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  • 90
    ATCC 2004 iums printed
    2004 Iums Printed, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology usp9x shrna
    Usp9x Shrna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology usp9x sirna
    ( A ) Cell viability between the cisplatin-alone and co-treatment groups was not different following <t>USP9X</t> knockdown. NSCLC cell viability was determined using CCK-8. ( B ) USP9X knockdown led to increased inhibition of cell viability after cisplatin administration. CCK-8 was used to determine NSCLC cell viability. ( C ) Western blotting verified the efficiency of USP9X knockdown. * P < 0.05 vs. Control.
    Usp9x Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/usp9x sirna/product/Santa Cruz Biotechnology
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    91
    Addgene inc lentiguide zsgreen1 expressing sgrna targeting gfpt2
    a and b, Sensitivity to GFPT1 ( a ) and <t>GFPT2</t> ( b ) silencing in K and KL cells (n = 6). c, Rescue effect of GlcNAc supplementation on GFPT2 silencing-induced cell death (n=6). d and e, Effects of a Dox-induced GFPT2 sgRNA (GFPT2) on anchorage independent growth of H460 ( d ) and H157 ( e ) (n=4 for H460, n = 3 for H157). NC is a Dox-inducible control sgRNA. f, Effects of Dox-induced GFPT2 KO on invasion capacity of H460 and H157 cells (n=4 for H460-sgNC−/+ Dox, n=8 for H460-sgGFPT2−/+Dox, n=12 for H157 GFPT2-Dox and H157-NC-Dox, n=8 for H157 GFPT2+Dox, n=7 for H157-NC+Dox). g, Abundance of GFPT1 and 2 in parental and GFPT2 KO H460 cells. Actin was used as a loading control. h, Effects of GFPT2 KO (two clones) on anchorage independent growth of H460. i, Effect of Dox-inducible GFPT2 KO H460 cells on cell surface L-PHA lectin binding. j, Abundance of hexosamine metabolites in Dox-inducible GFPT2 KO H460 cells (n=3). sgNC is used as a Dox-inducible sgRNA control. k, Effect of LKB1 on GFPT2 silencing-induced loss of viability (n=5). l, Effect of constitutively active AMPK (CA AMPK) on GFPT2 silencing-induced loss of viability (n=6).In b, d, e, f, h, i, j, and l, statistical significance was assessed using two-tailed Student’s t-test. In c and k , statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test was used. In c , *, p<0.05 comparing to sieGFP without GlcNAc; #, p<0.05 comparing to sieGFP with GlcNAc treatment; $, p<0.05 comparing to siGFPT2 with GlcNAc treatment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Western blot was repeated three times and all other experiments were performed twice.
    Lentiguide Zsgreen1 Expressing Sgrna Targeting Gfpt2, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ACell Inc mo 63198
    a and b, Sensitivity to GFPT1 ( a ) and <t>GFPT2</t> ( b ) silencing in K and KL cells (n = 6). c, Rescue effect of GlcNAc supplementation on GFPT2 silencing-induced cell death (n=6). d and e, Effects of a Dox-induced GFPT2 sgRNA (GFPT2) on anchorage independent growth of H460 ( d ) and H157 ( e ) (n=4 for H460, n = 3 for H157). NC is a Dox-inducible control sgRNA. f, Effects of Dox-induced GFPT2 KO on invasion capacity of H460 and H157 cells (n=4 for H460-sgNC−/+ Dox, n=8 for H460-sgGFPT2−/+Dox, n=12 for H157 GFPT2-Dox and H157-NC-Dox, n=8 for H157 GFPT2+Dox, n=7 for H157-NC+Dox). g, Abundance of GFPT1 and 2 in parental and GFPT2 KO H460 cells. Actin was used as a loading control. h, Effects of GFPT2 KO (two clones) on anchorage independent growth of H460. i, Effect of Dox-inducible GFPT2 KO H460 cells on cell surface L-PHA lectin binding. j, Abundance of hexosamine metabolites in Dox-inducible GFPT2 KO H460 cells (n=3). sgNC is used as a Dox-inducible sgRNA control. k, Effect of LKB1 on GFPT2 silencing-induced loss of viability (n=5). l, Effect of constitutively active AMPK (CA AMPK) on GFPT2 silencing-induced loss of viability (n=6).In b, d, e, f, h, i, j, and l, statistical significance was assessed using two-tailed Student’s t-test. In c and k , statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test was used. In c , *, p<0.05 comparing to sieGFP without GlcNAc; #, p<0.05 comparing to sieGFP with GlcNAc treatment; $, p<0.05 comparing to siGFPT2 with GlcNAc treatment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Western blot was repeated three times and all other experiments were performed twice.
    Mo 63198, supplied by ACell Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) Cell viability between the cisplatin-alone and co-treatment groups was not different following USP9X knockdown. NSCLC cell viability was determined using CCK-8. ( B ) USP9X knockdown led to increased inhibition of cell viability after cisplatin administration. CCK-8 was used to determine NSCLC cell viability. ( C ) Western blotting verified the efficiency of USP9X knockdown. * P < 0.05 vs. Control.

    Journal: Oncotarget

    Article Title: WP1130 attenuates cisplatin resistance by decreasing P53 expression in non–small cell lung carcinomas

    doi: 10.18632/oncotarget.16931

    Figure Lengend Snippet: ( A ) Cell viability between the cisplatin-alone and co-treatment groups was not different following USP9X knockdown. NSCLC cell viability was determined using CCK-8. ( B ) USP9X knockdown led to increased inhibition of cell viability after cisplatin administration. CCK-8 was used to determine NSCLC cell viability. ( C ) Western blotting verified the efficiency of USP9X knockdown. * P < 0.05 vs. Control.

    Article Snippet: NSCLC cells were transfected with USP9X siRNA (100 nM) or p53 siRNA (100 nM; Santa Cruz Biotechnology, Dallas, TX, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

    Techniques: CCK-8 Assay, Inhibition, Western Blot

    ( A ) MG132 reversed the decreased p53 expression in A549 and HCC827 cells co-treated with cisplatin and WP1130. Western blotting was performed to detect p53 expression in NSCLC cells. * P < 0.05 vs. cisplatin, # P < 0.05 vs. cisplatin+WP1130. ( B ) USP9X siRNA–transfected NSCLC cells were exposed to cisplatin alone or in combination with MG132 for 48 h followed by measurement of p53 expression in the cells. USP9X knockdown abolished the inhibitory effect of WP1130 on p53 expression in A549 and HCC827 cells treated with cisplatin plus MG132. ( C , D ) WP1130 and cisplatin were co-administered to NSCLC cell lines pretreated or untreated with tenovin-1. Cell viability was measured by CCK-8. Tenovin-1 increased the cisplatin resistance of A549 and HCC827 cells in the presence of WP1130.

    Journal: Oncotarget

    Article Title: WP1130 attenuates cisplatin resistance by decreasing P53 expression in non–small cell lung carcinomas

    doi: 10.18632/oncotarget.16931

    Figure Lengend Snippet: ( A ) MG132 reversed the decreased p53 expression in A549 and HCC827 cells co-treated with cisplatin and WP1130. Western blotting was performed to detect p53 expression in NSCLC cells. * P < 0.05 vs. cisplatin, # P < 0.05 vs. cisplatin+WP1130. ( B ) USP9X siRNA–transfected NSCLC cells were exposed to cisplatin alone or in combination with MG132 for 48 h followed by measurement of p53 expression in the cells. USP9X knockdown abolished the inhibitory effect of WP1130 on p53 expression in A549 and HCC827 cells treated with cisplatin plus MG132. ( C , D ) WP1130 and cisplatin were co-administered to NSCLC cell lines pretreated or untreated with tenovin-1. Cell viability was measured by CCK-8. Tenovin-1 increased the cisplatin resistance of A549 and HCC827 cells in the presence of WP1130.

    Article Snippet: NSCLC cells were transfected with USP9X siRNA (100 nM) or p53 siRNA (100 nM; Santa Cruz Biotechnology, Dallas, TX, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol.

    Techniques: Expressing, Western Blot, Transfection, CCK-8 Assay

    a and b, Sensitivity to GFPT1 ( a ) and GFPT2 ( b ) silencing in K and KL cells (n = 6). c, Rescue effect of GlcNAc supplementation on GFPT2 silencing-induced cell death (n=6). d and e, Effects of a Dox-induced GFPT2 sgRNA (GFPT2) on anchorage independent growth of H460 ( d ) and H157 ( e ) (n=4 for H460, n = 3 for H157). NC is a Dox-inducible control sgRNA. f, Effects of Dox-induced GFPT2 KO on invasion capacity of H460 and H157 cells (n=4 for H460-sgNC−/+ Dox, n=8 for H460-sgGFPT2−/+Dox, n=12 for H157 GFPT2-Dox and H157-NC-Dox, n=8 for H157 GFPT2+Dox, n=7 for H157-NC+Dox). g, Abundance of GFPT1 and 2 in parental and GFPT2 KO H460 cells. Actin was used as a loading control. h, Effects of GFPT2 KO (two clones) on anchorage independent growth of H460. i, Effect of Dox-inducible GFPT2 KO H460 cells on cell surface L-PHA lectin binding. j, Abundance of hexosamine metabolites in Dox-inducible GFPT2 KO H460 cells (n=3). sgNC is used as a Dox-inducible sgRNA control. k, Effect of LKB1 on GFPT2 silencing-induced loss of viability (n=5). l, Effect of constitutively active AMPK (CA AMPK) on GFPT2 silencing-induced loss of viability (n=6).In b, d, e, f, h, i, j, and l, statistical significance was assessed using two-tailed Student’s t-test. In c and k , statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test was used. In c , *, p<0.05 comparing to sieGFP without GlcNAc; #, p<0.05 comparing to sieGFP with GlcNAc treatment; $, p<0.05 comparing to siGFPT2 with GlcNAc treatment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Western blot was repeated three times and all other experiments were performed twice.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: a and b, Sensitivity to GFPT1 ( a ) and GFPT2 ( b ) silencing in K and KL cells (n = 6). c, Rescue effect of GlcNAc supplementation on GFPT2 silencing-induced cell death (n=6). d and e, Effects of a Dox-induced GFPT2 sgRNA (GFPT2) on anchorage independent growth of H460 ( d ) and H157 ( e ) (n=4 for H460, n = 3 for H157). NC is a Dox-inducible control sgRNA. f, Effects of Dox-induced GFPT2 KO on invasion capacity of H460 and H157 cells (n=4 for H460-sgNC−/+ Dox, n=8 for H460-sgGFPT2−/+Dox, n=12 for H157 GFPT2-Dox and H157-NC-Dox, n=8 for H157 GFPT2+Dox, n=7 for H157-NC+Dox). g, Abundance of GFPT1 and 2 in parental and GFPT2 KO H460 cells. Actin was used as a loading control. h, Effects of GFPT2 KO (two clones) on anchorage independent growth of H460. i, Effect of Dox-inducible GFPT2 KO H460 cells on cell surface L-PHA lectin binding. j, Abundance of hexosamine metabolites in Dox-inducible GFPT2 KO H460 cells (n=3). sgNC is used as a Dox-inducible sgRNA control. k, Effect of LKB1 on GFPT2 silencing-induced loss of viability (n=5). l, Effect of constitutively active AMPK (CA AMPK) on GFPT2 silencing-induced loss of viability (n=6).In b, d, e, f, h, i, j, and l, statistical significance was assessed using two-tailed Student’s t-test. In c and k , statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test was used. In c , *, p<0.05 comparing to sieGFP without GlcNAc; #, p<0.05 comparing to sieGFP with GlcNAc treatment; $, p<0.05 comparing to siGFPT2 with GlcNAc treatment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Western blot was repeated three times and all other experiments were performed twice.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Clone Assay, Binding Assay, Two Tailed Test, Western Blot

    a, Abundance of GFPT1 protein in cell lines transfected with a control esiRNA or esiRNA directed against GFPT1. Actin is used as a loading control. b, Abundance of GFPT2 protein in cell lines transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. c, Kaplan−Meier plot associating GFPT1 mRNA expression with survival. Dataset is from KM Plotter ( http://kmplot.com/analysis/index.php?p=service&cancer=lung ). d, Kaplan−Meier plot associating GFPT2 mRNA expression with survival. e, Abundance of GFPT1 in a panel of K and KL cells. Actin is used as a loading control. f, Abundance of GFPT2 in a panel of K and KL cells. Actin is used as a loading control. g, Effect of Dox-induced GFPT2 deletion on growth in a monolayer culture. Data are the average and SD of 6 replicates. h, Abundance of GFPT2 in Dox-inducible GFPT2 KO H157 (left) and H460 (right) cells with or without Dox induction. Actin is used as a loading control. i, Representative images of colonies grown in soft agar in . j, Effects of a GFPT2 KO on cell proliferation (H460 cells, n = 5). k, Effect of GlcNAc on anchorage-independent growth of GFPT2 KO cells. l, Global O-GlcNAcylation of GFPT2 WT, KO and KO treated with GlcNAc. m, Abundance of GlcNAc-6-P and ManNAc in Dox-inducible GFPT2 KO H157 cells (n=3). n, Effect of Dox-inducible GFPT2 KO H157 cells on cell surface L-PHA lectin binding. Statistical significance in g, j, m, and n was assessed using two-tailed Student’s t-tests. In j, to calculate significance on repeated measurements over time, a two-way ANOVA with Tukey’s post hoc test was used. In k, statistical significance was assessed using one-way ANOVA with Tukey post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Targeted metabolomics was performed once. Soft agar assay, monolayer cell growth, GFPT1 and 2, and O-GlcNAcylation western blotting were assayed twice. All other experiments were repeated three times or more.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: a, Abundance of GFPT1 protein in cell lines transfected with a control esiRNA or esiRNA directed against GFPT1. Actin is used as a loading control. b, Abundance of GFPT2 protein in cell lines transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. c, Kaplan−Meier plot associating GFPT1 mRNA expression with survival. Dataset is from KM Plotter ( http://kmplot.com/analysis/index.php?p=service&cancer=lung ). d, Kaplan−Meier plot associating GFPT2 mRNA expression with survival. e, Abundance of GFPT1 in a panel of K and KL cells. Actin is used as a loading control. f, Abundance of GFPT2 in a panel of K and KL cells. Actin is used as a loading control. g, Effect of Dox-induced GFPT2 deletion on growth in a monolayer culture. Data are the average and SD of 6 replicates. h, Abundance of GFPT2 in Dox-inducible GFPT2 KO H157 (left) and H460 (right) cells with or without Dox induction. Actin is used as a loading control. i, Representative images of colonies grown in soft agar in . j, Effects of a GFPT2 KO on cell proliferation (H460 cells, n = 5). k, Effect of GlcNAc on anchorage-independent growth of GFPT2 KO cells. l, Global O-GlcNAcylation of GFPT2 WT, KO and KO treated with GlcNAc. m, Abundance of GlcNAc-6-P and ManNAc in Dox-inducible GFPT2 KO H157 cells (n=3). n, Effect of Dox-inducible GFPT2 KO H157 cells on cell surface L-PHA lectin binding. Statistical significance in g, j, m, and n was assessed using two-tailed Student’s t-tests. In j, to calculate significance on repeated measurements over time, a two-way ANOVA with Tukey’s post hoc test was used. In k, statistical significance was assessed using one-way ANOVA with Tukey post hoc test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Targeted metabolomics was performed once. Soft agar assay, monolayer cell growth, GFPT1 and 2, and O-GlcNAcylation western blotting were assayed twice. All other experiments were repeated three times or more.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Transfection, esiRNA, Expressing, Binding Assay, Two Tailed Test, Soft Agar Assay, Western Blot

    a and b, Abundance of GlcNAc-6-P, ManNAc and GlcNAc-6-P in Dox-inducible NC KO H460 ( a ) and H157 ( b ) cells (n=3). c, Abundance of GFPT2 protein in EV and LKB1-expressing H460 transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. d, Left , Relative viability of EV and LKB1-expressing H2122 cells after GFPT2 silencing for 96hr. Right , Abundance of GFPT2 protein in EV and LKB1-expressing H2122 cells transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. e, Relative viability of EV and LKB1-expressing H460 ( Left ) and H2122 ( Right ) cells after GFPT1 silencing for 96hr. f, Abundance of GFPT1 protein in EV and LKB1-expressing H2122 cells transfected with a control esiRNA or esiRNA directed against GFPT1. Actin is used as a loading control. g, Abundance of GFPT2 protein in EV and constitutively active AMPK (CA AMPK)-expressing H460 cells transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: a and b, Abundance of GlcNAc-6-P, ManNAc and GlcNAc-6-P in Dox-inducible NC KO H460 ( a ) and H157 ( b ) cells (n=3). c, Abundance of GFPT2 protein in EV and LKB1-expressing H460 transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. d, Left , Relative viability of EV and LKB1-expressing H2122 cells after GFPT2 silencing for 96hr. Right , Abundance of GFPT2 protein in EV and LKB1-expressing H2122 cells transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control. e, Relative viability of EV and LKB1-expressing H460 ( Left ) and H2122 ( Right ) cells after GFPT1 silencing for 96hr. f, Abundance of GFPT1 protein in EV and LKB1-expressing H2122 cells transfected with a control esiRNA or esiRNA directed against GFPT1. Actin is used as a loading control. g, Abundance of GFPT2 protein in EV and constitutively active AMPK (CA AMPK)-expressing H460 cells transfected with a control esiRNA or esiRNA directed against GFPT2. Actin is used as a loading control.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Expressing, Transfection, esiRNA

    a , Left , Growth of A549 (left) and H460 (right) xenografts in presence and absence of azaserine (2.5mg/kg, qod for 6–7 times total, arrows indicate when azaserine was injected). Right , growth of Calu-1 (left) and Calu-6 (right) xenograft in presence and absence of azaserine. Mean tumor volume and SD are shown for each group (n=4 for A549, n=4 for H460, n=5 for both Calu-1 and Calu-6. Combined results from two independent H460 xenograft experiments (total n=8) are shown here). b, 15 N labeling in GlcNAc-6-P and UDP-HexNAc in mice bearing A549 treated (purple) or non-treated (turquoise) with azaserine. c and d, Abundance of hexosamine metabolites in A549 ( c ) and H460 ( d ) xenografts in Fig. 6a . AUC/TIC=Area under the curve/total ion count. Individual data points are shown with mean values and SD for 12 sections (three fragments per tumor). e, Growth of Dox-inducible GFPT2 KO H460 (left) and H157 (right) xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 per group). f, Full scan lung tissue images of non-treated KL mice (upper panel, total 7 mice) and azaserine treated mice (lower panel, total 6 mice). Scale bar, 4mm. g, Tumor area from Fig. 6f was quantified with ImageJ and % of tumor burden out of total lung was analyzed. h, Representative Ki67 staining images of the same mouse tissues used in Fig. 6f . Scale bar, 100μm. i, Ki67 positive cells and total cells within the area were quantified using ClickMaster2000. Three images per tissue were used for quantification. Statistical significance was assessed using two-tailed Student’s t-tests ( b, c, d, g and i ); two-way ANOVA with Sidak’s multiple comparisons test ( a,e );. *p<0.05; **p<0.01; ****p<0.0001. Tumor growth studies in f and h , in vivo infusion in b , and targeted metabolomics in c and d were performed once. Tumor growth study in a and d were repeated twice.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: a , Left , Growth of A549 (left) and H460 (right) xenografts in presence and absence of azaserine (2.5mg/kg, qod for 6–7 times total, arrows indicate when azaserine was injected). Right , growth of Calu-1 (left) and Calu-6 (right) xenograft in presence and absence of azaserine. Mean tumor volume and SD are shown for each group (n=4 for A549, n=4 for H460, n=5 for both Calu-1 and Calu-6. Combined results from two independent H460 xenograft experiments (total n=8) are shown here). b, 15 N labeling in GlcNAc-6-P and UDP-HexNAc in mice bearing A549 treated (purple) or non-treated (turquoise) with azaserine. c and d, Abundance of hexosamine metabolites in A549 ( c ) and H460 ( d ) xenografts in Fig. 6a . AUC/TIC=Area under the curve/total ion count. Individual data points are shown with mean values and SD for 12 sections (three fragments per tumor). e, Growth of Dox-inducible GFPT2 KO H460 (left) and H157 (right) xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 per group). f, Full scan lung tissue images of non-treated KL mice (upper panel, total 7 mice) and azaserine treated mice (lower panel, total 6 mice). Scale bar, 4mm. g, Tumor area from Fig. 6f was quantified with ImageJ and % of tumor burden out of total lung was analyzed. h, Representative Ki67 staining images of the same mouse tissues used in Fig. 6f . Scale bar, 100μm. i, Ki67 positive cells and total cells within the area were quantified using ClickMaster2000. Three images per tissue were used for quantification. Statistical significance was assessed using two-tailed Student’s t-tests ( b, c, d, g and i ); two-way ANOVA with Sidak’s multiple comparisons test ( a,e );. *p<0.05; **p<0.01; ****p<0.0001. Tumor growth studies in f and h , in vivo infusion in b , and targeted metabolomics in c and d were performed once. Tumor growth study in a and d were repeated twice.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Injection, Labeling, Staining, Two Tailed Test, In Vivo

    a , Global O-GlcNAcylation in A549 (left) and H460 (right) xenografts in presence and absence of azaserine. Actin was used as a loading control. b, Global O-GlcNAcylation in Calu-1 (left) and Calu-6 (right) xenografts in presence and absence of azaserine. Actin was used as a loading control. c and d, Abundance of GFPT2 protein in Dox-inducible GFPT2 KO H460 ( c ) and H157 ( d ) xenografts with or without Dox induction. Actin was used as a loading control. e and f, Growth of Dox-inducible NC KO H460 ( e ) and H157 ( f ) xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 per group). g, Growth of Dox-inducible GFPT2 KO Calu-1 xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 for GFPT2-Dox, n=4 for GFPT2+Dox). h and i, Abundance of GFPT2 protein in Dox-inducible NC KO H460 ( h ) and H157 ( i ) xenografts with or without Dox induction. Actin was used as a loading control. j, Abundance of GFPT2 protein in Dox-inducible GFPT2 KO Calu-1 xenografts with or without Dox induction. Actin was used as a loading control. k, Growth of H460 WT or GFPT2 KO (two different clones) xenografts. Mean tumor volume and SEM are shown for each group (n=5 per group). l, Abundance of GFPT2 protein in GFPT2 WT and KO (two independent clones) H460 xenografts with or without Dox induction. Note that only three mice bearing KO #2 cells developed tumors. Actin was used as a loading control. Statistical significance in f and g was assessed using two-way ANOVA followed by Sidak’s multiple comparisons test. In e and k , statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparisons test. Mouse experiments were performed once. Western blots were repeated twice.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: a , Global O-GlcNAcylation in A549 (left) and H460 (right) xenografts in presence and absence of azaserine. Actin was used as a loading control. b, Global O-GlcNAcylation in Calu-1 (left) and Calu-6 (right) xenografts in presence and absence of azaserine. Actin was used as a loading control. c and d, Abundance of GFPT2 protein in Dox-inducible GFPT2 KO H460 ( c ) and H157 ( d ) xenografts with or without Dox induction. Actin was used as a loading control. e and f, Growth of Dox-inducible NC KO H460 ( e ) and H157 ( f ) xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 per group). g, Growth of Dox-inducible GFPT2 KO Calu-1 xenografts in presence and absence of Dox. Mean tumor volume and SEM are shown for each group (n=5 for GFPT2-Dox, n=4 for GFPT2+Dox). h and i, Abundance of GFPT2 protein in Dox-inducible NC KO H460 ( h ) and H157 ( i ) xenografts with or without Dox induction. Actin was used as a loading control. j, Abundance of GFPT2 protein in Dox-inducible GFPT2 KO Calu-1 xenografts with or without Dox induction. Actin was used as a loading control. k, Growth of H460 WT or GFPT2 KO (two different clones) xenografts. Mean tumor volume and SEM are shown for each group (n=5 per group). l, Abundance of GFPT2 protein in GFPT2 WT and KO (two independent clones) H460 xenografts with or without Dox induction. Note that only three mice bearing KO #2 cells developed tumors. Actin was used as a loading control. Statistical significance in f and g was assessed using two-way ANOVA followed by Sidak’s multiple comparisons test. In e and k , statistical significance was assessed using two-way ANOVA with Tukey’s multiple comparisons test. Mouse experiments were performed once. Western blots were repeated twice.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Clone Assay, Western Blot

    Metabolic alterations mediated by concurrent mutations of KRAS and LKB1 render cells dependent on GFPT2 for hexosamine synthesis. Uptake of glucose and glutamine is elevated by mutant KRAS, establishing the environment favoring hexosamine synthesis. LKB1 loss in the context of KRAS mutation, which leads to loss of AMPK activation, then enhances HBP through GFPT2. Increased GFPT2 activity may also contribute to the glutamate pool for anaplerosis in the mitochondria. α KG, α -ketoglutaric acid.

    Journal: Nature metabolism

    Article Title: The hexosamine biosynthesis pathway is a targetable liability in KRAS/LKB1-mutant lung cancer

    doi: 10.1038/s42255-020-00316-0

    Figure Lengend Snippet: Metabolic alterations mediated by concurrent mutations of KRAS and LKB1 render cells dependent on GFPT2 for hexosamine synthesis. Uptake of glucose and glutamine is elevated by mutant KRAS, establishing the environment favoring hexosamine synthesis. LKB1 loss in the context of KRAS mutation, which leads to loss of AMPK activation, then enhances HBP through GFPT2. Increased GFPT2 activity may also contribute to the glutamate pool for anaplerosis in the mitochondria. α KG, α -ketoglutaric acid.

    Article Snippet: Stable integrants were then further infected by modified lentiGuide-puro plasmid (Addgene plasmid # 52963) whose original puro selection marker is replaced with ZSGreen1 which is amplified from pLVX-EF1a-IRES-zsGreen1 (Clontech: Catalog No. 631982). lentiGuide-ZSGreen1 expressing sgRNA targeting GFPT2 and scrambled sgRNA (sgNC) used in the CRISPR screening , and stable integrants were obtained by flow cytometry (FACS Aria II SORP).

    Techniques: Mutagenesis, Activation Assay, Activity Assay