ist5 002  (MedChemExpress)

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CWR22Pc, CWR22Rv1, and LAPC cells were treated with increasing concentrations of IST5-002 with DMSO (vehicle) as a control for 72 hours followed by the determination of AR mRNA levels by qRT-PCR. ( B ) IST5-002 suppresses the protein levels of AR-FL and AR-Vs but not the stability of the AR protein in PC cells. CWR22Rv1, CWR22Pc, and LAPC4 cells were treated for 96 hours with increasing concentrations of IST5-002. The cells were treated with CHX (30 μM) and MG132 (10 μM) for 24 hours, followed by the determination of the levels of the AR protein by WB of WCLs with actin blotting as a loading control. The levels of Stat5 activation were determined by the IP of Stat5 followed by WB for pStat5 and total Stat5. ( C ) Six localized PCs obtained from radical prostatectomies were cultured ex vivo in tumor explant cultures in the presence of vehicle (DMSO) or IST5-002 for 7 days followed by the determination of AR-FL, AR-V7, and AR-V9 mRNA levels by qRT-PCR. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Repeated measures adjustments were included for (C). Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Quantitative RT-PCR, Activation Assay, Cell Culture, Ex Vivo

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CA Stat5 or GFP was transduced to CWR22Pc and LAPC4 cells using lentivirus for 3 days before treatment of the cells with increasing concentrations of IST5-002. AR-FL, AR-V7, and AR-V9 mRNA levels were determined by qRT-PCR with shAR for 72 hours as a comparison. ( B ) Protein levels of AR-FL and AR-Vs were determined by WB with actin as a loading control. Stat5 was immunoprecipitated from the same lysates and blotted with antibodies against pStat5 and total Stat5. ( C ) Stat5 was suppressed by the lentiviral expression of shStat5 in CWR22Pc and LAPC4 cells for 3 days followed by treatment of the cells with increasing concentrations of IST5-002. AR-FL, AR-V7, and AR-V9 mRNA levels were determined by qRT-PCR with shAR for 72 hours as a comparison. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Quantitative RT-PCR, Comparison, Immunoprecipitation, Expressing

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CA Stat5 or GFP was lentivirally expressed in CWR22Pc and LAPC-4 cells for 6 hours followed by treatment with CHX (20 μM). AR-FL, AR-V7, and AR-V9 mRNA levels were determined by qRT-PCR. ( B ) CWR22Rv1 cells were transduced with lentivirus expressing Stat5 shRNA, ShCtrl, ( C ) CAStat5, and GFP for 24, 48, or 72 hours or ( D ) treated with IST5-002 (IST5) at increasing concentrations for 24 or 48 hours, as indicated. The nascent cellular mRNA was pulse-labeled with the ribonucleotide homolog ethylene uridine. Click-iT chemistry was used to biotinylate nascent transcripts, followed by streptavidin capture and quantification of spliced AR mRNA and nascent AR pre-mRNA levels by qRT-PCR. Control groups included shAR versus shCtrl for indicated time points or treatment of the cells with actinomycin D (ActD) (5 μM) for 24 hours. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels were determined in the same samples by qRT-PCR. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Quantitative RT-PCR, Transduction, Expressing, shRNA, Labeling

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) Experimental design for comparing transcriptome-wide effects of AR knockdown with shRNA (shAR-1 and shAR-2) versus Stat5 inhibition with IST5-002 (IST5) in CWR22Rv1 cells using RNA-seq. ( B ) Volcano plots of differentially expressed genes in CWR22Rv1 cells infected with shAR or shCtrl lentivirus (left) or CWR22Rv1 cells infected with shCtrl lentivirus and treated with 0.8 μM IST5 or DMSO (right). Colored dots reflect genes exceeding cutoffs of false discovery rate (FDR) < 0.05 and fold change > 2. The size of each dot reflects the log 2 of counts per million (cpm) measured for that gene. ( C ) Gene expression heatmap. Each row reflects the gene expression z -score for AR, STAT5A, STAT5B, 5 known STAT5 targets, and 17 AR target genes. ( D ) Violin plots comparing z -scores for each of the 17 AR target genes in (C) within indicated treatment groups. Gray lines indicate the median. P values are from the Student’s t test. ( E ) Normalized enrichment scores (NES) for the 50 HALLMARK gene sets derived from gene set enrichment analysis (GSEA) in the CWR22Rv1 RNA-seq data reflecting AR activity (shAR versus shCtrl, top) or STAT5 activity (IST5-002 versus DMSO, bottom). Dots are colored blue or red based on whether they were positively or negatively enriched in shAR versus shCtrl with an FDR < 0.05.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: shRNA, Inhibition, RNA Sequencing Assay, Infection, Expressing, Derivative Assay, Activity Assay

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CWR22Pc cells were treated with 800 nM IST5-002 (IST5) or DMSO up to 8 days, as indicated, and the fraction of live cells were determined by crystal violet staining of the attached surviving cells at each time-point and counted. ( B ) AR-FL, AR-V7, and AR-V9 mRNA levels were determined by qRT-PCR in cells from parallel wells at indicated time points with cells transduced with lentivirus expressing shAR, shStat5, or shCtrl for 72 hours as comparison. ( C ) AR protein levels evaluated by WB of WCLs with actin as a loading control. The levels of Stat5 activation were determined by the IP of Stat5 followed by WB for pStat5a/b and total Stat5. ( D ) CAStat5 or GFP was lentivirally expressed in CWR22Pc and LAPC4 cells for 3 days followed by treatment of the cells with IST5-002 for 8 days at indicated concentrations. The fractions of live attached cells in each treatment group were determined by crystal violet staining and counting. ( E ) Stat5 was suppressed by lentiviral expression of Stat5 shRNA or shCtrl for 3 days before treatment of the cells with increasing concentrations of IST5-002 followed by the determination of the fractions of viable cells in each treatment group. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Staining, Quantitative RT-PCR, Transduction, Expressing, Comparison, Activation Assay, shRNA

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) AR-FL or GFP was lentivirally expressed in CWR22Pc and LAPC4 cells for 3 days before treatment of the cells with increasing concentrations of IST5-002 for 4 or ( B ) 8 days. AR-FL mRNA and protein levels were determined by qRT-PCR and WB, respectively. Activation levels of Stat5 were evaluated by the IP of Stat5 followed by WB for pStat5 and total Stat5. (B) The fractions of live attached cells in each treatment group were determined by crystal violet staining and counting. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Quantitative RT-PCR, Activation Assay, Staining

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CWR22Pc and LAPC4 cells were treated with vehicle (DMSO), ADT (withdrawal of androgens), ENZ (20 μM), or IST5-002 (IST5) (800 and 1500 nM) for 12 days followed by the determination of the fractions of live attached PC cells by crystal violet staining and counting and ( B ) evaluation of AR-FL, AR-V7, and AR-V9 mRNA levels by qRT-PCR. ( C ) CWR22Pc cells were inoculated subcutaneously into flanks of castrated athymic nude mice supplied with sustained-release DHT pellets. Mice were surgically castrated or treated daily with vehicle, ENZ (30 mg/kg), or IST5-002 (50 mg/kg) for 32 days. AR-FL, AR-V7, and AR-V9 mRNA levels were evaluated by qRT-PCR from the endpoint tumors. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Staining, Quantitative RT-PCR

Journal: Science Advances

Article Title: Stat5 induces androgen receptor ( AR ) gene transcription in prostate cancer and offers a druggable pathway to target AR signaling

doi: 10.1126/sciadv.adi2742

Figure Lengend Snippet: ( A ) CWR22Pc and LAPC4 cells were treated with vehicle (DMSO), ADT (withdrawal of androgens), or ENZ (20 μM) for 3 days followed by continuation with DMSO, ADT, ENZ (20 μM), or IST5-002 (800 nM) for 8 days. The fractions of surviving attached cells were determined by crystal violet staining and counting. ( B ) AR-FL, AR-V7, and AR-V9 mRNA levels of cells in parallel wells were evaluated by qRT-PCR. ( C ) Experimental design for the sequential therapy of the xenograft tumors in vivo. A two-phase in vivo experiment using vehicle or ENZ as first-line therapy (phase 1, 13 days) and vehicle, ENZ, or IST5-002 as second-line therapy (phase 2, 18 days). On day 31, mice were euthanized and CWR22Pc xenograft tumors were collected for analyses. ( D ) AR-FL, AR-V7, and AR-V9 mRNA levels were determined in the endpoint tumors by qRT-PCR. Hypothesis tests were performed using ANOVA with post hoc testing using the Student’s t test with Bonferroni multiplicity corrections. Significance levels are indicated for relevant hypotheses as * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: ENZ was dissolved in 0.5% Tween 80 (Sigma-Aldrich)/phosphate-buffered saline (PBS), and IST5-002 in 0.3% hydroxypropyl cellulose (Sigma-Aldrich)/H 2 O.

Techniques: Staining, Quantitative RT-PCR, In Vivo