. (H) Quantitation of images shown in G. Heatmaps show area and compactness measurements as Z-score-normalised values (upper panels). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=3 with 4 replicates per condition. Between 1,693 and 2,435 acini quantified per condition. Cartoon, acini phenotype representative of each condition. (I) Phase images of PC3 acini described in F after 96 hours. Scr acini were also treated with IQSEC1-inhibiting compound NAV-2729. Scale bars, 100μm. (J) Quantitation of images shown in I. n=2 with 4 replicates/condition, 1,287-2,363 acini/condition. Cartoon, acini phenotype representative of each condition. (K) Schema, summarizes the relationship between location and level of cortical PIP 3 and 2D and 3D PC3 phenotype.

Techniques Used: Expressing, Staining, Quantitation Assay, Western Blot, shRNA

IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.
Figure Legend Snippet: IQSEC1-ARF controls cortical PI(4,5)P 2 generation, which is required for production of PIP 3. (A) PC3 cells expressing mNeonGreen tagged PH-PLCδ (PIP 2 ) or PH-Grp1 (PIP 3 ) and either Scr or IQSEC1 shRNA KD4 were fixed after 2 days. FIRE LUT is used to show localisation and intensity of GFP in magnified images of boxed regions. White arrowhead indicates localisation at protrusive tip. Scale bars, 20µm. (B-C) Quantitation of (B) PI(4,5)P 2 or (C) PIP 3 cortical enrichment in spindle, spread and round cells expressing either Scr or IQSEC1 KD4 shRNA is shown in box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=3, 4 replicates/condition. 1330/1049 and 2222/1182 cells analysed for Src/ IQSEC1 KD4 shRNA respectively in B and C. p-values (one-way ANOVA). (D) Cartoon, summary of cortical PI(4,5)P 2 and PIP 3 localization in different cell shapes. (E) Western blot of PC3 cells treated with LY294002 (pan PI3K), AZD8835 (PI3Kα), AZD8186 (PI3Kβ), AS605240 (PI3Kγ), Cal-101 (PI3Kδ) and AktII (Akt) inhibitors for 24 hours. Anti-phospho-S473 Akt, Akt and GAPDH antibodies were used. (F) Phase contrast images of PC3 acini described in E. Scale bars, 100μm. (G) Cartoon, depicts acini phenotype representative of each condition. (H) Quantitation of PC3 acini shown in F. Heatmaps show area and compactness measurements as Z-score-normalised values (upper heatmaps). p-values (one-way ANOVA): greyscale values as indicated (lower heatmaps). n=2, 4 replicates, minimum of 350 acini/condition. (I) Western blot of PC3 cells expressing Scr, PIP5K1α or PIP5K1β shRNA using anti-PIP5K, IQSEC1, phospho-S473 Akt, Akt and GAPDH (shown for Akt blots) antibodies. (J) Phase contrast images of PC3 acini expressing Scr, PIP5K1α or PIP5K1β shRNA at 96 hours. Scale bars, 100μm. (K) Quantitation of PC3 acini shown in J. n=2, 4 replicates, minimum of 1,348 acini/condition. Cartoon, depicts acini phenotype representative of each condition. (L) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained for pS473 Akt (black) and Hoechst /nuclei (magenta). Magnified images of boxed regions are shown in lower panels. Blue arrowheads indicate localisation of active Akt. Scale bars, 10μm. (M-N) Quantitation of pAkt (M) intensity or (N) area in spots in the juxtanuclear, cytoplasm and periphery of PC3 cells expressing either Scr or IQSEC1 KD4 shRNA. Box-and-whiskers plots: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. n=2, 4 replicates/condition. p values (Mann Whitney). (O) Cartoon, depicting phospho-Akt intensity, in the presence or absence of IQSEC1, at different subcellular locations. All p-values: n.s. not significant, *p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001.

Techniques Used: Expressing, shRNA, Quantitation Assay, Western Blot, Staining, MANN-WHITNEY

IQSEC1 is a regulator of cell elongation and collective invasion. (A) Graph generated using RNAseq data from CCLE comparing IQSEC2 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (B-C) Quantitation of IQSEC2 expression levels in either 52 normal or 487 prostate tumour samples (TCGA) (B) or in 29 normal, 131 primary or 19 metastatic prostate tumour tissues (GSE21034) (C) . Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA): n.s. not significant, and ****p≤0.0001. (D) IQSEC2 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Inset Heatmap shows clustering of expression values. p-values (Logrank) are annotated in plots. (E) Western blot of PC3 cells stably expressing Scrambled (Scr) or IQSEC1 (KD1, KD4) shRNAs using anti-IQSEC1 and GAPDH (loading control for IQSEC1 blot) antibodies. Arrowheads indicate reduction of three major bands. IQSEC1 intensity for all bands combined normalised to Scr control is shown. n=3 independent experiments. Values are mean ± s.d. (F) PC3 cells expressing Scr or IQSEC1 KD shRNA were imaged every hour for 3.5 days. Cell confluence was measured and normalised to control. Values are mean ± s.d. p-values (Students t-test) are annotated in plot. (G) Schematic of phenotypic analysis of PC3 cells in 2D. Machine learning applied to confocal images to classify and quantify cells into 3 categories based on shape: spindle, spread, round. (H) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained with whole cell stain (WCS) (red) and Hoechst/nuclei (blue) (left panels). Cells were classified into spindle, spread and round phenotypes shown in green, red and blue respectively (right panels). Scale bar, 100μm. (I-J) Proportion of PC3 cells with each phenotype (I) or classified into round, spindle or spread (J) is shown. Heatmap indicates log2 fold change of each phenotype over Scr (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=4 independent experiments, 10 replicates per condition, minimum 118,000 cells imaged/condition. (K-L) Schema, PC3 cells plated for 24 hours before the resultant monolayers were (K) wounded or (L) wounded and overlaid with 25% ECM for 1 hour prior to imaging using 10x objective. Phase contrast images are shown where the yellow dashed lines indicate initial scratch wound and red pseudo colour shows wound after 24 hours. Magnified images of boxed areas are shown for different time points. Relative wound density (RWD) was calculated at each time point and graphed (lower left panels). RWD at the time point where the Scr controls are 50% closed (t=Max 1/2 ) is also shown (lower right panels). Samples were normalised to the average of all Scr controls across experiments. n=3, 3 replicates/experiment in K and 4 in L. Values, mean ± s.d. p-values (Student’s t-test): *p≤0.05, **p≤0.01 and ****p≤0.0001. (M) Schema, depicts invasive ability of PC3 in ECM in the presence or absence of IQSEC1. (N) Schema, summarizes the effect IQSEC1 depletion has on 2D growth, shape and migration and on 3D invasion.
Figure Legend Snippet: IQSEC1 is a regulator of cell elongation and collective invasion. (A) Graph generated using RNAseq data from CCLE comparing IQSEC2 gene copy number and mRNA expression levels in multiple breast and prostate cancer and non-transformed cell lines. (B-C) Quantitation of IQSEC2 expression levels in either 52 normal or 487 prostate tumour samples (TCGA) (B) or in 29 normal, 131 primary or 19 metastatic prostate tumour tissues (GSE21034) (C) . Box-and-whiskers plot: 10–90 percentile; +, mean; dots, outliers; midline, median; boundaries, quartiles. p-values (one-way ANOVA): n.s. not significant, and ****p≤0.0001. (D) IQSEC2 gene expression from 487 primary prostate tumours (TCGA) was evaluated for outcome prediction from (recurrence-free) survival data. Patient samples were clustered into quartiles based on normalized gene expression. Highest expression, red, lowest expression, blue. Inset Heatmap shows clustering of expression values. p-values (Logrank) are annotated in plots. (E) Western blot of PC3 cells stably expressing Scrambled (Scr) or IQSEC1 (KD1, KD4) shRNAs using anti-IQSEC1 and GAPDH (loading control for IQSEC1 blot) antibodies. Arrowheads indicate reduction of three major bands. IQSEC1 intensity for all bands combined normalised to Scr control is shown. n=3 independent experiments. Values are mean ± s.d. (F) PC3 cells expressing Scr or IQSEC1 KD shRNA were imaged every hour for 3.5 days. Cell confluence was measured and normalised to control. Values are mean ± s.d. p-values (Students t-test) are annotated in plot. (G) Schematic of phenotypic analysis of PC3 cells in 2D. Machine learning applied to confocal images to classify and quantify cells into 3 categories based on shape: spindle, spread, round. (H) PC3 cells expressing Scr or IQSEC1 KD4 shRNA were stained with whole cell stain (WCS) (red) and Hoechst/nuclei (blue) (left panels). Cells were classified into spindle, spread and round phenotypes shown in green, red and blue respectively (right panels). Scale bar, 100μm. (I-J) Proportion of PC3 cells with each phenotype (I) or classified into round, spindle or spread (J) is shown. Heatmap indicates log2 fold change of each phenotype over Scr (upper heatmap). p-values (one-way ANOVA): greyscale values as indicated (lower heatmap). n=4 independent experiments, 10 replicates per condition, minimum 118,000 cells imaged/condition. (K-L) Schema, PC3 cells plated for 24 hours before the resultant monolayers were (K) wounded or (L) wounded and overlaid with 25% ECM for 1 hour prior to imaging using 10x objective. Phase contrast images are shown where the yellow dashed lines indicate initial scratch wound and red pseudo colour shows wound after 24 hours. Magnified images of boxed areas are shown for different time points. Relative wound density (RWD) was calculated at each time point and graphed (lower left panels). RWD at the time point where the Scr controls are 50% closed (t=Max 1/2 ) is also shown (lower right panels). Samples were normalised to the average of all Scr controls across experiments. n=3, 3 replicates/experiment in K and 4 in L. Values, mean ± s.d. p-values (Student’s t-test): *p≤0.05, **p≤0.01 and ****p≤0.0001. (M) Schema, depicts invasive ability of PC3 in ECM in the presence or absence of IQSEC1. (N) Schema, summarizes the effect IQSEC1 depletion has on 2D growth, shape and migration and on 3D invasion.

Techniques Used: Generated, Expressing, Transformation Assay, Quantitation Assay, Western Blot, Stable Transfection, shRNA, Staining, Imaging, Migration

2) Product Images from "Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression"

Article Title: Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression

Journal: Allergy, Asthma & Immunology Research

doi: 10.4168/aair.2016.8.2.161

PEDF ameliorates the expression of VEGF protein in lung tissue in chronic experimental asthma. (A) Total protein from lung tissue was extracted 24 hours after the final OVA challenge and subjected to Western blot analysis of VEGF. GAPDH was utilized as the standard control. (B) The band signal strength of VEGF expressed as a ratio to GAPDH. Data are presented as means±SEM (n=8 per group). * P
Figure Legend Snippet: PEDF ameliorates the expression of VEGF protein in lung tissue in chronic experimental asthma. (A) Total protein from lung tissue was extracted 24 hours after the final OVA challenge and subjected to Western blot analysis of VEGF. GAPDH was utilized as the standard control. (B) The band signal strength of VEGF expressed as a ratio to GAPDH. Data are presented as means±SEM (n=8 per group). * P

Techniques Used: Expressing, Western Blot

3) Product Images from "A PU.1 Suppressive Target Gene, Metallothionein 1G, Inhibits Retinoic Acid-Induced NB4 Cell Differentiation"

Article Title: A PU.1 Suppressive Target Gene, Metallothionein 1G, Inhibits Retinoic Acid-Induced NB4 Cell Differentiation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0103282

Establishment of NB4MTOE cells. (A) Expression of MT1G in NB4MTOE cells examined by western blotting. A rabbit polyclonal anti-MT antibody was used to detect exogenous MT1G. Equal amounts of soluble proteins were loaded in each lane and immunoblotted for MT and histone H3. The indicated numbers show the relative density, calculated with Image J 1.46 software, obtained as the density of each MT1G band divided by that of the corresponding histone H3 band. (B) The expression of MT1G was examined by real-time PCR (mean±SD; n.s., not significant). NB4MTOE cells and their control cells were cultured with or without 1 µM ATRA for 72 h, and then collected for analysis. Each gene transcript level was adjusted by the corresponding expression of GAPDH , and the relative levels are shown. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed by independent real-time PCR amplifications using different batches of cDNA.
Figure Legend Snippet: Establishment of NB4MTOE cells. (A) Expression of MT1G in NB4MTOE cells examined by western blotting. A rabbit polyclonal anti-MT antibody was used to detect exogenous MT1G. Equal amounts of soluble proteins were loaded in each lane and immunoblotted for MT and histone H3. The indicated numbers show the relative density, calculated with Image J 1.46 software, obtained as the density of each MT1G band divided by that of the corresponding histone H3 band. (B) The expression of MT1G was examined by real-time PCR (mean±SD; n.s., not significant). NB4MTOE cells and their control cells were cultured with or without 1 µM ATRA for 72 h, and then collected for analysis. Each gene transcript level was adjusted by the corresponding expression of GAPDH , and the relative levels are shown. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed by independent real-time PCR amplifications using different batches of cDNA.

Techniques Used: Expressing, Western Blot, Software, Real-time Polymerase Chain Reaction, Cell Culture, Polymerase Chain Reaction

4) Product Images from "Arsenic trioxide induces autophagic degradation of the FLT3-ITD mutated protein in FLT3-ITD acute myeloid leukemia cells"

Article Title: Arsenic trioxide induces autophagic degradation of the FLT3-ITD mutated protein in FLT3-ITD acute myeloid leukemia cells

Journal: Journal of Cancer

doi: 10.7150/jca.29751

ATO induced autophagy in the FLT3-ITD AML cell line. (A) MV4-11 cells were treated with ATO for 24 h, and the expression levels of LC3, ATG5 and ATG7 were detected by western blot analysis. GAPDH expression was used as a loading control. (B) MV4-11 cells transfected with mRFP-GFP-LC3 plasmids were treated with ATO for 0, 12, and 24 h and then observed by confocal fluorescence microscopy. Scale bar=5 µm. (C) MV4-11 cells were treated with ATO for 0, 12, and 24 h. The number of autophagosomes was observed by transmission electron microscopy and calculated (each group had 30 views, * P
Figure Legend Snippet: ATO induced autophagy in the FLT3-ITD AML cell line. (A) MV4-11 cells were treated with ATO for 24 h, and the expression levels of LC3, ATG5 and ATG7 were detected by western blot analysis. GAPDH expression was used as a loading control. (B) MV4-11 cells transfected with mRFP-GFP-LC3 plasmids were treated with ATO for 0, 12, and 24 h and then observed by confocal fluorescence microscopy. Scale bar=5 µm. (C) MV4-11 cells were treated with ATO for 0, 12, and 24 h. The number of autophagosomes was observed by transmission electron microscopy and calculated (each group had 30 views, * P

Techniques Used: Expressing, Western Blot, Transfection, Fluorescence, Microscopy, Transmission Assay, Electron Microscopy

Degradation of FLT3-ITD by ATO was reversed by inhibition of autophagy (A). MV4-11 cells were treated with ATO for 0, 12, and 24 h, and immunofluorescence staining of p-FLT3 was analyzed. (B). MV4-11 cells were treated with ATO alone or in combination with bafilomycin A (BafA) for 24 h, and p-FLT3 and LC3-I to LC3-II conversion were assessed by western blot analysis. (C). MV4-11 cells were transfected with siRNAs targeting Atg5 or Atg7 for 48 h and then treated with or without ATO for 24 h. Then, p-FLT3, ATG5 and ATG7 were assessed by western blotting. GAPDH expression was used as a loading control.
Figure Legend Snippet: Degradation of FLT3-ITD by ATO was reversed by inhibition of autophagy (A). MV4-11 cells were treated with ATO for 0, 12, and 24 h, and immunofluorescence staining of p-FLT3 was analyzed. (B). MV4-11 cells were treated with ATO alone or in combination with bafilomycin A (BafA) for 24 h, and p-FLT3 and LC3-I to LC3-II conversion were assessed by western blot analysis. (C). MV4-11 cells were transfected with siRNAs targeting Atg5 or Atg7 for 48 h and then treated with or without ATO for 24 h. Then, p-FLT3, ATG5 and ATG7 were assessed by western blotting. GAPDH expression was used as a loading control.

Techniques Used: Inhibition, Immunofluorescence, Staining, Western Blot, Transfection, Expressing

5) Product Images from "Zα2 domain of ZBP1 is a molecular switch regulating Influenza-induced PANoptosis and perinatal lethality during development"

Article Title: Zα2 domain of ZBP1 is a molecular switch regulating Influenza-induced PANoptosis and perinatal lethality during development

Journal: bioRxiv

doi: 10.1101/2020.04.05.026542

Zα2 domain of ZBP1 is critical for triggering IAV-induced NLRP3 inflammasome activation and PANoptosis A, Immunoblot analysis of ZBP1, caspase-1 (CASP1), gasdermin D (GSDMD), IAV-NS1, and GAPDH in WT and Zbp1 ΔZα2/ΔZα2 BMDMs infected with IAV (mouse adapted, influenza A/Puerto Rico/8/34 [PR8; H1N1]). B and C, Cell death as measured by the number of SYTOX Green + cells. BMDMs were infected with IAV, and cell death was monitored at regular intervals. **** P
Figure Legend Snippet: Zα2 domain of ZBP1 is critical for triggering IAV-induced NLRP3 inflammasome activation and PANoptosis A, Immunoblot analysis of ZBP1, caspase-1 (CASP1), gasdermin D (GSDMD), IAV-NS1, and GAPDH in WT and Zbp1 ΔZα2/ΔZα2 BMDMs infected with IAV (mouse adapted, influenza A/Puerto Rico/8/34 [PR8; H1N1]). B and C, Cell death as measured by the number of SYTOX Green + cells. BMDMs were infected with IAV, and cell death was monitored at regular intervals. **** P

Techniques Used: Activation Assay, Infection

6) Product Images from "Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies"

Article Title: Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0633-9

Immunohistochemistry analysis of TK1 expression in lung cancer tissue. Lung cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of lung cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in ~ 53% of the lung adenocarcinoma tissues and in ~ 58% of the lung squamous cell carcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Images showing lung adenocarcinoma tissue positive and weakly positive for TK1, which we classified as TK1− with gray value quantification. The yellow circle in adenocarcinoma TK1+ image corresponds to lung papillary adenocarcinoma formed by abnormal proliferation of glanduli form structures of papillary disposition. The adenocarcinoma TK1− image shows pulmonary adenocarcinoma tissue with acinar pattern conformed by cells of convoluted nuclei, irregular membrane, and prominent central macronucleoli. The yellow arrows correspond to a diffuse weak positive nuclear staining for TK1. c Images showing lung squamous cell carcinoma tissue positive and weakly positive for TK1, which we classified as TK1− with gray value quantification. In the squamous cell carcinoma TK1+ image, the green line shows strong diffuse positive nuclear staining for TK1. The green arrow shows cytoplasmic background where there is evidence of infiltration in the underlying stroma shown by positive immunostaining against TK1. In the squamous cell carcinoma TK1− image, the red circle and arrows show weakly positive nuclear focal staining in poorly differentiated lung squamous carcinoma with solid pattern. ***P ≤ 0.001; ns = P > 0.05
Figure Legend Snippet: Immunohistochemistry analysis of TK1 expression in lung cancer tissue. Lung cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of lung cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in ~ 53% of the lung adenocarcinoma tissues and in ~ 58% of the lung squamous cell carcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Images showing lung adenocarcinoma tissue positive and weakly positive for TK1, which we classified as TK1− with gray value quantification. The yellow circle in adenocarcinoma TK1+ image corresponds to lung papillary adenocarcinoma formed by abnormal proliferation of glanduli form structures of papillary disposition. The adenocarcinoma TK1− image shows pulmonary adenocarcinoma tissue with acinar pattern conformed by cells of convoluted nuclei, irregular membrane, and prominent central macronucleoli. The yellow arrows correspond to a diffuse weak positive nuclear staining for TK1. c Images showing lung squamous cell carcinoma tissue positive and weakly positive for TK1, which we classified as TK1− with gray value quantification. In the squamous cell carcinoma TK1+ image, the green line shows strong diffuse positive nuclear staining for TK1. The green arrow shows cytoplasmic background where there is evidence of infiltration in the underlying stroma shown by positive immunostaining against TK1. In the squamous cell carcinoma TK1− image, the red circle and arrows show weakly positive nuclear focal staining in poorly differentiated lung squamous carcinoma with solid pattern. ***P ≤ 0.001; ns = P > 0.05

Techniques Used: Immunohistochemistry, Expressing, Staining, Light Microscopy, Immunostaining

Immunohistochemistry analysis of TK1 expression in breast cancer tissue. Breast cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. GAPDH was used as a positive control to account for housekeeping gene expression. The isotype antibody was used to account for background noise and non-specific binding. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of breast cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in 20% of the ductal carcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Image showing breast ductal carcinoma positive for TK1. The yellow circle encloses a malignant gland structure corresponding to a moderately differentiated ductal carcinoma, and the arrow shows strong nuclear staining against TK1 in approximately 25% of the cells. c Image showing breast ductal carcinoma negative for TK1. The green circle shows an atypical gland structure corresponding to moderately differentiated ductal carcinoma negative for staining against TK1. The green arrow shows the tumor stroma conformed by fibrotic tissue also negative for TK1. Overall, the tissues shown in Fig. 5 b, c show what we observed in the tissue’s average gray values represented in Fig. 5 a, that some ductal carcinoma showed strong TK1 staining and some showed negative TK1 staining. ***P ≤ 0.001; ns = P > 0.05
Figure Legend Snippet: Immunohistochemistry analysis of TK1 expression in breast cancer tissue. Breast cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. GAPDH was used as a positive control to account for housekeeping gene expression. The isotype antibody was used to account for background noise and non-specific binding. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of breast cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in 20% of the ductal carcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Image showing breast ductal carcinoma positive for TK1. The yellow circle encloses a malignant gland structure corresponding to a moderately differentiated ductal carcinoma, and the arrow shows strong nuclear staining against TK1 in approximately 25% of the cells. c Image showing breast ductal carcinoma negative for TK1. The green circle shows an atypical gland structure corresponding to moderately differentiated ductal carcinoma negative for staining against TK1. The green arrow shows the tumor stroma conformed by fibrotic tissue also negative for TK1. Overall, the tissues shown in Fig. 5 b, c show what we observed in the tissue’s average gray values represented in Fig. 5 a, that some ductal carcinoma showed strong TK1 staining and some showed negative TK1 staining. ***P ≤ 0.001; ns = P > 0.05

Techniques Used: Immunohistochemistry, Expressing, Staining, Positive Control, Binding Assay, Light Microscopy

Immunohistochemistry analysis of TK1 expression in colon cancer tissue. Colon cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. GAPDH was used as a positive control to account for housekeeping gene expression. The isotype antibody was used to account for background noise and non-specific binding. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of colorectal cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in ~ 74% of the colon adenocarcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Image showing colorectal adenocarcinoma positive for TK1. The yellow circle encloses an atypical glandular structure positive for TK1 in over 90% of the cells. c Image showing colorectal adenocarcinoma negative for TK1. The circle an atypical glandular structure negative for TK1. Overall, the tissues shown in ( b , c ) show what we observed in the tissue’s average gray values represented in a , that some colorectal adenocarcinoma tissues showed strong TK1 staining and some showed negative TK1 staining. ***P ≤ 0.001; ns = P > 0.05
Figure Legend Snippet: Immunohistochemistry analysis of TK1 expression in colon cancer tissue. Colon cancer tissue arrays were stained with anti-TK1 antibody ab91651, GAPDH, or isotype antibody. GAPDH was used as a positive control to account for housekeeping gene expression. The isotype antibody was used to account for background noise and non-specific binding. Tissues were imaged in a light microscope at 20×. Analysis was conducted using a gray scale. The lower the gray value, the darker the staining. a Quantitative analysis of colorectal cancer IHC staining. The top graph shows that there is a statistically significant expression of TK1 in ~ 74% of the colon adenocarcinoma tissues. The bottom graph shows the TK1 expression next to GAPDH and isotype controls. Background levels show no statistical difference between malignant and normal healthy tissues. Malignant and normal healthy tissue showed non-statistical difference in GAPDH expression, whereas TK1 expression did show a statically significant difference between TK1+ and TK1− tissues. b Image showing colorectal adenocarcinoma positive for TK1. The yellow circle encloses an atypical glandular structure positive for TK1 in over 90% of the cells. c Image showing colorectal adenocarcinoma negative for TK1. The circle an atypical glandular structure negative for TK1. Overall, the tissues shown in ( b , c ) show what we observed in the tissue’s average gray values represented in a , that some colorectal adenocarcinoma tissues showed strong TK1 staining and some showed negative TK1 staining. ***P ≤ 0.001; ns = P > 0.05

Techniques Used: Immunohistochemistry, Expressing, Staining, Positive Control, Binding Assay, Light Microscopy

7) Product Images from "Ubiquitin-specific protease 7 is a drug-able target that promotes hepatocellular carcinoma and chemoresistance"

Article Title: Ubiquitin-specific protease 7 is a drug-able target that promotes hepatocellular carcinoma and chemoresistance

Journal: Cancer Cell International

doi: 10.1186/s12935-020-1109-2

P22077 regulated multiple essential biological processes in Huh7 cell. Huh7 cells were treated with P22077 for 24 h. The treated and non-treated cells were harvested. Total protein was extracted and subjected for mass spectrometry analysis. a The identified down-regulated protein was further analyzed by pathway enrichment to identified significantly affected pathways. b The identified up-regulated protein after P22077 treatment was analyzed for pathway enrichment. c The expression of H3 and H3K4me 2 was examined by Western Blot. d Cells were treated with P22077 and samples were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes for immunoblotting assays to detected the expression of H3, H3K4me 2 , BAX was probed to detect apoptosis. GAPDH was a loading control
Figure Legend Snippet: P22077 regulated multiple essential biological processes in Huh7 cell. Huh7 cells were treated with P22077 for 24 h. The treated and non-treated cells were harvested. Total protein was extracted and subjected for mass spectrometry analysis. a The identified down-regulated protein was further analyzed by pathway enrichment to identified significantly affected pathways. b The identified up-regulated protein after P22077 treatment was analyzed for pathway enrichment. c The expression of H3 and H3K4me 2 was examined by Western Blot. d Cells were treated with P22077 and samples were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes for immunoblotting assays to detected the expression of H3, H3K4me 2 , BAX was probed to detect apoptosis. GAPDH was a loading control

Techniques Used: Mass Spectrometry, Expressing, Western Blot, SDS Page

8) Product Images from "RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells"

Article Title: RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

Journal: Pflugers Archiv

doi: 10.1007/s00424-019-02338-4

RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control
Figure Legend Snippet: RBP2 interaction with Cav1.3 C-terminal splice variants. a Schematic representation of the Cav1.3 C-terminal GST-fusion proteins: GST-Cav1.3 42 C-term (GST-42), GST-Cav1.3 42A C-term (GST-42A), and GST-Cav1.3 43S C-term (GST-43S) including the binding position for the anti-Cav1.3α1 2022–2138 antibody (anti-42) in the full-length C terminus. Numbers indicate the amino acid position in the Cav1.3 protein (GenBank™ accession number NM_000720). b GST pull-down of whole-cell extracts prepared from HEK293 cells transfected with HA-RBP2 with the indicated Cav1.3 C termini coupled to GST; 1 of 4 similar experiments is illustrated. Bound HA-RBP2 was visualized by western blotting using anti-HA. Anti-GAPDH staining served as a negative control. Input—0.5, 0.25, and 0.1% of the lysate. GST, GST-RIIβ, and GST-max p14 were control peptides not binding to HA-RBP2. Migration of molecular mass markers is indicated. c Left: Ponceau staining of GST-fusion proteins. Arrows indicate the migration of the full-length construct. Despite the partial degradation of GST-fusion proteins GST-42 and GST-42A, we observed selective protein–protein interactions between GST-42 and RBP2. Right: Immunoblot from panel b was stripped and the presence of GST-Cav1.3 42 C-term was verified by immunoblotting using anti-Cav1.3α1 2022–2138 antibody directed against an epitope present only in the long C-terminal splice variant as illustrated in panel a . d Confirmation of HA-RBP2 interaction with the long Cav1.3 C terminus by co-immunoprecipitation of HA-rRBP2 expressed in tsA-201 cells with YFP-tagged long Cav1.3 C terminus (YFP-Cav1.3 42 C-term; YFP-42). Top: Verification of the presence of YFP-Cav1.3 42 C-term by immunoblotting using an YFP antibody. Bottom: Specific immunoprecipitation of RBP2 by Cav1.3 42 C-term (detection by anti-RBP2-1318). Input control—1 and 0.5% of the lysate. Mock: untransfected control

Techniques Used: Binding Assay, Transfection, Western Blot, Staining, Negative Control, Migration, Construct, Variant Assay, Immunoprecipitation

9) Product Images from "Simvastatin Inhibits Toll-like Receptor 8 (TLR8) Signaling in Primary Human Monocytes and Spontaneous Tumor Necrosis Factor Production from Rheumatoid Synovial Membrane Cultures"

Article Title: Simvastatin Inhibits Toll-like Receptor 8 (TLR8) Signaling in Primary Human Monocytes and Spontaneous Tumor Necrosis Factor Production from Rheumatoid Synovial Membrane Cultures

Journal: Molecular Medicine

doi: 10.2119/molmed.2015.00154

Inhibitory effects of simvastatin on TLR8 signaling are mediated by inhibition of NF-κB but not AP-1. (A) Inhibition of NF-κB–induced luciferase production in HEK Blue TLR8 cells in response to 16 h incubation with 5 μg/mL R-848 after a 30-min incubation with simvastatin. (B) HEK Blue TLR8 cells were treated with 10 μg/mL simvastatin for 30 min ± 10 μmol/L GGPP before treatment with 5 μg/mL R-848 for 16 h. Cell lysates were examined for phosphorylation of p38 by Western blotting. (C) Primary human monocytes were treated with 10 μg/mL simvastatin ± 10 μmol/L GGPP for 30 min before treatment with 2 μg/mL R-848 for 16 h. Cell lysates were examined for phosphorylation of p38, IKKα/β and TAK-1 by Western blotting. GAPDH was used as a loading control. These blots are representative of three independent experiments. (D) Densitometric analysis of Western blots for phosphorylated IKKα/β. Data are expressed as arbitrary units and are the mean ± SE (n = 3). * p
Figure Legend Snippet: Inhibitory effects of simvastatin on TLR8 signaling are mediated by inhibition of NF-κB but not AP-1. (A) Inhibition of NF-κB–induced luciferase production in HEK Blue TLR8 cells in response to 16 h incubation with 5 μg/mL R-848 after a 30-min incubation with simvastatin. (B) HEK Blue TLR8 cells were treated with 10 μg/mL simvastatin for 30 min ± 10 μmol/L GGPP before treatment with 5 μg/mL R-848 for 16 h. Cell lysates were examined for phosphorylation of p38 by Western blotting. (C) Primary human monocytes were treated with 10 μg/mL simvastatin ± 10 μmol/L GGPP for 30 min before treatment with 2 μg/mL R-848 for 16 h. Cell lysates were examined for phosphorylation of p38, IKKα/β and TAK-1 by Western blotting. GAPDH was used as a loading control. These blots are representative of three independent experiments. (D) Densitometric analysis of Western blots for phosphorylated IKKα/β. Data are expressed as arbitrary units and are the mean ± SE (n = 3). * p

Techniques Used: Inhibition, Luciferase, Incubation, Western Blot

10) Product Images from "Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway"

Article Title: Downregulation of Pim-2 induces cell cycle arrest in the G0/G1 phase via the p53-non-dependent p21 signaling pathway

Journal: Oncology Letters

doi: 10.3892/ol.2018.7865

Effect of inhibition of Pim-2 in K562, RPMI-8226, H1299 and A549 cells on the cell cycle regulatory protein expression levels. Western blot analysis of cell cycle regulatory proteins P53, P21, CDK2, pRb and Rb in control and pim-2 silenced cells. p21 was highly expressed in all the cell lines. GAPDH was used as endogenous control. CDK2, cell dependent kinase 2; Rb, retinoblastoma; p, phosphorylated; siRNA, short interfering RNA.
Figure Legend Snippet: Effect of inhibition of Pim-2 in K562, RPMI-8226, H1299 and A549 cells on the cell cycle regulatory protein expression levels. Western blot analysis of cell cycle regulatory proteins P53, P21, CDK2, pRb and Rb in control and pim-2 silenced cells. p21 was highly expressed in all the cell lines. GAPDH was used as endogenous control. CDK2, cell dependent kinase 2; Rb, retinoblastoma; p, phosphorylated; siRNA, short interfering RNA.

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

11) Product Images from "Human CYP2C8 Is Post-Transcriptionally Regulated by MicroRNAs 103 and 107 in Human Liver"

Article Title: Human CYP2C8 Is Post-Transcriptionally Regulated by MicroRNAs 103 and 107 in Human Liver

Journal: Molecular Pharmacology

doi: 10.1124/mol.112.078386

Regulation of human CYP2C8 protein levels through ectopic overexpression or silencing of miR-103 and miR-107 in primary human hepatocytes. A and C, cells were transfected with 10 or 50 pmol of precursors for miR-103 and miR-107 individually (A) or 10 or 50 pmol of AsOs (C) or the respective control (Con). After 72 h, total RNA and microsomal membranes were prepared as described. Mature miRNA levels were determined through real-time reverse transcription-PCR analysis after transfection with precursors or AsOs. Values were normalized with respect to RNU44 levels and expressed relative to control values. Microsomal human CYP2C8 and GAPDH levels were determined through immunoblot analysis. B and D, primary hepatocytes from one donor were transfected with 10 or 50 pmol of precursors (B) or 10 or 50 pmol of AsOs (D). Lanes represent results for one of three replicate wells. The relative CYP2C8 protein levels were analyzed through densitometric image analysis and expressed relative to GAPDH levels. Values are the mean ± S.E. of triplicate observations. *, significantly different from control, P
Figure Legend Snippet: Regulation of human CYP2C8 protein levels through ectopic overexpression or silencing of miR-103 and miR-107 in primary human hepatocytes. A and C, cells were transfected with 10 or 50 pmol of precursors for miR-103 and miR-107 individually (A) or 10 or 50 pmol of AsOs (C) or the respective control (Con). After 72 h, total RNA and microsomal membranes were prepared as described. Mature miRNA levels were determined through real-time reverse transcription-PCR analysis after transfection with precursors or AsOs. Values were normalized with respect to RNU44 levels and expressed relative to control values. Microsomal human CYP2C8 and GAPDH levels were determined through immunoblot analysis. B and D, primary hepatocytes from one donor were transfected with 10 or 50 pmol of precursors (B) or 10 or 50 pmol of AsOs (D). Lanes represent results for one of three replicate wells. The relative CYP2C8 protein levels were analyzed through densitometric image analysis and expressed relative to GAPDH levels. Values are the mean ± S.E. of triplicate observations. *, significantly different from control, P

Techniques Used: Over Expression, Transfection, Polymerase Chain Reaction

Correlation between CYP2C8 mRNA and protein levels in 31 human liver samples (A) and predicted target sequence for miR-103/miR-107 in human CYP2C8 mRNA (B). A, CYP2C8 mRNA levels were determined with real-time quantitative PCR assays and were normalized with respect to GAPDH mRNA levels. Microsomal CYP2C8 protein contents were determined through immunoblotting with an antibody specific for CYP2C8 and were normalized with respect to GAPDH protein levels. As determined through Spearman correlation analysis, there was no significant correlation between CYP2C8 mRNA levels and protein levels. B, the predicted target sequence for miR-103/miR-107 in human CYP2C8 mRNA was identified. The numbering refers to the ATG in translation starting with A as 1; the coding region continues to position 1470, with a stop codon at positions 1471 to 1473. The sequence of the putative 2C8MRE (gray box) is located 197 to 219 bp downstream from the stop codon in the 3′-UTR of human CYP2C8 mRNA.
Figure Legend Snippet: Correlation between CYP2C8 mRNA and protein levels in 31 human liver samples (A) and predicted target sequence for miR-103/miR-107 in human CYP2C8 mRNA (B). A, CYP2C8 mRNA levels were determined with real-time quantitative PCR assays and were normalized with respect to GAPDH mRNA levels. Microsomal CYP2C8 protein contents were determined through immunoblotting with an antibody specific for CYP2C8 and were normalized with respect to GAPDH protein levels. As determined through Spearman correlation analysis, there was no significant correlation between CYP2C8 mRNA levels and protein levels. B, the predicted target sequence for miR-103/miR-107 in human CYP2C8 mRNA was identified. The numbering refers to the ATG in translation starting with A as 1; the coding region continues to position 1470, with a stop codon at positions 1471 to 1473. The sequence of the putative 2C8MRE (gray box) is located 197 to 219 bp downstream from the stop codon in the 3′-UTR of human CYP2C8 mRNA.

Techniques Used: Sequencing, Real-time Polymerase Chain Reaction

Effects of miR-103/miR-107 precursors or AsOs on expression of other CYP2C proteins. A and B, for each treatment, three separate wells of primary hepatocytes from a single donor were transfected with 50 pmol of control, miR-103, or miR-107 precursor miRNAs (A) or 50 pmol of control, miR-103, or miR-107 AsOs (B) for 48 h, and microsomal membranes were prepared from each set of donor hepatocytes. The membrane proteins were separated on large 10% SDS-polyacrylamide gels, with BD Gentest human liver microsomes (HLM) and recombinant CYP2Cs expressed in yeast as controls, and were transferred to nitrocellulose membranes. The membranes were probed with in-house-generated antibodies to CYP2C19 (antibody 1590), a specific CYP2C8 peptide (antibody 1937), and GAPDH. The antibody to CYP2C19 also recognized CYP2C9 and weakly recognized CYP2C8; therefore, the individual proteins could be identified only on the basis of their electrophoretic mobilities on large gels. C–H, the images were scanned and quantitated through densitometric analysis; data represent the means from three wells. C, effects of precursor miRNAs on CYP2C8 protein levels. D, effects of AsOs on CYP2C8 protein levels. E, effects of precursor miRNAs on CYP2C9 levels. F, effects of AsOs on CYP2C9 levels. G, effects of precursor miRNAs on CYP2C19 levels. H, effects of AsOs on CYP2C19 levels. **, significantly different from precursor or AsO control, P
Figure Legend Snippet: Effects of miR-103/miR-107 precursors or AsOs on expression of other CYP2C proteins. A and B, for each treatment, three separate wells of primary hepatocytes from a single donor were transfected with 50 pmol of control, miR-103, or miR-107 precursor miRNAs (A) or 50 pmol of control, miR-103, or miR-107 AsOs (B) for 48 h, and microsomal membranes were prepared from each set of donor hepatocytes. The membrane proteins were separated on large 10% SDS-polyacrylamide gels, with BD Gentest human liver microsomes (HLM) and recombinant CYP2Cs expressed in yeast as controls, and were transferred to nitrocellulose membranes. The membranes were probed with in-house-generated antibodies to CYP2C19 (antibody 1590), a specific CYP2C8 peptide (antibody 1937), and GAPDH. The antibody to CYP2C19 also recognized CYP2C9 and weakly recognized CYP2C8; therefore, the individual proteins could be identified only on the basis of their electrophoretic mobilities on large gels. C–H, the images were scanned and quantitated through densitometric analysis; data represent the means from three wells. C, effects of precursor miRNAs on CYP2C8 protein levels. D, effects of AsOs on CYP2C8 protein levels. E, effects of precursor miRNAs on CYP2C9 levels. F, effects of AsOs on CYP2C9 levels. G, effects of precursor miRNAs on CYP2C19 levels. H, effects of AsOs on CYP2C19 levels. **, significantly different from precursor or AsO control, P

Techniques Used: Expressing, Transfection, Recombinant, Generated, Allele-specific Oligonucleotide

12) Product Images from "Notch-Mediated Epigenetic Regulation of Voltage-Gated Potassium Currents"

Article Title: Notch-Mediated Epigenetic Regulation of Voltage-Gated Potassium Currents

Journal: Circulation research

doi: 10.1161/CIRCRESAHA.116.309877

Notch activation dysregulates expression of Kv channel subunits (A) Notch GOF upregulates expression of the Purkinje cell marker Cntn2 8-fold, Scn5a 9-fold as well as ventricle-enriched Notch targets Hrt2 (9-fold) and Hes1 (3-fold), while the atrial-enriched Notch target Hrt1 was unchanged (n = 6) compared to littermate controls. (B) Notch GOF represses expression of transcripts encoding potassium channel subunits comprising I to,f ( Kcnd3 2-fold, Kcnip2 5-fold); IK slow ( Kcnb1 and Kcna5 2-fold) while subunits encoding IK 1 ( Kcnj2 ) are not changed (n = 6) compared with littermate controls. (C) Western blot showing decreased protein levels of KChIP2 (35kDa doublet) and Kv2.1 (114kDa) in Notch GOF when compared with GAPDH protein levels. (D) Quantification of protein levels based on band density shows significantly reduced levels of KChIP2 (3-fold) and of Kv2.1 (2-fold) in Notch GOF compared with littermate controls (n = 4). Equal variance, two-tailed Students' t-test was performed to determine statistical significance. * p
Figure Legend Snippet: Notch activation dysregulates expression of Kv channel subunits (A) Notch GOF upregulates expression of the Purkinje cell marker Cntn2 8-fold, Scn5a 9-fold as well as ventricle-enriched Notch targets Hrt2 (9-fold) and Hes1 (3-fold), while the atrial-enriched Notch target Hrt1 was unchanged (n = 6) compared to littermate controls. (B) Notch GOF represses expression of transcripts encoding potassium channel subunits comprising I to,f ( Kcnd3 2-fold, Kcnip2 5-fold); IK slow ( Kcnb1 and Kcna5 2-fold) while subunits encoding IK 1 ( Kcnj2 ) are not changed (n = 6) compared with littermate controls. (C) Western blot showing decreased protein levels of KChIP2 (35kDa doublet) and Kv2.1 (114kDa) in Notch GOF when compared with GAPDH protein levels. (D) Quantification of protein levels based on band density shows significantly reduced levels of KChIP2 (3-fold) and of Kv2.1 (2-fold) in Notch GOF compared with littermate controls (n = 4). Equal variance, two-tailed Students' t-test was performed to determine statistical significance. * p

Techniques Used: Activation Assay, Expressing, Marker, Western Blot, Two Tailed Test

13) Product Images from "Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells"

Article Title: Molecular interplay between T-Antigen and splicing factor, arginine/serine-rich 1 (SRSF1) controls JC virus gene expression in glial cells

Journal: Virology Journal

doi: 10.1186/s12985-015-0426-x

T-Antigen suppresses SRSF1 expression in glial cells. a . PHFA cells were transfected with increasing concentration of T-antigen expression plasmid. After 48 h post-transfection, whole cell protein extracts were collected. Western blot analyses were performed to detect levels of SRSF1, T-antigen, and small t antigen expressions. Grb2 served as a loading control. b . Bar graph representation of relative SRSF1 expression from panel a . Quantification of the intensity of the bands of SRSF1 from panel a were normalized to Grb2 band intensities and used to calculate relative expression. c . T98G cells were transfected with increasing concentration of T-antigen expression plasmid. After 48 h post-transfections, whole cell protein extracts were collected. Western blot analyses were performed to detect levels of SRSF1, T-antigen, and small t antigen expressions. GAPDH served as a loading control. d . Bar graph representation of relative SRSF1 expression from panel c . Quantification of the intensity of the bands of SRSF1 were normalized to GAPDH band intensities and used to calculate relative SRSF1 expression levels. e . Whole cell protein lysates from PHFA and T98G cells were prepared and analyzed by Western blotting for the detection of SRSF1 expression. GAPDH served as a loading control. Band intensity of the bands of SRSF1 were quantified, normalized to GAPDH, and shown as bar graph
Figure Legend Snippet: T-Antigen suppresses SRSF1 expression in glial cells. a . PHFA cells were transfected with increasing concentration of T-antigen expression plasmid. After 48 h post-transfection, whole cell protein extracts were collected. Western blot analyses were performed to detect levels of SRSF1, T-antigen, and small t antigen expressions. Grb2 served as a loading control. b . Bar graph representation of relative SRSF1 expression from panel a . Quantification of the intensity of the bands of SRSF1 from panel a were normalized to Grb2 band intensities and used to calculate relative expression. c . T98G cells were transfected with increasing concentration of T-antigen expression plasmid. After 48 h post-transfections, whole cell protein extracts were collected. Western blot analyses were performed to detect levels of SRSF1, T-antigen, and small t antigen expressions. GAPDH served as a loading control. d . Bar graph representation of relative SRSF1 expression from panel c . Quantification of the intensity of the bands of SRSF1 were normalized to GAPDH band intensities and used to calculate relative SRSF1 expression levels. e . Whole cell protein lysates from PHFA and T98G cells were prepared and analyzed by Western blotting for the detection of SRSF1 expression. GAPDH served as a loading control. Band intensity of the bands of SRSF1 were quantified, normalized to GAPDH, and shown as bar graph

Techniques Used: Expressing, Transfection, Concentration Assay, Plasmid Preparation, Western Blot

14) Product Images from "Supplemental Treatment for Huntington’s Disease with miR-132 that Is Deficient in Huntington’s Disease Brain"

Article Title: Supplemental Treatment for Huntington’s Disease with miR-132 that Is Deficient in Huntington’s Disease Brain

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.01.007

Mutant HTT Expression Total RNAs used in Figure 4 B were examined by RT-semiquantitative PCR to see mutant HTT expression. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Gapdh was also examined as an internal control.
Figure Legend Snippet: Mutant HTT Expression Total RNAs used in Figure 4 B were examined by RT-semiquantitative PCR to see mutant HTT expression. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining. Gapdh was also examined as an internal control.

Techniques Used: Mutagenesis, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

15) Product Images from "Hematopoietic lineage cell-specific protein 1 (HS1), a hidden player in migration, invasion, and tumor formation, is over-expressed in ovarian carcinoma cells"

Article Title: Hematopoietic lineage cell-specific protein 1 (HS1), a hidden player in migration, invasion, and tumor formation, is over-expressed in ovarian carcinoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.25975

HS1 is expressed in highly invasive cells and is localized in invadopodia ( A ) Cells were lysed and immunoblot analysis was performed with an antibody against HS1. GAPDH was used as a loading control. ( B ) Total RNA was isolated from cells and used to synthesize cDNA. Quantitative PCR was used to measure the expression levels of the HS1 gene. GAPDH was used for normalization. ( C ) Human ovarian surface epithelial cell (OSE) total RNA was purchased from Cosmo Bio. ( D ) Lysates extracted from NOS2-AcGFP-Mock and -AcGFP-HS1 WT were used to perform immunoblot analysis with an antibody against HS1. GAPDH was used as a loading control. ( E ) NOS2-AcGFP-HS1 WT cells were cultured overnight on gelatin-coated coverslips; the cells were then fixed and stained with phalloidin-594. A FluoView (Olympus) confocal microscope was used to acquire the photographs and reconstruct 3D images. The yellow arrow shows an invadopodium.
Figure Legend Snippet: HS1 is expressed in highly invasive cells and is localized in invadopodia ( A ) Cells were lysed and immunoblot analysis was performed with an antibody against HS1. GAPDH was used as a loading control. ( B ) Total RNA was isolated from cells and used to synthesize cDNA. Quantitative PCR was used to measure the expression levels of the HS1 gene. GAPDH was used for normalization. ( C ) Human ovarian surface epithelial cell (OSE) total RNA was purchased from Cosmo Bio. ( D ) Lysates extracted from NOS2-AcGFP-Mock and -AcGFP-HS1 WT were used to perform immunoblot analysis with an antibody against HS1. GAPDH was used as a loading control. ( E ) NOS2-AcGFP-HS1 WT cells were cultured overnight on gelatin-coated coverslips; the cells were then fixed and stained with phalloidin-594. A FluoView (Olympus) confocal microscope was used to acquire the photographs and reconstruct 3D images. The yellow arrow shows an invadopodium.

Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Staining, Microscopy

Phosphorylation of tyrosine residues may contribute to the impact of HS1 on cell migration and invasion abilities ( A ) A portion of ES2 cell lysates was set aside as input, shown as (−). The remainders of the lysates were subjected to precipitation with an anti-HS1 antibody (CST). The precipitates were immunoblotted with anti-HS1 (BD) or anti-pTyr (Millipore) antibodies. ( B ) The amino acid sequence of HS1 is divided into five domains, specifically N (NTA domain, interaction with Arp2/3), R1–R3 (interaction with F-actin), CC (coiled-coil region), Pro (proline-rich region), and SH3 (SH3 domain) [ 58 ]. The construction of HS1 WT and HS1 3YF : HS1 WT has three tyrosine residues (Y) that are phosphorylation sites, and these residues were mutated to phenylalanine (F) in HS1 3YF . ( C ) After transfection with siRNA HS1-1788 (20 nM) or control (20 nM), cell lysates were used for immunoblot analysis to confirm knock-down of endogenous HS1. GAPDH was used as a loading control. ( D ) Following transfection with siRNAs, transwell migration and invasion assays were performed. After 22 hours, cells were fixed and stained and then photographs were acquired. The migrating and invading cells were counted using ImageJ software, and the relative migration or invasive abilities are shown (cells transfected with control siRNA were used as a reference, with a value of 1). Each experiment was performed in triplicate. The bars indicate the mean ± s.d. * P
Figure Legend Snippet: Phosphorylation of tyrosine residues may contribute to the impact of HS1 on cell migration and invasion abilities ( A ) A portion of ES2 cell lysates was set aside as input, shown as (−). The remainders of the lysates were subjected to precipitation with an anti-HS1 antibody (CST). The precipitates were immunoblotted with anti-HS1 (BD) or anti-pTyr (Millipore) antibodies. ( B ) The amino acid sequence of HS1 is divided into five domains, specifically N (NTA domain, interaction with Arp2/3), R1–R3 (interaction with F-actin), CC (coiled-coil region), Pro (proline-rich region), and SH3 (SH3 domain) [ 58 ]. The construction of HS1 WT and HS1 3YF : HS1 WT has three tyrosine residues (Y) that are phosphorylation sites, and these residues were mutated to phenylalanine (F) in HS1 3YF . ( C ) After transfection with siRNA HS1-1788 (20 nM) or control (20 nM), cell lysates were used for immunoblot analysis to confirm knock-down of endogenous HS1. GAPDH was used as a loading control. ( D ) Following transfection with siRNAs, transwell migration and invasion assays were performed. After 22 hours, cells were fixed and stained and then photographs were acquired. The migrating and invading cells were counted using ImageJ software, and the relative migration or invasive abilities are shown (cells transfected with control siRNA were used as a reference, with a value of 1). Each experiment was performed in triplicate. The bars indicate the mean ± s.d. * P

Techniques Used: Migration, Sequencing, Transfection, Staining, Software

HS1 plays an important role in tumorigenesis in ovarian cancer cells ( A ) Lysates extracted from ES2-shRNA-Control and -shRNA-HS1 were used to perform immunoblot analysis with an antibody against HS1. GAPDH was used as a loading control. ( B ) Cells were cultured overnight in a Culture-Insert 2 Well; at 0 and 6 hours after removal from the well, photographs were acquired by microscopy. ( C ) The percentages of original wound areas that had healed were measured by ImageJ software based on the acquired photographs, and are shown in the bar graph. ( D ) Cells were transduced with shRNA and then transwell migration and invasion assays were performed. After 22 hours, cells were fixed and stained and then photographs were acquired. The migrating and invading cells were counted using ImageJ software, and the relative migration or invasive abilities are shown (the abilities of cells transfected with control shRNA were used as a reference, with a value of 1). Each experiment was performed in triplicate. The bars indicate the mean ± s.d. * P
Figure Legend Snippet: HS1 plays an important role in tumorigenesis in ovarian cancer cells ( A ) Lysates extracted from ES2-shRNA-Control and -shRNA-HS1 were used to perform immunoblot analysis with an antibody against HS1. GAPDH was used as a loading control. ( B ) Cells were cultured overnight in a Culture-Insert 2 Well; at 0 and 6 hours after removal from the well, photographs were acquired by microscopy. ( C ) The percentages of original wound areas that had healed were measured by ImageJ software based on the acquired photographs, and are shown in the bar graph. ( D ) Cells were transduced with shRNA and then transwell migration and invasion assays were performed. After 22 hours, cells were fixed and stained and then photographs were acquired. The migrating and invading cells were counted using ImageJ software, and the relative migration or invasive abilities are shown (the abilities of cells transfected with control shRNA were used as a reference, with a value of 1). Each experiment was performed in triplicate. The bars indicate the mean ± s.d. * P

Techniques Used: shRNA, Cell Culture, Microscopy, Software, Transduction, Migration, Staining, Transfection

16) Product Images from "Impairments of spatial learning and memory following intrahippocampal injection in rats of 3-mercaptopropionic acid-modified CdTe quantum dots and molecular mechanisms"

Article Title: Impairments of spatial learning and memory following intrahippocampal injection in rats of 3-mercaptopropionic acid-modified CdTe quantum dots and molecular mechanisms

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S104985

Effects of 3.5 nm MPA-capped CdTe QD exposure on the protein expression of Akt, ERK1/2, their corresponding phosphorylated (p-) proteins, and c-FOS on rat hippocampus. Notes: ( A ) Representative Western blot of Akt, p-Akt, and GAPDH (reference protein). The density of each band was measured, and the ratios of Akt:GAPDH and p-Akt:Akt were calculated. ( B ) Representative Western blot of ERK1/2, p-ERK1/2, and GAPDH. The density of each band was measured, and the ratios of ERK1/2:GAPDH and p-ERK1/2:ERK1/2 were calculated. ( C ) Representative Western blot of c-Fos and GAPDH. The density of each band was measured, and the ratio of c-Fos:GAPDH was calculated. Data shown are mean ± SD (n=3). One-way analysis of variance followed by Dunnett’s post hoc test used for statistical analysis at each testing time point. * P
Figure Legend Snippet: Effects of 3.5 nm MPA-capped CdTe QD exposure on the protein expression of Akt, ERK1/2, their corresponding phosphorylated (p-) proteins, and c-FOS on rat hippocampus. Notes: ( A ) Representative Western blot of Akt, p-Akt, and GAPDH (reference protein). The density of each band was measured, and the ratios of Akt:GAPDH and p-Akt:Akt were calculated. ( B ) Representative Western blot of ERK1/2, p-ERK1/2, and GAPDH. The density of each band was measured, and the ratios of ERK1/2:GAPDH and p-ERK1/2:ERK1/2 were calculated. ( C ) Representative Western blot of c-Fos and GAPDH. The density of each band was measured, and the ratio of c-Fos:GAPDH was calculated. Data shown are mean ± SD (n=3). One-way analysis of variance followed by Dunnett’s post hoc test used for statistical analysis at each testing time point. * P

Techniques Used: Expressing, Western Blot

17) Product Images from "Emodin Attenuates Bleomycin-Induced Pulmonary Fibrosis via Anti-Inflammatory and Anti-Oxidative Activities in Rats"

Article Title: Emodin Attenuates Bleomycin-Induced Pulmonary Fibrosis via Anti-Inflammatory and Anti-Oxidative Activities in Rats

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.905496

Emodin suppressed inflammatory infiltration in BLM-induced pulmonary tissues. ( A ) Total number of cells and number of differentiate cell types in 1 mL BALFs was counted (n=5). ( B ) IL-1β, IL-6, and TNF-α level in cell-free BALFs supernatant were measured with ELISA kits. ( C ) The levels of protein expression of p-IκBα and nuclear translocation of NF-κB p65 were determined by Western blot analysis. ( D ) Quantitation of Western blot signal intensities. The study used endogenous GAPDH as a control for p-IκBα, and Histone H3 as a control for p65. All data are presented as mean ±SD (n=5), * P
Figure Legend Snippet: Emodin suppressed inflammatory infiltration in BLM-induced pulmonary tissues. ( A ) Total number of cells and number of differentiate cell types in 1 mL BALFs was counted (n=5). ( B ) IL-1β, IL-6, and TNF-α level in cell-free BALFs supernatant were measured with ELISA kits. ( C ) The levels of protein expression of p-IκBα and nuclear translocation of NF-κB p65 were determined by Western blot analysis. ( D ) Quantitation of Western blot signal intensities. The study used endogenous GAPDH as a control for p-IκBα, and Histone H3 as a control for p65. All data are presented as mean ±SD (n=5), * P

Techniques Used: Enzyme-linked Immunosorbent Assay, Expressing, Translocation Assay, Western Blot, Quantitation Assay

Emodin has anti-oxidative function and stimulates Nrf2 signaling in BLM-treated pulmonary tissues of rats. Activities of MDA, GSH ( A ), SOD, and GSH-Px ( B ) in control group, BLM group, and BLM + emodin group were measured using commercial kits. ( C ) Protein expression levels of nuclear Nrf2 and HO-1 were assessed by Western blot assay. ( D ) Quantitation of Western blot signal intensities. Histone H3 was used as a control for Nrf2 and GAPDH for HO-1. All data are presented as mean ±SD (n=5), ** P
Figure Legend Snippet: Emodin has anti-oxidative function and stimulates Nrf2 signaling in BLM-treated pulmonary tissues of rats. Activities of MDA, GSH ( A ), SOD, and GSH-Px ( B ) in control group, BLM group, and BLM + emodin group were measured using commercial kits. ( C ) Protein expression levels of nuclear Nrf2 and HO-1 were assessed by Western blot assay. ( D ) Quantitation of Western blot signal intensities. Histone H3 was used as a control for Nrf2 and GAPDH for HO-1. All data are presented as mean ±SD (n=5), ** P

Techniques Used: Multiple Displacement Amplification, Expressing, Western Blot, Quantitation Assay

18) Product Images from "Identification and characterization of Dicer1e, a Dicer1 protein variant, in oral cancer cells"

Article Title: Identification and characterization of Dicer1e, a Dicer1 protein variant, in oral cancer cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-13-190

The cellular localization of Dicer1e protein variant is predominantly nuclear. (A) Western blot analysis of Dicer1 and Dicer1e protein levels in nuclear (N) and cytoplasmic (C) fractions and whole (W) cell lysates of human OSCC cell lines (CAL 27, SCC-4, SCC-9, SCC-15, and SCC-25) and normal HOKs. The histone deacetylase class 1 (HDAC1) and GAPDH were used as nuclear and cytoplasmic markers, respectively. (B) HeLa cells transiently transfected with FLAG-Dicer1e. Representative images are shown of recombinant Dicer1e localization in cells (upper panels i-iii). Cells with predominantly nuclear (arrows), nuclear and cytoplasmic (arrowheads), or cytoplasmic (double-arrowheads) Dicer1e localizations are indicated. Nuclei were counterstained with DAPI (lower panels i-iii). Scale bar: 20 μm.
Figure Legend Snippet: The cellular localization of Dicer1e protein variant is predominantly nuclear. (A) Western blot analysis of Dicer1 and Dicer1e protein levels in nuclear (N) and cytoplasmic (C) fractions and whole (W) cell lysates of human OSCC cell lines (CAL 27, SCC-4, SCC-9, SCC-15, and SCC-25) and normal HOKs. The histone deacetylase class 1 (HDAC1) and GAPDH were used as nuclear and cytoplasmic markers, respectively. (B) HeLa cells transiently transfected with FLAG-Dicer1e. Representative images are shown of recombinant Dicer1e localization in cells (upper panels i-iii). Cells with predominantly nuclear (arrows), nuclear and cytoplasmic (arrowheads), or cytoplasmic (double-arrowheads) Dicer1e localizations are indicated. Nuclei were counterstained with DAPI (lower panels i-iii). Scale bar: 20 μm.

Techniques Used: Variant Assay, Western Blot, Histone Deacetylase Assay, Transfection, Recombinant

Silencing of Dicer1e induces apoptosis and/or G2/M cell cycle arrest in oral cancer cells. (A) Western blot analysis of PARP and caspase-3 cleavage levels in human OSCC cell lines (CAL 27, SCC-4, and SCC-25) 48 hours post-transfection with either control non-targeting siRNA (siNT) or siRNA targeting Dicer1e (siDicer1e). Dicer1e and GAPDH levels were monitored to ensure knockdown and equal loading of samples, respectively. (B) Cell cycle analyses of SCC-4 and SCC-25 cells after treatment with siDicer1e compared to control siNT-treated cells. Data are mean ± SEM of three independent experiments, where *P
Figure Legend Snippet: Silencing of Dicer1e induces apoptosis and/or G2/M cell cycle arrest in oral cancer cells. (A) Western blot analysis of PARP and caspase-3 cleavage levels in human OSCC cell lines (CAL 27, SCC-4, and SCC-25) 48 hours post-transfection with either control non-targeting siRNA (siNT) or siRNA targeting Dicer1e (siDicer1e). Dicer1e and GAPDH levels were monitored to ensure knockdown and equal loading of samples, respectively. (B) Cell cycle analyses of SCC-4 and SCC-25 cells after treatment with siDicer1e compared to control siNT-treated cells. Data are mean ± SEM of three independent experiments, where *P

Techniques Used: Western Blot, Transfection

19) Product Images from "Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity"

Article Title: Na,K-ATPase β1-subunit is a target of sonic hedgehog signaling and enhances medulloblastoma tumorigenicity

Journal: Molecular Cancer

doi: 10.1186/s12943-015-0430-1

Inverse relationship between Bmi1 and Na,K-ATPase β 1 -subunit levels in medulloblastoma and CGP cells. a . Protein extracts obtained from four Smo/Smo medulloblastoma and cerebellum of four wild type mice were immunoblotted with the indicated antibodies. α-tubulin was used as loading control. b . mRNA levels of Bmi1 and β 1 -subunit in medulloblastoma and cerebellum as determined by qRT-PCR. c . Protein levels of Bmi1 in differentiating CGP cells. GAPDH was used to ensure equal loading. d . mRNA levels of Bmi1 and β 1 -subunit in CGP cells differentiating in vitro . The difference in β 1 -subunit mRNA expression was significant during CGP differentiation (** = p
Figure Legend Snippet: Inverse relationship between Bmi1 and Na,K-ATPase β 1 -subunit levels in medulloblastoma and CGP cells. a . Protein extracts obtained from four Smo/Smo medulloblastoma and cerebellum of four wild type mice were immunoblotted with the indicated antibodies. α-tubulin was used as loading control. b . mRNA levels of Bmi1 and β 1 -subunit in medulloblastoma and cerebellum as determined by qRT-PCR. c . Protein levels of Bmi1 in differentiating CGP cells. GAPDH was used to ensure equal loading. d . mRNA levels of Bmi1 and β 1 -subunit in CGP cells differentiating in vitro . The difference in β 1 -subunit mRNA expression was significant during CGP differentiation (** = p

Techniques Used: Mouse Assay, Quantitative RT-PCR, In Vitro, Expressing

Na,K-ATPase subunits in medulloblastoma and CGP cells. a . Na,K-ATPase α 1 - and β 1 -subunit expression in cerebellum from 6 month old WT C57BL6/J mice (WT) and tumors from age-matched Smo/Smo mice. An immunoblot for GAPDH confirmed equal loading of protein. b . Na,K-ATPase α 1 -subunit and β 1 -subunit mRNA levels in WT and medulloblastoma cerebellum normalized to beta-2 microglobulin. For both α 1 - and β 1 -subunit the difference between WT and medulloblastoma cerebellum is statistically significant ( p
Figure Legend Snippet: Na,K-ATPase subunits in medulloblastoma and CGP cells. a . Na,K-ATPase α 1 - and β 1 -subunit expression in cerebellum from 6 month old WT C57BL6/J mice (WT) and tumors from age-matched Smo/Smo mice. An immunoblot for GAPDH confirmed equal loading of protein. b . Na,K-ATPase α 1 -subunit and β 1 -subunit mRNA levels in WT and medulloblastoma cerebellum normalized to beta-2 microglobulin. For both α 1 - and β 1 -subunit the difference between WT and medulloblastoma cerebellum is statistically significant ( p

Techniques Used: Expressing, Mouse Assay

20) Product Images from "Netrin-1 suppresses the MEK/ERK pathway and ITGB4 in pancreatic cancer"

Article Title: Netrin-1 suppresses the MEK/ERK pathway and ITGB4 in pancreatic cancer

Journal: Oncotarget

doi: 10.18632/oncotarget.8348

Netrin-1 down-regulates integrin β4 expression through the UNC5b receptor and the activation of FAK ( A ) Real-time PCR analysis for the expression of netrin-1 receptors in MiaPaCa II cells. GAPDH was used as an internal control. ( B – C ) The anti-UNC5b antibody selectively blocks the integrin β4-suppressing effect of netrin-1. The receptors on the MiaPaCa II cells were blocked by their respective antibodies before the cells were treated with the indicated concentrations netrin-1; integrin β4 expression in the cells was then detected by real-time RT-PCR (*** P
Figure Legend Snippet: Netrin-1 down-regulates integrin β4 expression through the UNC5b receptor and the activation of FAK ( A ) Real-time PCR analysis for the expression of netrin-1 receptors in MiaPaCa II cells. GAPDH was used as an internal control. ( B – C ) The anti-UNC5b antibody selectively blocks the integrin β4-suppressing effect of netrin-1. The receptors on the MiaPaCa II cells were blocked by their respective antibodies before the cells were treated with the indicated concentrations netrin-1; integrin β4 expression in the cells was then detected by real-time RT-PCR (*** P

Techniques Used: Expressing, Activation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Netrin-1 inhibits PDAC growth by decreasing integrin β4 expression ( A – B ) Real-time RT-PCR (A) and western blotting (B) analyses of integrin β4 expression in control (ctrl) and netrin-1-over-expressing MiaPaCa II cells (ntn1+). GAPDH was used as the internal control for both analyses. ( C – D ) Real-time RT-PCR (C) and western blotting (D) analyses of integrin β4 expression in MiaPaCa II cells treated with the indicated concentrations of netrin-1 for 48 hr. GAPDH was used as the internal control for both analyses. ( E ) Western blotting analysis of integrin β4 expression in control GFP RNAi (RiGFP) and ITGB4 RNAi (RiITGB4) MiaPaCa II cells. GAPDH was used as the internal control. ( F ) Representative xenograft tumors formed by GFP RNAi (asterisk) and ITGB4 RNAi (arrowhead) MiaPaCa II cells in SCID-beige mice (bar, 1 cm). ( G ) Statistics for the weights of the xenograft tumors formed by the control RiGFP ( N = 7) and RiITGB4 ( N = 7) MiaPaCa II cells (*** P
Figure Legend Snippet: Netrin-1 inhibits PDAC growth by decreasing integrin β4 expression ( A – B ) Real-time RT-PCR (A) and western blotting (B) analyses of integrin β4 expression in control (ctrl) and netrin-1-over-expressing MiaPaCa II cells (ntn1+). GAPDH was used as the internal control for both analyses. ( C – D ) Real-time RT-PCR (C) and western blotting (D) analyses of integrin β4 expression in MiaPaCa II cells treated with the indicated concentrations of netrin-1 for 48 hr. GAPDH was used as the internal control for both analyses. ( E ) Western blotting analysis of integrin β4 expression in control GFP RNAi (RiGFP) and ITGB4 RNAi (RiITGB4) MiaPaCa II cells. GAPDH was used as the internal control. ( F ) Representative xenograft tumors formed by GFP RNAi (asterisk) and ITGB4 RNAi (arrowhead) MiaPaCa II cells in SCID-beige mice (bar, 1 cm). ( G ) Statistics for the weights of the xenograft tumors formed by the control RiGFP ( N = 7) and RiITGB4 ( N = 7) MiaPaCa II cells (*** P

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Mouse Assay

21) Product Images from "Decorin reduces hypertrophic scarring through inhibition of the TGF-β1/Smad signaling pathway in a rat osteomyelitis model"

Article Title: Decorin reduces hypertrophic scarring through inhibition of the TGF-β1/Smad signaling pathway in a rat osteomyelitis model

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2016.3591

(A) Western blot analysis in the control and osteomyelitis groups at weeks (W) 1, 2 and 4 after surgery. The expression levels of (B) TGF-β1, (C) TβRI, (D) TβRII, (E) pSmad2 and (F) pSmad3 are shown as the integrated density against GAPDH and Smad2/3. *P
Figure Legend Snippet: (A) Western blot analysis in the control and osteomyelitis groups at weeks (W) 1, 2 and 4 after surgery. The expression levels of (B) TGF-β1, (C) TβRI, (D) TβRII, (E) pSmad2 and (F) pSmad3 are shown as the integrated density against GAPDH and Smad2/3. *P

Techniques Used: Western Blot, Expressing

(A) Immunoblotting analysis in the control, osteomyelitis and decorin-treated groups at weeks (W) 2 and 4 after surgery. The expression levels of (B) TGF-β1, (C) pSmad2, (D) pSmad3 and (E) collagen I are shown as the integrated density against GAPDH or Smad2/3. # P
Figure Legend Snippet: (A) Immunoblotting analysis in the control, osteomyelitis and decorin-treated groups at weeks (W) 2 and 4 after surgery. The expression levels of (B) TGF-β1, (C) pSmad2, (D) pSmad3 and (E) collagen I are shown as the integrated density against GAPDH or Smad2/3. # P

Techniques Used: Expressing

22) Product Images from "Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP"

Article Title: Cab45S inhibits the ER stress-induced IRE1-JNK pathway and apoptosis via GRP78/BiP

Journal: Cell Death & Disease

doi: 10.1038/cddis.2014.193

Cab45S increases the GRP78/BiP protein level. ( a and d ) Western blots of GRP78/BiP and two other ER molecular chaperones, PDI and calnexin, in stable Cab45S-knockdown ( a ) or Cab45S-overexpressed ( d ) PANC-1 cell lines. Numbers represent different cell lines. ( b and c ) Western blots ( b ) and quantification ( c ) of GRP78/BiP in Cab45S-knockdown and control (shNC, scrambled shRNA) HeLa cells treated with TM (2 μ g/ml) for the indicated periods. GAPDH was used as a loading control. ( e ) Quantitative real-time PCR of the relative GRP78/BiP mRNA expression levels in Cab45S-knockdown and control HeLa cells treated with TM for the indicated times ( n =3). ( f ) Western blots of GRP78/BiP in Cab45S-knockdown and control HeLa cells treated with TM (2 μ g/ml, 4 h) followed by cycloheximide (Chx; 100 μ M) for the indicated periods. For c and e , data are presented as mean±S.E.M. ** P
Figure Legend Snippet: Cab45S increases the GRP78/BiP protein level. ( a and d ) Western blots of GRP78/BiP and two other ER molecular chaperones, PDI and calnexin, in stable Cab45S-knockdown ( a ) or Cab45S-overexpressed ( d ) PANC-1 cell lines. Numbers represent different cell lines. ( b and c ) Western blots ( b ) and quantification ( c ) of GRP78/BiP in Cab45S-knockdown and control (shNC, scrambled shRNA) HeLa cells treated with TM (2 μ g/ml) for the indicated periods. GAPDH was used as a loading control. ( e ) Quantitative real-time PCR of the relative GRP78/BiP mRNA expression levels in Cab45S-knockdown and control HeLa cells treated with TM for the indicated times ( n =3). ( f ) Western blots of GRP78/BiP in Cab45S-knockdown and control HeLa cells treated with TM (2 μ g/ml, 4 h) followed by cycloheximide (Chx; 100 μ M) for the indicated periods. For c and e , data are presented as mean±S.E.M. ** P

Techniques Used: Western Blot, shRNA, Real-time Polymerase Chain Reaction, Expressing

The IRE1-JNK signaling is required for activating ER stress-induced apoptosis in Cab45S-depleted cells. ( a ) Quantitative real-time PCR of relative mRNA expression levels of XBP1S in control (shNC, scrambled shRNA) and Cab45S-knockdown HeLa cell lines treated with TM (2 μ g/ml) for the indicated periods ( n =3). ( b ) Western blots of CHOP, IRE1 and p-IRE1 in control and Cab45S-knockdown HeLa cell lines after treatment with TM (2 μ g/ml) at the indicated time points. ( c ) Quantification of p-IRE1 in b . GAPDH was used as a loading control. ( d ) MTS assay of viable HeLa cells transfected with vectors expressing shNC, shCab45S and shNC, shCab45S and shPERK, and shCab45S and shIRE1 with TM (2 μ g/ml) treatment ( n =3). ( e ) Representative photomicrographs of apoptotic cells in transfected HeLa cells in which Cab45S or both Cab45S and IRE1 were depleted with TM (2 μ g/ml, 48 h) treatment (TUNEL assay). Scale bar, 100 μ m. ( f ) Quantification of TUNEL-positive cells as in e ( n =3; > 100 cells per experiment). ( g ) Western blots of cleaved caspase-3 and p-JNK in control and Cab45S-knockdown HeLa cell lines after treatment with TM (2 μ g/ml) at the indicated time points. ( h ) Western blots of cleaved caspase-3 and p-JNK in control and Cab45S-knockdown HeLa cell lines after 24 h treatment with indicated drugs. TM, 2 μ g/ml; sp600125, 20 μ M. ( i ) Quantitative real-time PCR of relative mRNA expression levels of BAX in control and Cab45S-knockdown HeLa cell lines treated with TM (2 μ g/ml) for the indicated periods ( n =3). For a , c , d , f and i , data are presented as mean±S.E.M. * P
Figure Legend Snippet: The IRE1-JNK signaling is required for activating ER stress-induced apoptosis in Cab45S-depleted cells. ( a ) Quantitative real-time PCR of relative mRNA expression levels of XBP1S in control (shNC, scrambled shRNA) and Cab45S-knockdown HeLa cell lines treated with TM (2 μ g/ml) for the indicated periods ( n =3). ( b ) Western blots of CHOP, IRE1 and p-IRE1 in control and Cab45S-knockdown HeLa cell lines after treatment with TM (2 μ g/ml) at the indicated time points. ( c ) Quantification of p-IRE1 in b . GAPDH was used as a loading control. ( d ) MTS assay of viable HeLa cells transfected with vectors expressing shNC, shCab45S and shNC, shCab45S and shPERK, and shCab45S and shIRE1 with TM (2 μ g/ml) treatment ( n =3). ( e ) Representative photomicrographs of apoptotic cells in transfected HeLa cells in which Cab45S or both Cab45S and IRE1 were depleted with TM (2 μ g/ml, 48 h) treatment (TUNEL assay). Scale bar, 100 μ m. ( f ) Quantification of TUNEL-positive cells as in e ( n =3; > 100 cells per experiment). ( g ) Western blots of cleaved caspase-3 and p-JNK in control and Cab45S-knockdown HeLa cell lines after treatment with TM (2 μ g/ml) at the indicated time points. ( h ) Western blots of cleaved caspase-3 and p-JNK in control and Cab45S-knockdown HeLa cell lines after 24 h treatment with indicated drugs. TM, 2 μ g/ml; sp600125, 20 μ M. ( i ) Quantitative real-time PCR of relative mRNA expression levels of BAX in control and Cab45S-knockdown HeLa cell lines treated with TM (2 μ g/ml) for the indicated periods ( n =3). For a , c , d , f and i , data are presented as mean±S.E.M. * P

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, shRNA, Western Blot, MTS Assay, Transfection, TUNEL Assay

Cab45S inhibits IRE1 activation via GRP78/BiP. ( a and b ) Western blots ( a ) and quantification ( b ) of p-IRE1 in HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( c and d ) Western blots ( c ) and quantification ( d ) of p-IRE1 in HeLa cells expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( e ) Extracts of HEK293T cells overexpressing the indicated vectors treated with TM (1 μ g/ml, 24 h) were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag, anti-EGFP or anti-Cab45S antibody. ( f ) Working model of the mechanism by which Cab45S controls ER stress-induced apoptosis. Cab45S interacts with the NBD of GRP78/BiP, which prevents its disassociation from IRE1 and increases the protein level of GRP78/BiP. These effects lead to inhibition of the IRE1-JNK pathway and ER stress-induced apoptosis. For b and d , data are presented as mean±S.E.M. ** P
Figure Legend Snippet: Cab45S inhibits IRE1 activation via GRP78/BiP. ( a and b ) Western blots ( a ) and quantification ( b ) of p-IRE1 in HeLa cells transfected with vectors expressing 3 × Flag and shNC (scrambled shRNA), 3 × Flag-Cab45S and shNC, 3 × Flag and shGRP78/BiP, or 3 × Flag-Cab45S and shGRP78/BiP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( c and d ) Western blots ( c ) and quantification ( d ) of p-IRE1 in HeLa cells expressing shNC and EGFP, shCab45S and EGFP, shNC and GRP78/BiP-EGFP, or shCab45S and GRP78/BiP-EGFP after TM treatment (2 μ g/ml, 48 h). GAPDH was used as a loading control. ( e ) Extracts of HEK293T cells overexpressing the indicated vectors treated with TM (1 μ g/ml, 24 h) were immunoprecipitated with anti-Flag antibody. The immunoprecipitates were immunoblotted with anti-Flag, anti-EGFP or anti-Cab45S antibody. ( f ) Working model of the mechanism by which Cab45S controls ER stress-induced apoptosis. Cab45S interacts with the NBD of GRP78/BiP, which prevents its disassociation from IRE1 and increases the protein level of GRP78/BiP. These effects lead to inhibition of the IRE1-JNK pathway and ER stress-induced apoptosis. For b and d , data are presented as mean±S.E.M. ** P

Techniques Used: Activation Assay, Western Blot, Transfection, Expressing, shRNA, Immunoprecipitation, Inhibition

23) Product Images from "Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells"

Article Title: Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells

Journal: Molecular & Cellular Proteomics : MCP

doi: 10.1074/mcp.RA118.000735

Validation of altered proteins by immunoblotting assay. SDS-PAGE was performed on treated and untreated lysates, followed by membrane transfer and incubation with anti-OAS3, anti-SAMHD1, anti-STAT1, and anti-GAPDH overnight. The proteins of interest were visualized using the LI-COR Odyssey system. Consistent with proteomic results, the expression of OAS3, SAMHD1 and STAT1 increased after IFN-λ3 treatment. These experiments were performed in triplicate.
Figure Legend Snippet: Validation of altered proteins by immunoblotting assay. SDS-PAGE was performed on treated and untreated lysates, followed by membrane transfer and incubation with anti-OAS3, anti-SAMHD1, anti-STAT1, and anti-GAPDH overnight. The proteins of interest were visualized using the LI-COR Odyssey system. Consistent with proteomic results, the expression of OAS3, SAMHD1 and STAT1 increased after IFN-λ3 treatment. These experiments were performed in triplicate.

Techniques Used: SDS Page, Incubation, Expressing

24) Product Images from "Arginine rich short linear motif of HIV-1 regulatory proteins inhibits Dicer dependent RNA interference"

Article Title: Arginine rich short linear motif of HIV-1 regulatory proteins inhibits Dicer dependent RNA interference

Journal: Retrovirology

doi: 10.1186/1742-4690-10-97

RNAi affects HIV-1 replication. Latently infected T cells (J1.1) were treated with TNF-α (10 ng/ml) and enoxacin (50 μM) for 48 hours. (A) shows p24 levels by western blot on enoxacin and TNF-α treatment. (B) shows use of 10-23 DNAzyme in sequence specific knockdown of RLC components PACT, TRBP, Dicer and Ago2. (C) shows the levels of p24 on Dicer, RHA, PACT, TRBP and Ago2 knockdown (DNAzyme)/Overexpression and both. GAPDH was used as a loading control.
Figure Legend Snippet: RNAi affects HIV-1 replication. Latently infected T cells (J1.1) were treated with TNF-α (10 ng/ml) and enoxacin (50 μM) for 48 hours. (A) shows p24 levels by western blot on enoxacin and TNF-α treatment. (B) shows use of 10-23 DNAzyme in sequence specific knockdown of RLC components PACT, TRBP, Dicer and Ago2. (C) shows the levels of p24 on Dicer, RHA, PACT, TRBP and Ago2 knockdown (DNAzyme)/Overexpression and both. GAPDH was used as a loading control.

Techniques Used: Infection, Western Blot, Sequencing, Over Expression

25) Product Images from "IL-9 promotes the pathogenesis of ulcerative colitis through STAT3/SOCS3 signaling"

Article Title: IL-9 promotes the pathogenesis of ulcerative colitis through STAT3/SOCS3 signaling

Journal: Bioscience Reports

doi: 10.1042/BSR20181521

Overexpression of SOCS3 inhibits IL-9/STAT3-mediated colonic mucosal injury and effect to intestinal barrier related gene expression ( A ) SW480 cells were pretreated with SOCS3 overexpression lentivirus (OE), with or without IL-9 stimulation 24 h later. Expression of occludin, claudin-3, and claudin-2 were extracted for analysis by Western blot (Left). SOCS3, STAT3, and pSTAT3 expression were quantitated (Right). GAPDH was used as a control. Each experiment was performed for three times, and data are shown as mean ± S.E.M. (* P
Figure Legend Snippet: Overexpression of SOCS3 inhibits IL-9/STAT3-mediated colonic mucosal injury and effect to intestinal barrier related gene expression ( A ) SW480 cells were pretreated with SOCS3 overexpression lentivirus (OE), with or without IL-9 stimulation 24 h later. Expression of occludin, claudin-3, and claudin-2 were extracted for analysis by Western blot (Left). SOCS3, STAT3, and pSTAT3 expression were quantitated (Right). GAPDH was used as a control. Each experiment was performed for three times, and data are shown as mean ± S.E.M. (* P

Techniques Used: Over Expression, Expressing, Western Blot

26) Product Images from "Decrease in membrane phospholipids unsaturation correlates with myocardial diastolic dysfunction"

Article Title: Decrease in membrane phospholipids unsaturation correlates with myocardial diastolic dysfunction

Journal: PLoS ONE

doi: 10.1371/journal.pone.0208396

SFA-rich HFD increased fatty acid saturation in cardiac membrane phospholipids. (A) Relative microRNA (miR) expression. (n = 6). (B) Top, Representative immunoblots of phospho-AMPK(Thr172)(P-AMPK), AMPK and GAPDH. Bottom, Quantification of P-AMPK to AMPK ratio by densitometry analysis. (n = 3). (C) Top, representative immunoblots of Sirt1 and GAPDH. Bottom, quantification of Sirt1 by densitometry analysis. (n = 4). (D) Relative Sirt1 deacetylase activity. (n = 6). (E) Relative gene expression of mitochondrial biogenesis, TG turnover, PPARα-target genes in hearts. (n = 6). (F) Triglyceride (TG) content in hearts. (n = 4). (G) Ceramide content in hearts. (n = 4). (H) Diacylglycerol (DAG) content in hearts. (n = 4). (I) FA composition (mol% of phospholipids) of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and the SFA/MUFA ratio. (n = 4). (J) Relative expression of UPR signaling genes. (n = 5). sXBP1 indicates spliced-XBP1. (K) Top, representative immunoblots of SCD1 and GAPDH. Bottom, quantification of SCD1 by densitometry analysis. (n = 4). Data were presented as mean ± SEM. * P
Figure Legend Snippet: SFA-rich HFD increased fatty acid saturation in cardiac membrane phospholipids. (A) Relative microRNA (miR) expression. (n = 6). (B) Top, Representative immunoblots of phospho-AMPK(Thr172)(P-AMPK), AMPK and GAPDH. Bottom, Quantification of P-AMPK to AMPK ratio by densitometry analysis. (n = 3). (C) Top, representative immunoblots of Sirt1 and GAPDH. Bottom, quantification of Sirt1 by densitometry analysis. (n = 4). (D) Relative Sirt1 deacetylase activity. (n = 6). (E) Relative gene expression of mitochondrial biogenesis, TG turnover, PPARα-target genes in hearts. (n = 6). (F) Triglyceride (TG) content in hearts. (n = 4). (G) Ceramide content in hearts. (n = 4). (H) Diacylglycerol (DAG) content in hearts. (n = 4). (I) FA composition (mol% of phospholipids) of saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and the SFA/MUFA ratio. (n = 4). (J) Relative expression of UPR signaling genes. (n = 5). sXBP1 indicates spliced-XBP1. (K) Top, representative immunoblots of SCD1 and GAPDH. Bottom, quantification of SCD1 by densitometry analysis. (n = 4). Data were presented as mean ± SEM. * P

Techniques Used: Expressing, Western Blot, Histone Deacetylase Assay, Activity Assay

27) Product Images from "The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication"

Article Title: The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication

Journal: Nature Communications

doi: 10.1038/s41467-019-08299-7

REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)
Figure Legend Snippet: REV-ERB activation perturbs fatty acid metabolism. a REV-ERB agonist SR9009 perturbs metabolic pathways. Huh-7 cells were treated with SR9009 (20 µM) or vehicle control for 24 h and gene expression analysed by human genome microarray (biological replicates n = 3). Differentially expressed genes were assessed by KEGG pathway enrichment analysis. b Metabolic genes perturbed by SR9009. Relative expression of metabolic genes involved in lipogenesis, cholesterol and bile acid metabolism following SR9009 (20 µM) treatment. c REV-ERB agonist inhibits SCD promoter activity and protein expression. Huh-7 cells were treated with REV-ERB agonist SR9009 (20 µM) for 24 h and SCD promoter activity (mean ± S.E.M., n = 4, Kruskal–Wallis ANOVA with Dunn’s test), transcript levels and protein expression assessed by western blotting and mass spectrometric analysis. d REV-ERBs bind SCD1 promoter. Analyses of available ChIP-seq data in mouse livers 30 demonstrate REV-ERB peaks in SCD1 promoter. Read densities for REV-ERBα tracks are represented by height on the y axis. e Overexpressing REV-ERBα inhibits SCD expression. HCV A2-Luc replicon cells were transfected with an increasing dose of REV-ERBα expression plasmid. Cell lysates were collected 48 h post-transfection and assessed for SCD expression, together with housekeeping GAPDH by western blotting. f Oleic acid partially restores anti-viral activity of REV-ERB agonist SR9009. HCV replicon cells were treated with SR9009 at 15 µM (left) or SCD inhibitor A939572 (SCDi) at 10 µM (right) alone or in combination with oleic acid (OA) at 100 µM. HCV RNA replication was monitored at 30 min intervals for 24 h. g A role for SCD in REV-ERB agonist inhibition of HCV replication. HCV replicon cells were transfected with CRISPRs targeting exons 2 and 3 of SCD or a scrambled guide RNA and 24 h later treated with SR9009 or SCD inhibitor A939572. SCD expression was assessed by western blotting and the dose of REV-ERB agonist or SCD inhibitor required to inhibit HCV RNA replication by 50% (IC 50 ) in control or knock-down (KD) cells determined (mean ± S.E.M., n = 5, Mann–Whitney statistical test)

Techniques Used: Activation Assay, Expressing, Microarray, Activity Assay, Western Blot, Chromatin Immunoprecipitation, Transfection, Plasmid Preparation, Inhibition, MANN-WHITNEY

28) Product Images from "Honokiol inhibits c-Met-HO-1 tumor-promoting pathway and its cross-talk with calcineurin inhibitor-mediated renal cancer growth"

Article Title: Honokiol inhibits c-Met-HO-1 tumor-promoting pathway and its cross-talk with calcineurin inhibitor-mediated renal cancer growth

Journal: Scientific Reports

doi: 10.1038/s41598-017-05455-1

HNK treatment down-regulates HGF-induced c-Met phosphorylation; and it inhibits both HGF- and CNI-mediated Ras activation and HO-1 over-expression. ( A ) 786-O cells were pre-treated with either HNK (20 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the cell lysates were used to measure the levels of phospho-c-Met, c-Met, and β-actin (internal control) by Western blot analysis. ( B ) 786-O cells were treated as described in ( A ). Following treatment, cell lysates were prepared utilizing a Ras activation kit as described under “Materials and Methods” section, and the expression of GTP-bound Ras was subsequently analyzed by Western blot. ( C ). 786-O cells were pre-treated with either CsA (5 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the expression of GTP-bound Ras was analyzed by Western blot. ( D ) 786-O cells were treated with either CsA (5 μM) or vehicle alone for 30 min and cell lysates were utilized to measure the levels of phospho-c-Met, c-Met, and β-actin by Western blot analysis. ( E ) 786-O cells were pre-treated with either HNK (20 μM) or vehicle alone for 2 h and then treated with different combinations of HGF (50 ng/ml), CsA (5 μM) or vehicle alone for 24 h. Following treatment, the cell lysates were used to measure the expression of HO-1 and β-actin. F. 786-0 cells were treated as described in ( E ), and following treatment nuclear and cytoplasmic fractions were isolated from cells, and Western blot analysis was performed to measure the expression of Nrf-2 in each of the fractions. The purities of nuclear and cytoplasmic fractions were evaluated by the expression of SP-1 and GAPDH respectively. ( A to F ) are representative of three independent experiments. For all Western blots ( A – F ), the bands from adjacent lanes were cropped from the same blot (full-length blots are included in a supplementary file).
Figure Legend Snippet: HNK treatment down-regulates HGF-induced c-Met phosphorylation; and it inhibits both HGF- and CNI-mediated Ras activation and HO-1 over-expression. ( A ) 786-O cells were pre-treated with either HNK (20 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the cell lysates were used to measure the levels of phospho-c-Met, c-Met, and β-actin (internal control) by Western blot analysis. ( B ) 786-O cells were treated as described in ( A ). Following treatment, cell lysates were prepared utilizing a Ras activation kit as described under “Materials and Methods” section, and the expression of GTP-bound Ras was subsequently analyzed by Western blot. ( C ). 786-O cells were pre-treated with either CsA (5 μM)/vehicle alone for 2 h, and then treated with either HGF (50 ng/ml) or vehicle alone for 30 min. Following treatment, the expression of GTP-bound Ras was analyzed by Western blot. ( D ) 786-O cells were treated with either CsA (5 μM) or vehicle alone for 30 min and cell lysates were utilized to measure the levels of phospho-c-Met, c-Met, and β-actin by Western blot analysis. ( E ) 786-O cells were pre-treated with either HNK (20 μM) or vehicle alone for 2 h and then treated with different combinations of HGF (50 ng/ml), CsA (5 μM) or vehicle alone for 24 h. Following treatment, the cell lysates were used to measure the expression of HO-1 and β-actin. F. 786-0 cells were treated as described in ( E ), and following treatment nuclear and cytoplasmic fractions were isolated from cells, and Western blot analysis was performed to measure the expression of Nrf-2 in each of the fractions. The purities of nuclear and cytoplasmic fractions were evaluated by the expression of SP-1 and GAPDH respectively. ( A to F ) are representative of three independent experiments. For all Western blots ( A – F ), the bands from adjacent lanes were cropped from the same blot (full-length blots are included in a supplementary file).

Techniques Used: Activation Assay, Over Expression, Western Blot, Expressing, Isolation

29) Product Images from "Sex-specific control of CNS autoimmunity by p38 MAPK signaling in myeloid cells"

Article Title: Sex-specific control of CNS autoimmunity by p38 MAPK signaling in myeloid cells

Journal: Annals of neurology

doi: 10.1002/ana.24020

Macrophage responses to TLR stimulation in p38CKO Lysm mice Female and male WT and p38CKO Lysm mice were immunized using the 2×MOG 35-55 /CFA protocol. On day 6 thioglycolate was injected i.p., and elicited peritoneal macrophages were isolated on day 10 post-immunization. Adherent macrophages were stimulated for 30 min with 100 ng/ml LPS ( A ) or 50 μg/ml heat killed MTB ( B ), lysed, and analyzed by immunoblot for phosphorylation of p38 MAPK (p-p38). GAPDH is shown as a loading control. Panel ( B ) is a composite image of two different parts of the same membrane image, as indicated by the lines, treated identically and shown at the same exposure. Alternatively, adherent macrophages were stimulated for 4 or 24 hrs (as indicated) with LPS ( C and D ) or MTB ( E and F ), supernatants were collected and analyzed by ELISA for the presence of TNFα or IL-6. Data are representative of 3 independent experiments. * ≤ 0.05; ** ≤ 0.01. WT female (n=6), p38CKO Lysm female (n=9), WT male (n=8), p38CKO Lysm male (n=8).
Figure Legend Snippet: Macrophage responses to TLR stimulation in p38CKO Lysm mice Female and male WT and p38CKO Lysm mice were immunized using the 2×MOG 35-55 /CFA protocol. On day 6 thioglycolate was injected i.p., and elicited peritoneal macrophages were isolated on day 10 post-immunization. Adherent macrophages were stimulated for 30 min with 100 ng/ml LPS ( A ) or 50 μg/ml heat killed MTB ( B ), lysed, and analyzed by immunoblot for phosphorylation of p38 MAPK (p-p38). GAPDH is shown as a loading control. Panel ( B ) is a composite image of two different parts of the same membrane image, as indicated by the lines, treated identically and shown at the same exposure. Alternatively, adherent macrophages were stimulated for 4 or 24 hrs (as indicated) with LPS ( C and D ) or MTB ( E and F ), supernatants were collected and analyzed by ELISA for the presence of TNFα or IL-6. Data are representative of 3 independent experiments. * ≤ 0.05; ** ≤ 0.01. WT female (n=6), p38CKO Lysm female (n=9), WT male (n=8), p38CKO Lysm male (n=8).

Techniques Used: Mouse Assay, Injection, Isolation, Enzyme-linked Immunosorbent Assay

30) Product Images from "Tazarotene-Induced Gene 1 Interacts with DNAJC8 and Regulates Glycolysis in Cervical Cancer Cells"

Article Title: Tazarotene-Induced Gene 1 Interacts with DNAJC8 and Regulates Glycolysis in Cervical Cancer Cells

Journal: Molecules and Cells

doi: 10.14348/molcells.2018.2347

TIG1 inhibits DNAJC8 expression and the DNAJC8-mediated increase in glycolysis Lysates were prepared from HtTA cells transfected with 0.5 μg (A) or 0.5–1.5 μg (B) of DNAJC8-flag expression vector along with 0.5–1.5 μg (A) or 0.5 μg (B) of TIG1-myc expression vector. The levels of TIG1, DNAJC8, and Actin were determined by immunoblotting. The nuclear and cytosolic fractions of HtTA cells transfected with TIG1-myc and DNAJC8-flag vectors were prepared, and the levels of TIG1 and DNAJC8 were determined by immunoblotting. GAPDH and Lamin B1 were markers of the cytosolic and nuclear fractions, respectively (C). The media were collected from HtTA cells transfected with TIG1-myc and DNAJC8-flag vectors, and glucose consumption (D) and lactate production (E) were determined using enzyme immunoassays. HtTA cells were transfected with TIG1-myc and DNAJC8-Flag vectors, and the cell numbers were counted at 24 to 96 h (F).
Figure Legend Snippet: TIG1 inhibits DNAJC8 expression and the DNAJC8-mediated increase in glycolysis Lysates were prepared from HtTA cells transfected with 0.5 μg (A) or 0.5–1.5 μg (B) of DNAJC8-flag expression vector along with 0.5–1.5 μg (A) or 0.5 μg (B) of TIG1-myc expression vector. The levels of TIG1, DNAJC8, and Actin were determined by immunoblotting. The nuclear and cytosolic fractions of HtTA cells transfected with TIG1-myc and DNAJC8-flag vectors were prepared, and the levels of TIG1 and DNAJC8 were determined by immunoblotting. GAPDH and Lamin B1 were markers of the cytosolic and nuclear fractions, respectively (C). The media were collected from HtTA cells transfected with TIG1-myc and DNAJC8-flag vectors, and glucose consumption (D) and lactate production (E) were determined using enzyme immunoassays. HtTA cells were transfected with TIG1-myc and DNAJC8-Flag vectors, and the cell numbers were counted at 24 to 96 h (F).

Techniques Used: Expressing, Transfection, Plasmid Preparation, Enzyme Immunoassay

31) Product Images from "DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome"

Article Title: DDX3X acts as a live-or-die checkpoint in stressed cells by regulating NLRP3 inflammasome

Journal: Nature

doi: 10.1038/s41586-019-1551-2

DDX3X interacts with NLRP3 but not ASC and CASP1, and its loss does not lead to a decrease in the levels of core components of the NLRP3 inflammasome. a , Immunoblot analysis of the levels of NLRP3, ASC, CASP1, pro-IL-1β, NEK7, DDX3X and GAPDH proteins in Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs after LPS priming. Representative blots ( n = 3 biologically independent experiments). b , Immunoblot analysis of NLRP3 and DDX3X immunoprecipitation from HEK293T cells transfected with Flag–NLRP3 and DDX3X–mCherry expression constructs. S.E., short exposure; L.E., long exposure. Representative blots ( n = 3 biologically independent experiments). c , Immunoblot analysis of ASC and DDX3X immunoprecipitates from HEK293T cells transfected with ASC and DDX3X–mCherry expression constructs. Representative blots ( n = 3 biologically independent experiments). d , Immunoblot analysis of CASP1 and DDX3X immunoprecipitates from HEK293T cells transfected with CASP1 and DDX3X–mCherry expression constructs. Representative blots ( n = 2 biologically independent experiments). e , Quantification of the percentage of cells that contain ASC specks in Ddx3x fl/fl and Ddx3x fl/fl LysM cre BMDMs. **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). f , Quantification of the percentage of cells that contain ASC specks in wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages (PMs). **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). g , Confocal microscopy imaging of wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages treated with LPS and nigericin to visualize ASC and DDX3X. Scale bar, 10 μm.
Figure Legend Snippet: DDX3X interacts with NLRP3 but not ASC and CASP1, and its loss does not lead to a decrease in the levels of core components of the NLRP3 inflammasome. a , Immunoblot analysis of the levels of NLRP3, ASC, CASP1, pro-IL-1β, NEK7, DDX3X and GAPDH proteins in Ddx3x fl/fl , Ddx3x fl/fl LysM cre and Nlrp3 −/− BMDMs after LPS priming. Representative blots ( n = 3 biologically independent experiments). b , Immunoblot analysis of NLRP3 and DDX3X immunoprecipitation from HEK293T cells transfected with Flag–NLRP3 and DDX3X–mCherry expression constructs. S.E., short exposure; L.E., long exposure. Representative blots ( n = 3 biologically independent experiments). c , Immunoblot analysis of ASC and DDX3X immunoprecipitates from HEK293T cells transfected with ASC and DDX3X–mCherry expression constructs. Representative blots ( n = 3 biologically independent experiments). d , Immunoblot analysis of CASP1 and DDX3X immunoprecipitates from HEK293T cells transfected with CASP1 and DDX3X–mCherry expression constructs. Representative blots ( n = 2 biologically independent experiments). e , Quantification of the percentage of cells that contain ASC specks in Ddx3x fl/fl and Ddx3x fl/fl LysM cre BMDMs. **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). f , Quantification of the percentage of cells that contain ASC specks in wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages (PMs). **** P ≤ 0.0001 (unpaired two-sided t- test). Data are mean ± s.e.m. ( n = 48). g , Confocal microscopy imaging of wild-type and Ddx3x fl/fl LysM cre peritoneal macrophages treated with LPS and nigericin to visualize ASC and DDX3X. Scale bar, 10 μm.

Techniques Used: Immunoprecipitation, Transfection, Expressing, Construct, Confocal Microscopy, Imaging

32) Product Images from "The nonreceptor tyrosine kinase SYK drives caspase-8/NLRP3 inflammasome-mediated autoinflammatory osteomyelitis"

Article Title: The nonreceptor tyrosine kinase SYK drives caspase-8/NLRP3 inflammasome-mediated autoinflammatory osteomyelitis

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA119.010623

SYK is involved in regulating the levels of pro-IL-1β and NF-κB in Pstpip2 cmo mice. A , immunoblot analysis of pro-IL-1β, caspase-8 (Casp-8), caspase-1 (Casp-1), phospho-SYK (p-SYK), total SYK (t-SYK), and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre footpad lysates. B , immunoblot analysis of pro-IL-1β, Casp-8, Casp-1, p-SYK, t-SYK, and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs at the indicated time points after LPS treatment. C , immunoblot analysis of phospho-IκBα (p-IκBα), total IκBα (t-IκBα), phospho-ERK (p-ERK), total ERK (t-ERK), phospho-JNK (p-JNK), total JNK (t-JNK), phospho-p38 (p-p38), total p38 (t-p38), and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre footpad lysates. D , immunoblot analysis of activated (cleaved) Casp-1, Casp-8, and GSDMD in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs treated with LPS + ATP or left untreated with medium. E , immunoblot analysis of the inflammasome components pro-IL-1β, NLRP3, ASC, and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs treated with LPS + ATP or left untreated with medium. Representative blots from three independent experiments are shown.
Figure Legend Snippet: SYK is involved in regulating the levels of pro-IL-1β and NF-κB in Pstpip2 cmo mice. A , immunoblot analysis of pro-IL-1β, caspase-8 (Casp-8), caspase-1 (Casp-1), phospho-SYK (p-SYK), total SYK (t-SYK), and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre footpad lysates. B , immunoblot analysis of pro-IL-1β, Casp-8, Casp-1, p-SYK, t-SYK, and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs at the indicated time points after LPS treatment. C , immunoblot analysis of phospho-IκBα (p-IκBα), total IκBα (t-IκBα), phospho-ERK (p-ERK), total ERK (t-ERK), phospho-JNK (p-JNK), total JNK (t-JNK), phospho-p38 (p-p38), total p38 (t-p38), and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre footpad lysates. D , immunoblot analysis of activated (cleaved) Casp-1, Casp-8, and GSDMD in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs treated with LPS + ATP or left untreated with medium. E , immunoblot analysis of the inflammasome components pro-IL-1β, NLRP3, ASC, and GAPDH in WT, Pstpip2 cmo , and Pstpip2 cmo Syk fl/fl LysM cre BMDMs treated with LPS + ATP or left untreated with medium. Representative blots from three independent experiments are shown.

Techniques Used: Mouse Assay

33) Product Images from "Suppression of STIM1 inhibits the migration and invasion of human prostate cancer cells and is associated with PI3K/Akt signaling inactivation"

Article Title: Suppression of STIM1 inhibits the migration and invasion of human prostate cancer cells and is associated with PI3K/Akt signaling inactivation

Journal: Oncology Reports

doi: 10.3892/or.2017.5961

STIM1 is upregulated in PCa. (A) The expression of STIM1 in BPH tissues and prostate cancer tissues was detected by immunohistochemistry. High expression levels of STIM1 were observed in prostate cancer tissues. The expression of STIM1 was low in BPH tissues. (B and C) The expression of STIM1 in normal prostate cell line RWPE-1 and prostate cancer cell lines LNCap, C4-2, PC-3 and DU-145 were determined by (B) RT-PCR at the mRNA level and (C) western blotting at the protein level. GAPDH was used as an internal control. (D) The expression of STIM1 in samples from the Oncomine database; *P
Figure Legend Snippet: STIM1 is upregulated in PCa. (A) The expression of STIM1 in BPH tissues and prostate cancer tissues was detected by immunohistochemistry. High expression levels of STIM1 were observed in prostate cancer tissues. The expression of STIM1 was low in BPH tissues. (B and C) The expression of STIM1 in normal prostate cell line RWPE-1 and prostate cancer cell lines LNCap, C4-2, PC-3 and DU-145 were determined by (B) RT-PCR at the mRNA level and (C) western blotting at the protein level. GAPDH was used as an internal control. (D) The expression of STIM1 in samples from the Oncomine database; *P

Techniques Used: Expressing, Immunohistochemistry, Reverse Transcription Polymerase Chain Reaction, Western Blot

STIM1 knockdown inactivates PI3K-Akt. (A) Two independent shSTIM1s and shControl were transfected into PC-3 and DU-145 cells for 3 days and whole cell extracts were isolated. The expression of p-Akt, t-Akt and STIM1 was determined by western blotting. (B) shSTIM1 and shControl were transfected into PC-3 and DU-145 cells for 2 days, and then the culture medium was replaced with fresh medium with/without 10 µM LY294002. Protein was harvested after 24 h of treatment and the expression of p-Akt, t-Akt and STIM1 was detected by western blotting. GAPDH was used as a loading control. STIM1, stromal-interacting molecule 1.
Figure Legend Snippet: STIM1 knockdown inactivates PI3K-Akt. (A) Two independent shSTIM1s and shControl were transfected into PC-3 and DU-145 cells for 3 days and whole cell extracts were isolated. The expression of p-Akt, t-Akt and STIM1 was determined by western blotting. (B) shSTIM1 and shControl were transfected into PC-3 and DU-145 cells for 2 days, and then the culture medium was replaced with fresh medium with/without 10 µM LY294002. Protein was harvested after 24 h of treatment and the expression of p-Akt, t-Akt and STIM1 was detected by western blotting. GAPDH was used as a loading control. STIM1, stromal-interacting molecule 1.

Techniques Used: Transfection, Isolation, Expressing, Western Blot

34) Product Images from "Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase"

Article Title: Human melanoma cells resistant to MAPK inhibitors can be effectively targeted by inhibition of the p90 ribosomal S6 kinase

Journal: Oncotarget

doi: 10.18632/oncotarget.16204

RSK inhibition induces a G2/M arrest and apoptotic cell death in resistant melanoma cells ( A ) Semi-quantification of P S102 -YB-1 fluorescence signal intensities following confocal immunofluorescence analysis in vemurafenib resistant melanoma cells. The signals in mitotic cells were normalized to those of interphase cells ( n = 4). ( B ) Confocal immunofluorescence analysis for P S102 -YB-1 (Cy5-labelled, blue) in mitotic vemurafenib resistant cells. Nuclei were stained with YOPRO-1 (green). Scale bars represent 25 μm. ( C , D ) Flow cytometric cell cycle analysis following treatment with signalling pathway inhibitors. Vemurafenib resistant cells were treated with vemurafenib (2 μM) or BI-D1870 (3 μM, 10 μM) for 3 d (C). SKMel28 RR cells were treated with vemurafenib (5 μM), trametinib (50 nM) and BI-D1870 (5 μM) either alone or in combination for 3 d (top panel) or for 7 d (bottom panel). Two independent experiments were performed and representative data shown (mean ± SD, n = 3) (D). ( E ) Western Blot analysis examining cleavage of the effector caspase 3 and its target PARP in double resistant SKMel28 RR after treatment with signalling pathway inhibitors for 7 d. GAPDH was detected as a loading control.
Figure Legend Snippet: RSK inhibition induces a G2/M arrest and apoptotic cell death in resistant melanoma cells ( A ) Semi-quantification of P S102 -YB-1 fluorescence signal intensities following confocal immunofluorescence analysis in vemurafenib resistant melanoma cells. The signals in mitotic cells were normalized to those of interphase cells ( n = 4). ( B ) Confocal immunofluorescence analysis for P S102 -YB-1 (Cy5-labelled, blue) in mitotic vemurafenib resistant cells. Nuclei were stained with YOPRO-1 (green). Scale bars represent 25 μm. ( C , D ) Flow cytometric cell cycle analysis following treatment with signalling pathway inhibitors. Vemurafenib resistant cells were treated with vemurafenib (2 μM) or BI-D1870 (3 μM, 10 μM) for 3 d (C). SKMel28 RR cells were treated with vemurafenib (5 μM), trametinib (50 nM) and BI-D1870 (5 μM) either alone or in combination for 3 d (top panel) or for 7 d (bottom panel). Two independent experiments were performed and representative data shown (mean ± SD, n = 3) (D). ( E ) Western Blot analysis examining cleavage of the effector caspase 3 and its target PARP in double resistant SKMel28 RR after treatment with signalling pathway inhibitors for 7 d. GAPDH was detected as a loading control.

Techniques Used: Inhibition, Fluorescence, Immunofluorescence, Staining, Flow Cytometry, Cell Cycle Assay, Western Blot

35) Product Images from "Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis"

Article Title: Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20160791

IRE1α controls CD-like inflammation. (A) Representative images of IRE1α immunohistochemistry (brown) on ileal sections of 10-wk-old Atg16l1 ΔIEC , Xbp1 ΔIEC , and Atg16l1;Xbp1 ΔIEC mice. n = 3. Hematoxylin is in blue. Bars, 20 µm. (B) Representative confocal images of IRE1α immunofluorescence (green) and Paneth cell–derived lysozyme (red) on ileal sections from the indicated genotypes at 10 wk of age. Note the perinuclear cytoplasmic IRE1α immunoreactivity of Paneth cells from Atg16l1 ΔIEC mice that are lysozyme positive on the luminally oriented apex. n = 3. DAPI is in blue. Bars, 20 µm. (C) Representative confocal images of IRE1α immunofluorescence (green) and the Paneth and goblet cell–type–specific lectin UEA-1 (red) on ileal sections from the indicated genotypes at 10 wk of age. n = 3. DAPI is in blue. Bars, 20 µm. (D) Representative confocal images of IRE1β immunofluorescence (green) of the indicated genotypes at 10 wk of age. Note the specific IRE1β immunoreactivity of crypts (insets, bottom) and villus IECs from Atg16l1;Xbp1 ΔIEC and Xbp1 ΔIEC mice. DAPI is in blue. n = 3. Bars: (top) 50 µm; (bottom) 20 µm. (E) IRE1β immunoprecipitation (IP) and immunoblot (IB) as well as pIRE1β immunoblot in crypt lysates. n = 3. GAPDH was used as the loading control. (F and G) Representative H E images (F) and their histological score (G) of 10-wk-old mice. n = 28/13/13/9. The median is shown. One-way ANOVA with Bonferroni’s correction was used. Bars, 100 µm. (H) Enteritis histology score of the indicated genotypes housed at the mouse norovirus–positive SPF animal facility (ZVTA) of the Medical University of Innsbruck. n = 12/15/13. The median is shown. Unpaired two-tailed Student’s t test was used. (I and J) Representative images of TUNEL-labeled IECs (brown; I) and quantification (J). n = 5/7/6. The mean ± SEM is shown. Mann–Whitney U test was used. Hematoxylin is in blue. Bars, 50 µm. (K and L) Representative confocal images of lysozyme immunofluorescence in crypt IECs (green; K) and quantification (L). n = 4/3/5. The mean ± SEM is shown. A Mann–Whitney U test was used. DAPI is in blue. Bars, 20 µm. *, P
Figure Legend Snippet: IRE1α controls CD-like inflammation. (A) Representative images of IRE1α immunohistochemistry (brown) on ileal sections of 10-wk-old Atg16l1 ΔIEC , Xbp1 ΔIEC , and Atg16l1;Xbp1 ΔIEC mice. n = 3. Hematoxylin is in blue. Bars, 20 µm. (B) Representative confocal images of IRE1α immunofluorescence (green) and Paneth cell–derived lysozyme (red) on ileal sections from the indicated genotypes at 10 wk of age. Note the perinuclear cytoplasmic IRE1α immunoreactivity of Paneth cells from Atg16l1 ΔIEC mice that are lysozyme positive on the luminally oriented apex. n = 3. DAPI is in blue. Bars, 20 µm. (C) Representative confocal images of IRE1α immunofluorescence (green) and the Paneth and goblet cell–type–specific lectin UEA-1 (red) on ileal sections from the indicated genotypes at 10 wk of age. n = 3. DAPI is in blue. Bars, 20 µm. (D) Representative confocal images of IRE1β immunofluorescence (green) of the indicated genotypes at 10 wk of age. Note the specific IRE1β immunoreactivity of crypts (insets, bottom) and villus IECs from Atg16l1;Xbp1 ΔIEC and Xbp1 ΔIEC mice. DAPI is in blue. n = 3. Bars: (top) 50 µm; (bottom) 20 µm. (E) IRE1β immunoprecipitation (IP) and immunoblot (IB) as well as pIRE1β immunoblot in crypt lysates. n = 3. GAPDH was used as the loading control. (F and G) Representative H E images (F) and their histological score (G) of 10-wk-old mice. n = 28/13/13/9. The median is shown. One-way ANOVA with Bonferroni’s correction was used. Bars, 100 µm. (H) Enteritis histology score of the indicated genotypes housed at the mouse norovirus–positive SPF animal facility (ZVTA) of the Medical University of Innsbruck. n = 12/15/13. The median is shown. Unpaired two-tailed Student’s t test was used. (I and J) Representative images of TUNEL-labeled IECs (brown; I) and quantification (J). n = 5/7/6. The mean ± SEM is shown. Mann–Whitney U test was used. Hematoxylin is in blue. Bars, 50 µm. (K and L) Representative confocal images of lysozyme immunofluorescence in crypt IECs (green; K) and quantification (L). n = 4/3/5. The mean ± SEM is shown. A Mann–Whitney U test was used. DAPI is in blue. Bars, 20 µm. *, P

Techniques Used: Immunohistochemistry, Mouse Assay, Immunofluorescence, Derivative Assay, Immunoprecipitation, Two Tailed Test, TUNEL Assay, Labeling, MANN-WHITNEY

36) Product Images from "MiR-146a suppresses hepatocellular carcinoma by downregulating TRAF6"

Article Title: MiR-146a suppresses hepatocellular carcinoma by downregulating TRAF6

Journal: American Journal of Cancer Research

doi:

Over expression TRAF6 rescues growth and invasion inhibitory effect of over-expression miR-146a. (A) A Western blot analysis was used to determine relative levels of TRAF6 protein in HepG2 cells infected with the indicated lentivirus. GAPDH was used as
Figure Legend Snippet: Over expression TRAF6 rescues growth and invasion inhibitory effect of over-expression miR-146a. (A) A Western blot analysis was used to determine relative levels of TRAF6 protein in HepG2 cells infected with the indicated lentivirus. GAPDH was used as

Techniques Used: Over Expression, Western Blot, Infection

37) Product Images from "AML1/ETO proteins control POU4F1/BRN3A expression and function in t(8;21) acute myeloid leukaemia"

Article Title: AML1/ETO proteins control POU4F1/BRN3A expression and function in t(8;21) acute myeloid leukaemia

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-09-3604

AML1/ETO promotes BRN3A/Brn3a expression A ) Immunoblot analysis of AML1/ETO, BRN3A and GAPDH in Patient M2 t(8;21) blasts electroporated on day 0 with siRNAs targeting AML1/ETO (A/E) or control (con) and harvested at indicated timepoints. BRN3A immunoblots show BRN3A(L). An additional independent t(8;21) patient showed similar results. B ) Densitometric analysis of immunoblots as in A , mean ± S.E. of two experiments C ) Real-time PCR analyses of transcripts as indicated relative to 18s in murine progenitor cells transduced with retroviruses encoding GFP (GFP) or AML1/ETO (A/E) or AML1/ETO9a (9a), and sorted for GFP positivity prior to lysis 48 hrs later. Data are pooled from three separate experiments using cells from both day E12.5 (2 expts) and E13.5 (1 expt) foetal liver, each data point represents one of two experimental replicates, relative to the first GFP sample in each experiment. Comparisons with Actin and Gapdh gave similar results (data not shown). D ) In similar experiments to those described in C , GFP, AML1/ETO9a, AML1b and the indicated AML1/ETO9a mutants were expressed in progenitors and Brn3a expression monitored in sorted cells harvested at 48 hrs (graph). Expression of AML1 proteins was confirmed by anti-HA immunoblotting of producer cell lysates (bottom), leftmost lane from GFP alone transfected.
Figure Legend Snippet: AML1/ETO promotes BRN3A/Brn3a expression A ) Immunoblot analysis of AML1/ETO, BRN3A and GAPDH in Patient M2 t(8;21) blasts electroporated on day 0 with siRNAs targeting AML1/ETO (A/E) or control (con) and harvested at indicated timepoints. BRN3A immunoblots show BRN3A(L). An additional independent t(8;21) patient showed similar results. B ) Densitometric analysis of immunoblots as in A , mean ± S.E. of two experiments C ) Real-time PCR analyses of transcripts as indicated relative to 18s in murine progenitor cells transduced with retroviruses encoding GFP (GFP) or AML1/ETO (A/E) or AML1/ETO9a (9a), and sorted for GFP positivity prior to lysis 48 hrs later. Data are pooled from three separate experiments using cells from both day E12.5 (2 expts) and E13.5 (1 expt) foetal liver, each data point represents one of two experimental replicates, relative to the first GFP sample in each experiment. Comparisons with Actin and Gapdh gave similar results (data not shown). D ) In similar experiments to those described in C , GFP, AML1/ETO9a, AML1b and the indicated AML1/ETO9a mutants were expressed in progenitors and Brn3a expression monitored in sorted cells harvested at 48 hrs (graph). Expression of AML1 proteins was confirmed by anti-HA immunoblotting of producer cell lysates (bottom), leftmost lane from GFP alone transfected.

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Transduction, Lysis, Transfection

BRN3A protein is highly expressed in nuclei of t(8;21) cells A ) Real-time PCR analysis of BRN3A relative to 18S expression in patient blasts from bone marrow (BM) and peripheral blood (PB), expression relative to highest expressing sample in a representative experiment is shown. Non-t(8;21) (other) include both marrow and blood samples. B ) Immunoblot analysis of BRN3A and GAPDH in AML patient samples. The lowest band (asterisk) represents a non-specific cross-reactive protein as BRN3A transcripts are absent from the non-t(8;21) samples shown. The 45 kDa long (L) isoform of BRN3A is predominant whilst the 32 kDa short (S) isoform is also present in t(8;21) samples. C ) Indirect immunofluorescence of M2 t(8;21) and M3 t(15;17) patient blasts and Kasumi-1 cells using specific anti-sera or IgG control as indicated, nuclei visualised using DNA counterstain (DAPI). One representative experiment of three is shown. D ) Real-time PCR analysis of BRN3A relative to GAPDH and 18S expression in cell lines, a representative patient sample and cord blood (CB), relative to Kasumi-1. BRN3A(L) expression in ND7 cells (neuronal positive control), haematopoietic cell lines and primary AML samples (as indicated) was determined by immunoblot (inset).
Figure Legend Snippet: BRN3A protein is highly expressed in nuclei of t(8;21) cells A ) Real-time PCR analysis of BRN3A relative to 18S expression in patient blasts from bone marrow (BM) and peripheral blood (PB), expression relative to highest expressing sample in a representative experiment is shown. Non-t(8;21) (other) include both marrow and blood samples. B ) Immunoblot analysis of BRN3A and GAPDH in AML patient samples. The lowest band (asterisk) represents a non-specific cross-reactive protein as BRN3A transcripts are absent from the non-t(8;21) samples shown. The 45 kDa long (L) isoform of BRN3A is predominant whilst the 32 kDa short (S) isoform is also present in t(8;21) samples. C ) Indirect immunofluorescence of M2 t(8;21) and M3 t(15;17) patient blasts and Kasumi-1 cells using specific anti-sera or IgG control as indicated, nuclei visualised using DNA counterstain (DAPI). One representative experiment of three is shown. D ) Real-time PCR analysis of BRN3A relative to GAPDH and 18S expression in cell lines, a representative patient sample and cord blood (CB), relative to Kasumi-1. BRN3A(L) expression in ND7 cells (neuronal positive control), haematopoietic cell lines and primary AML samples (as indicated) was determined by immunoblot (inset).

Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Immunofluorescence, Positive Control

38) Product Images from "Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/mTOR/ERK1/2 signaling pathways"

Article Title: Synergistic effects of snail and quercetin on renal cell carcinoma Caki-2 by altering AKT/mTOR/ERK1/2 signaling pathways

Journal: International Journal of Clinical and Experimental Pathology

doi:

Inhibition of Snail and quercetin treatment alters the Akt/mTOR/ERK signaling pathways. ccRCC Caki-2 cells were treated as described above. The expression of E-cadherin, COX2, HIF-1a, VEGF/VEGFR2, CD147, and the phosphorylated or total Akt, mTOR, ERK1/2 proteins was detected by Western blot. GAPDH was used as a control for sample loading.
Figure Legend Snippet: Inhibition of Snail and quercetin treatment alters the Akt/mTOR/ERK signaling pathways. ccRCC Caki-2 cells were treated as described above. The expression of E-cadherin, COX2, HIF-1a, VEGF/VEGFR2, CD147, and the phosphorylated or total Akt, mTOR, ERK1/2 proteins was detected by Western blot. GAPDH was used as a control for sample loading.

Techniques Used: Inhibition, Expressing, Western Blot

39) Product Images from "Actinidia chinensis planch polysaccharide protects against hypoxia-induced apoptosis of cardiomyocytes in vitro"

Article Title: Actinidia chinensis planch polysaccharide protects against hypoxia-induced apoptosis of cardiomyocytes in vitro

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.8953

ACP inhibited the phosphorylation of ERK1/2 and PI3K/AKT in H9c2 cells. Western blot analysis of (A) p-ERK1/2, (B) p-PIK3 and p-AKT. GAPDH was used as the loading control. (C) Quantitative analysis of western blots. The different ACP pretreatment concentrations of ACP applied were 1.25 and 2.5 mg/ml. Data were presented as the mean ± standard deviation (n=4). *P
Figure Legend Snippet: ACP inhibited the phosphorylation of ERK1/2 and PI3K/AKT in H9c2 cells. Western blot analysis of (A) p-ERK1/2, (B) p-PIK3 and p-AKT. GAPDH was used as the loading control. (C) Quantitative analysis of western blots. The different ACP pretreatment concentrations of ACP applied were 1.25 and 2.5 mg/ml. Data were presented as the mean ± standard deviation (n=4). *P

Techniques Used: Western Blot, Standard Deviation

40) Product Images from "PINK1/Parkin-mediated mitophagy is activated in cisplatin nephrotoxicity to protect against kidney injury"

Article Title: PINK1/Parkin-mediated mitophagy is activated in cisplatin nephrotoxicity to protect against kidney injury

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-1152-2

Mitophagy is inhibited in PINK1-KO mice during cisplatin nephrotoxicity. a , c , e Kidney cortex tissues was collected from PINK1-KO mice and PINK1-WT littermates for immunoblot analysis of Parkin, PINK1, TOM20, TIM23, HSP60, and GAPDH. b Representative images of PCR-based genotyping. Genomic DNA was extracted from ear biopsy and amplified to detect wild-type (WT) and PINK1-KO and PINK1 heterozygote (HZ) allele as indicated. d , f Densitometry of TOM20, TIM23, and HSP60 signals. g , h After cisplatin treatment, renal cortex was collected and extracted mitochondrial (mito) fractions followed by immunoblot analysis of Parkin, COX IV (mitochondrial marker). g Representative blots. h Densitometry of Parkin signals. Mean ± SD. n = 4, * P
Figure Legend Snippet: Mitophagy is inhibited in PINK1-KO mice during cisplatin nephrotoxicity. a , c , e Kidney cortex tissues was collected from PINK1-KO mice and PINK1-WT littermates for immunoblot analysis of Parkin, PINK1, TOM20, TIM23, HSP60, and GAPDH. b Representative images of PCR-based genotyping. Genomic DNA was extracted from ear biopsy and amplified to detect wild-type (WT) and PINK1-KO and PINK1 heterozygote (HZ) allele as indicated. d , f Densitometry of TOM20, TIM23, and HSP60 signals. g , h After cisplatin treatment, renal cortex was collected and extracted mitochondrial (mito) fractions followed by immunoblot analysis of Parkin, COX IV (mitochondrial marker). g Representative blots. h Densitometry of Parkin signals. Mean ± SD. n = 4, * P

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Marker

PINK1/Parkin-mediated mitophagy is induced during cisplatin treatment in BUMPT cells. BUMPT cells were treated with 20 μM cisplatin for 0–24 h. a Representative images of cellular and nuclear morphologies. After treatment, cells were stained with Hoechst33342 to examine by phase-contrast and fluorescence microscopy. Scale bar, 50 μm. b Apoptosis percentage. Apoptosis was evaluated to determine the cells with typical apoptotic morphology. c Whole cell lysate was collected for immunoblot analysis for PINK1, Parkin, TOM20, TIM23, LC3 II/I, and GAPDH (loading control). d Densitometry analysis of proteins signals on immunoblots The proteins signals were divided by GAPDH signal of the same samples to determine the ratios. e Co-localization of autophagosomes with mitochondria upon cisplatin treatment. BUMPT cells transiently transfected with LC3-GFP were subjected to control or cisplatin treatment. Mitochondria in these cells were then labled with MitoTracker Red FM. The cells were examined by confocal microscopy to show the co-localiztion of autophagosomes (green LC3-GFP puncta) and mitochondria (red). Scale bar: 20 μm. Data in b and d are expressed as mean ± SD. n = 3. * P
Figure Legend Snippet: PINK1/Parkin-mediated mitophagy is induced during cisplatin treatment in BUMPT cells. BUMPT cells were treated with 20 μM cisplatin for 0–24 h. a Representative images of cellular and nuclear morphologies. After treatment, cells were stained with Hoechst33342 to examine by phase-contrast and fluorescence microscopy. Scale bar, 50 μm. b Apoptosis percentage. Apoptosis was evaluated to determine the cells with typical apoptotic morphology. c Whole cell lysate was collected for immunoblot analysis for PINK1, Parkin, TOM20, TIM23, LC3 II/I, and GAPDH (loading control). d Densitometry analysis of proteins signals on immunoblots The proteins signals were divided by GAPDH signal of the same samples to determine the ratios. e Co-localization of autophagosomes with mitochondria upon cisplatin treatment. BUMPT cells transiently transfected with LC3-GFP were subjected to control or cisplatin treatment. Mitochondria in these cells were then labled with MitoTracker Red FM. The cells were examined by confocal microscopy to show the co-localiztion of autophagosomes (green LC3-GFP puncta) and mitochondria (red). Scale bar: 20 μm. Data in b and d are expressed as mean ± SD. n = 3. * P

Techniques Used: Staining, Fluorescence, Microscopy, Western Blot, Transfection, Confocal Microscopy

Mitophagy is suppressed in Parkin-KO mice during cisplatin nephrotoxicity. Whole tissue lysate of kidney cortex was collected from Parkin-KO and wild-type (Parkin-WT) littermate mice. a , c , e Immunoblot analysis of Parkin, PINK1, TOM20, TIM23, HSP60, and GAPDH. b Representative images of PCR-based genotyping. Genomic DNA was extracted from ear biopsy and amplified to detect wild-type (WT), Parkin KO, or Parkin heterozygote (HZ) alleles as indicated. d , f Densitometry analysis of TOM20, TIM23, and HSP60 signals on immunoblots. Mean ± SD. n = 4, * P
Figure Legend Snippet: Mitophagy is suppressed in Parkin-KO mice during cisplatin nephrotoxicity. Whole tissue lysate of kidney cortex was collected from Parkin-KO and wild-type (Parkin-WT) littermate mice. a , c , e Immunoblot analysis of Parkin, PINK1, TOM20, TIM23, HSP60, and GAPDH. b Representative images of PCR-based genotyping. Genomic DNA was extracted from ear biopsy and amplified to detect wild-type (WT), Parkin KO, or Parkin heterozygote (HZ) alleles as indicated. d , f Densitometry analysis of TOM20, TIM23, and HSP60 signals on immunoblots. Mean ± SD. n = 4, * P

Techniques Used: Mouse Assay, Polymerase Chain Reaction, Amplification, Western Blot

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other:

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Article Title: mTORC1 induced HK1-dependent glycolysis regulates NLRP3 inflammasome activation
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    Cell Signaling Technology Inc anti gapdh antibody
    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on <t>JNK</t> phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; <t>GAPDH</t> antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p
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    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Journal: Neurochemistry International

    Article Title: Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    doi: 10.1016/j.neuint.2012.04.015

    Figure Lengend Snippet: Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Article Snippet: Phospho-JNK antibody, anti-GAPDH antibody and secondary antibody for Western blots were obtained from Cell Signaling (Danvers, MA) and Western lightning plus substrate to develop immunoblots was from Perkin Elmer (Waltham, MA).

    Techniques: Western Blot

    PEDF ameliorates the expression of VEGF protein in lung tissue in chronic experimental asthma. (A) Total protein from lung tissue was extracted 24 hours after the final OVA challenge and subjected to Western blot analysis of VEGF. GAPDH was utilized as the standard control. (B) The band signal strength of VEGF expressed as a ratio to GAPDH. Data are presented as means±SEM (n=8 per group). * P

    Journal: Allergy, Asthma & Immunology Research

    Article Title: Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression

    doi: 10.4168/aair.2016.8.2.161

    Figure Lengend Snippet: PEDF ameliorates the expression of VEGF protein in lung tissue in chronic experimental asthma. (A) Total protein from lung tissue was extracted 24 hours after the final OVA challenge and subjected to Western blot analysis of VEGF. GAPDH was utilized as the standard control. (B) The band signal strength of VEGF expressed as a ratio to GAPDH. Data are presented as means±SEM (n=8 per group). * P

    Article Snippet: The membrane was blocked for 1 hour with Tris-buffered saline containing 0.05% Tween 20 (TBST) plus 5% skim milk and then incubated with 1:1,000 anti-VEGF (Abcam, Cambridge, UK) or 1:1,000 anti-GAPDH (Cell Signaling Technology Inc., Beverly, MA, USA) overnight at 4℃.

    Techniques: Expressing, Western Blot

    Establishment of NB4MTOE cells. (A) Expression of MT1G in NB4MTOE cells examined by western blotting. A rabbit polyclonal anti-MT antibody was used to detect exogenous MT1G. Equal amounts of soluble proteins were loaded in each lane and immunoblotted for MT and histone H3. The indicated numbers show the relative density, calculated with Image J 1.46 software, obtained as the density of each MT1G band divided by that of the corresponding histone H3 band. (B) The expression of MT1G was examined by real-time PCR (mean±SD; n.s., not significant). NB4MTOE cells and their control cells were cultured with or without 1 µM ATRA for 72 h, and then collected for analysis. Each gene transcript level was adjusted by the corresponding expression of GAPDH , and the relative levels are shown. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed by independent real-time PCR amplifications using different batches of cDNA.

    Journal: PLoS ONE

    Article Title: A PU.1 Suppressive Target Gene, Metallothionein 1G, Inhibits Retinoic Acid-Induced NB4 Cell Differentiation

    doi: 10.1371/journal.pone.0103282

    Figure Lengend Snippet: Establishment of NB4MTOE cells. (A) Expression of MT1G in NB4MTOE cells examined by western blotting. A rabbit polyclonal anti-MT antibody was used to detect exogenous MT1G. Equal amounts of soluble proteins were loaded in each lane and immunoblotted for MT and histone H3. The indicated numbers show the relative density, calculated with Image J 1.46 software, obtained as the density of each MT1G band divided by that of the corresponding histone H3 band. (B) The expression of MT1G was examined by real-time PCR (mean±SD; n.s., not significant). NB4MTOE cells and their control cells were cultured with or without 1 µM ATRA for 72 h, and then collected for analysis. Each gene transcript level was adjusted by the corresponding expression of GAPDH , and the relative levels are shown. The data presented were obtained from three independent PCR amplifications, and the reproducibility was confirmed by independent real-time PCR amplifications using different batches of cDNA.

    Article Snippet: To verify equal loading of proteins in each lane, anti-histone H3 (for nuclear extracts) and anti-GAPDH (for total cellular extracts) rabbit polyclonal antibodies (Cell Signaling Technology) were also employed.

    Techniques: Expressing, Western Blot, Software, Real-time Polymerase Chain Reaction, Cell Culture, Polymerase Chain Reaction

    Caspase-3 mediates C-terminal cleavage of GIRK channels in cultured hippocampal neurons upon prolonged seizure activity. ( a , b ) Surface biotinylation was performed after APV control (ctl) or APV withdrawal (wd) in cultured hippocampal neurons pretreated for 2 h with vehicle control (VC, 0.1% DMSO), pan caspase inhibitor ZVAD-Fmk (I, 100 μM) ( a ), caspase-1 inhibitor YVAD-cmk (I-1, 20 μM), or caspase-3 inhibitor DEVD-fmk (I-3, 20 μM) ( b ). Surface and Total GIRK1 and GIRK2 proteins were examined by immunoblotting with antibodies recognizing intracellular N-termini of GIRK1 and GIRK2. ( c , d ) Quantitative immunoblot analyses of total GIRK2 ( c ) and GIRK1 ( d ) expression after APV control (ctl) or APV withdrawal (wd) for 120 min in cultured hippocampal neurons which were pretreated for 2 h with vehicle control (VC), ZVAD-Fmk (I), YVAD-cmk (I-1), or DEVD-fmk (I-3). GAPDH served as a loading control. Pretreatment with ZVAD-Fmk and DEVD-fmk but not YVAD-cmk blocked C-terminal cleavages of GIRK1 and GIRK2 induced by prolonged APV withdrawal. Data shown represent the mean ± SEM (n = 4 per treatment). * p

    Journal: Scientific Reports

    Article Title: Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels

    doi: 10.1038/s41598-017-12508-y

    Figure Lengend Snippet: Caspase-3 mediates C-terminal cleavage of GIRK channels in cultured hippocampal neurons upon prolonged seizure activity. ( a , b ) Surface biotinylation was performed after APV control (ctl) or APV withdrawal (wd) in cultured hippocampal neurons pretreated for 2 h with vehicle control (VC, 0.1% DMSO), pan caspase inhibitor ZVAD-Fmk (I, 100 μM) ( a ), caspase-1 inhibitor YVAD-cmk (I-1, 20 μM), or caspase-3 inhibitor DEVD-fmk (I-3, 20 μM) ( b ). Surface and Total GIRK1 and GIRK2 proteins were examined by immunoblotting with antibodies recognizing intracellular N-termini of GIRK1 and GIRK2. ( c , d ) Quantitative immunoblot analyses of total GIRK2 ( c ) and GIRK1 ( d ) expression after APV control (ctl) or APV withdrawal (wd) for 120 min in cultured hippocampal neurons which were pretreated for 2 h with vehicle control (VC), ZVAD-Fmk (I), YVAD-cmk (I-1), or DEVD-fmk (I-3). GAPDH served as a loading control. Pretreatment with ZVAD-Fmk and DEVD-fmk but not YVAD-cmk blocked C-terminal cleavages of GIRK1 and GIRK2 induced by prolonged APV withdrawal. Data shown represent the mean ± SEM (n = 4 per treatment). * p

    Article Snippet: IP was performed with rabbit anti-GIRK2 N-terminal antibodies (5 μg, Cell Signaling) and analyzed by immunoblotting with anti-HA, anti-GIRK2 N-term, anti-GAPDH antibodies (1:500–1:1,000) (Cell Signaling).

    Techniques: Cell Culture, Activity Assay, CTL Assay, Expressing

    Kainate-induced status epilepticus in rats induces C-terminal cleavage of GIRK1 and GIRK2 in the hippocampus. ( a ) Schematic workflow of an experiment from kainate (KA)-mediated induction of status epilepticus in rats to collection of their hippocampi. ( b , c ) Immunoblot analysis of total hippocampal lysates from Sprague Dawley rats injected with kainate (9 mg/kg) and vehicle control (H 2 O) with antibodies against GIRK2 N-terminus, c-Jun (marker for seizure induction), and β-tubulin (loading control). Kainate-injected rats developed stage 4–5 seizures and status epilepticus (n = 3) whereas H 2 O-injected rats did not display seizures (n = 3). Representative immunoblots ( b ) and quantification ( c ) showed that status epilepticus induces C-terminal cleavage of GIRK2 in the rat hippocampi at 8 h post injection with 9 mg/kg kainate. ( d , e ) Immunoblot analysis of total hippocampal lysate from CD001 rats injected with kainate (20 and 30 mg/kg) or vehicle control (saline) with antibodies against GIRK1 and GIRK2 N-terminus, c-Fos (marker for seizure induction), and GAPDH (loading control). In one pair of CD001 rats (set #1), the rat injected with 20 mg/kg kainate developed a short duration of stage 2–3 seizures. In 4 pairs of rats (set #2–5), rats injected with 30 mg/kg kainate developed stage 4–5 seizures and status epilepticus. Saline-injected rats did not display seizures. Representative immunoblots ( d ) and quantification ( e ) showed that status epilepticus induced C-terminal cleavage of GIRK1 in the rat hippocampus within 3 h post injection with 30 mg/kg kainate (n = 4). The cropped gray-scale blots are displayed. Full-length blots are included in the Supplementary Fig. S7 . Data shown represent the mean ± SEM. * p

    Journal: Scientific Reports

    Article Title: Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels

    doi: 10.1038/s41598-017-12508-y

    Figure Lengend Snippet: Kainate-induced status epilepticus in rats induces C-terminal cleavage of GIRK1 and GIRK2 in the hippocampus. ( a ) Schematic workflow of an experiment from kainate (KA)-mediated induction of status epilepticus in rats to collection of their hippocampi. ( b , c ) Immunoblot analysis of total hippocampal lysates from Sprague Dawley rats injected with kainate (9 mg/kg) and vehicle control (H 2 O) with antibodies against GIRK2 N-terminus, c-Jun (marker for seizure induction), and β-tubulin (loading control). Kainate-injected rats developed stage 4–5 seizures and status epilepticus (n = 3) whereas H 2 O-injected rats did not display seizures (n = 3). Representative immunoblots ( b ) and quantification ( c ) showed that status epilepticus induces C-terminal cleavage of GIRK2 in the rat hippocampi at 8 h post injection with 9 mg/kg kainate. ( d , e ) Immunoblot analysis of total hippocampal lysate from CD001 rats injected with kainate (20 and 30 mg/kg) or vehicle control (saline) with antibodies against GIRK1 and GIRK2 N-terminus, c-Fos (marker for seizure induction), and GAPDH (loading control). In one pair of CD001 rats (set #1), the rat injected with 20 mg/kg kainate developed a short duration of stage 2–3 seizures. In 4 pairs of rats (set #2–5), rats injected with 30 mg/kg kainate developed stage 4–5 seizures and status epilepticus. Saline-injected rats did not display seizures. Representative immunoblots ( d ) and quantification ( e ) showed that status epilepticus induced C-terminal cleavage of GIRK1 in the rat hippocampus within 3 h post injection with 30 mg/kg kainate (n = 4). The cropped gray-scale blots are displayed. Full-length blots are included in the Supplementary Fig. S7 . Data shown represent the mean ± SEM. * p

    Article Snippet: IP was performed with rabbit anti-GIRK2 N-terminal antibodies (5 μg, Cell Signaling) and analyzed by immunoblotting with anti-HA, anti-GIRK2 N-term, anti-GAPDH antibodies (1:500–1:1,000) (Cell Signaling).

    Techniques: Injection, Marker, Western Blot

    GIRK2A truncated at 349 YEVD 352 motif shows decreased binding to Gβγ and GIRK1. ( a ) Quantitative immunoblot analyses of the HEK293T cells transfected with equal amounts of plasmids containing GIRK1 with extracellular HA tag (HA-GIRK1) and GIRK2A wild type (WT) or GIRK2A-Y353X (n = 7–8 each), GIRK2A WT alone, or GIRK2A-Y353X alone (n = 6–7 each). GAPDH served as a loading control. GIRK2A-Y353X expression was significantly lower than GIRK2A WT expression. Coexpression of GIRK2A-Y353X decreased HA-GIRK1 expression compared to coexpression of GIRK2A WT. ( b ) Immunoprecipitation (IP) with anti-GFP antibodies was performed from untransfected HEK293T cells (none, n = 4), or the cells transfected with YFP-Gβ 1 and YFP-Gγ 2 alone (n = 4) or together with GIRK2A WT (n = 6) or GIRK2A-Y353X (n = 6). To increase GIRK2A-Y353X expression to a similar extent as GIRK2A-WT expression, we doubled the amount of GIRK2A-Y353X plasmid for transfection compared to GIRK2A-WT plasmids. Coimmunoprecipitation of GIRK2-Y353X with Gβ 1 γ 2 was decreased compared to GIRK2A WT. ( c ) IP with anti-GIRK2 N-terminal antibodies was performed from untransfected HEK293T cells (none), or the cells expressing HA-GIRK1 alone or together with GIRK2A WT or GIRK2A-Y353X (n = 3 each). To increase GIRK2A-Y353X expression to a similar extent as GIRK2AWT expression, we doubled the amount of GIRK2A-Y353X plasmid for transfection compared to GIRK2A-WT plasmids. Coimmunoprecipitation of HA-GIRK1 with GIRK2A-Y353X was reduced compared to GIRK2A WT. In ( b , c ), *points at IgG bands. Data shown represent the mean ± SEM (** p

    Journal: Scientific Reports

    Article Title: Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels

    doi: 10.1038/s41598-017-12508-y

    Figure Lengend Snippet: GIRK2A truncated at 349 YEVD 352 motif shows decreased binding to Gβγ and GIRK1. ( a ) Quantitative immunoblot analyses of the HEK293T cells transfected with equal amounts of plasmids containing GIRK1 with extracellular HA tag (HA-GIRK1) and GIRK2A wild type (WT) or GIRK2A-Y353X (n = 7–8 each), GIRK2A WT alone, or GIRK2A-Y353X alone (n = 6–7 each). GAPDH served as a loading control. GIRK2A-Y353X expression was significantly lower than GIRK2A WT expression. Coexpression of GIRK2A-Y353X decreased HA-GIRK1 expression compared to coexpression of GIRK2A WT. ( b ) Immunoprecipitation (IP) with anti-GFP antibodies was performed from untransfected HEK293T cells (none, n = 4), or the cells transfected with YFP-Gβ 1 and YFP-Gγ 2 alone (n = 4) or together with GIRK2A WT (n = 6) or GIRK2A-Y353X (n = 6). To increase GIRK2A-Y353X expression to a similar extent as GIRK2A-WT expression, we doubled the amount of GIRK2A-Y353X plasmid for transfection compared to GIRK2A-WT plasmids. Coimmunoprecipitation of GIRK2-Y353X with Gβ 1 γ 2 was decreased compared to GIRK2A WT. ( c ) IP with anti-GIRK2 N-terminal antibodies was performed from untransfected HEK293T cells (none), or the cells expressing HA-GIRK1 alone or together with GIRK2A WT or GIRK2A-Y353X (n = 3 each). To increase GIRK2A-Y353X expression to a similar extent as GIRK2AWT expression, we doubled the amount of GIRK2A-Y353X plasmid for transfection compared to GIRK2A-WT plasmids. Coimmunoprecipitation of HA-GIRK1 with GIRK2A-Y353X was reduced compared to GIRK2A WT. In ( b , c ), *points at IgG bands. Data shown represent the mean ± SEM (** p

    Article Snippet: IP was performed with rabbit anti-GIRK2 N-terminal antibodies (5 μg, Cell Signaling) and analyzed by immunoblotting with anti-HA, anti-GIRK2 N-term, anti-GAPDH antibodies (1:500–1:1,000) (Cell Signaling).

    Techniques: Binding Assay, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation

    Prolonged seizure activity induces C-terminal cleavage of GIRK channels in cultured hippocampal neurons. ( a ) Whole-cell patch clamp recording of spontaneous action potentials in cultured hippocampal neurons (12–13 DIV, pretreated with 200 μM DL-APV for 2–3 days at 10–11 DIV) before and after APV control (left representative trace) and APV withdrawal (right representative trace). Activation of synaptic NMDAR upon APV withdrawal induced high frequency burst firing of action potentials and sustained depolarization. ( b ) Surface biotinylation of cultured hippocampal neurons after APV control (ctl) or APV withdrawal (wd) was analyzed by immunoblotting with antibodies recognizing intracellular N-termini of GIRK1 and GIRK2. Prolonged APV withdrawal for 30–90 min resulted in the C-terminal cleavage of GIRK1 and GIRK2 proteins that were biotinylated (Surface) and in the lysates (Total). ( c , d ) Quantitative immunoblot analyses of total GIRK2 ( c ) and GIRK1 ( d ) expression in cultured hippocampal neurons after APV control for 90 min (ctl, n = 4–5) or APV withdrawal (wd) for 30–90 min (n = 3–4 each time point). GAPDH served as a loading control. Data shown represent the mean ± SEM. * p

    Journal: Scientific Reports

    Article Title: Prolonged seizure activity causes caspase dependent cleavage and dysfunction of G-protein activated inwardly rectifying potassium channels

    doi: 10.1038/s41598-017-12508-y

    Figure Lengend Snippet: Prolonged seizure activity induces C-terminal cleavage of GIRK channels in cultured hippocampal neurons. ( a ) Whole-cell patch clamp recording of spontaneous action potentials in cultured hippocampal neurons (12–13 DIV, pretreated with 200 μM DL-APV for 2–3 days at 10–11 DIV) before and after APV control (left representative trace) and APV withdrawal (right representative trace). Activation of synaptic NMDAR upon APV withdrawal induced high frequency burst firing of action potentials and sustained depolarization. ( b ) Surface biotinylation of cultured hippocampal neurons after APV control (ctl) or APV withdrawal (wd) was analyzed by immunoblotting with antibodies recognizing intracellular N-termini of GIRK1 and GIRK2. Prolonged APV withdrawal for 30–90 min resulted in the C-terminal cleavage of GIRK1 and GIRK2 proteins that were biotinylated (Surface) and in the lysates (Total). ( c , d ) Quantitative immunoblot analyses of total GIRK2 ( c ) and GIRK1 ( d ) expression in cultured hippocampal neurons after APV control for 90 min (ctl, n = 4–5) or APV withdrawal (wd) for 30–90 min (n = 3–4 each time point). GAPDH served as a loading control. Data shown represent the mean ± SEM. * p

    Article Snippet: IP was performed with rabbit anti-GIRK2 N-terminal antibodies (5 μg, Cell Signaling) and analyzed by immunoblotting with anti-HA, anti-GIRK2 N-term, anti-GAPDH antibodies (1:500–1:1,000) (Cell Signaling).

    Techniques: Activity Assay, Cell Culture, Patch Clamp, Activation Assay, CTL Assay, Expressing