stat3 reporter kit  (BPS Bioscience)

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2 impairs IL6‐induced STAT3 activation at high cell density. A‐F, Immunoblotting analyses were performed using indicated antibodies. Immunoprecipitation with anti‐STAT3 or anti‐YAP1 antibody was performed. STAT3 transcription activity was measured. Data represent the mean and SD from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. A, DU145 and LNCaP cells at high (~80%) or low (~10%) density were treated with 50 ng/ml IL6 for 30 min. B, DU145 and LNCaP cells at high (~80%) density were treated with 5 μM XMU‐MP‐1 for 2 h, and then treated with 50 ng/ml IL6 for 30 min. C, DU145 cells with expression of MST2 shRNAs at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. D, DU145 cells with expression of LATS1/2 shRNAs at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. E, DU145 cells with expression of MST1 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. F, DU145 cells with expression of YAP1 and TAZ at high (~80%) density were treated with 50 ng/ml IL6 for 30 min

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Immunoprecipitation, Activity Assay, Expressing, shRNA

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2 phosphorylates STAT3 at T622. A, B, D‐G, Immunoblotting analyses were performed using indicated antibodies. A, Purified GST‐STAT3 protein was incubated with purified WT His‐MST2 or His‐MST2 K56R proteins for an in vitro kinase assay. A GST pulldown assay was performed. B, Purified WT GST‐STAT3 or indicated mutant protein was incubated with purified WT His‐MST2 for an in vitro kinase assay. A GST pulldown assay was performed. C, Alignment analyses of STAT3 T622 among indicated species were performed. D, DU145 cells with expression of Flag‐STAT3 at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. Immunoblotting analysis was performed in the presence or absence of the blocking peptide. E, DU145 cells with expression of Flag‐STAT3, MST2 shRNA, or SAV1 shRNA at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. F, DU145 cells with expression of WT Flag‐STAT3 or Flag‐STAT3 T622A at high (H, ~80%) or low (L, ~10%) density were harvested and lysed. Immunoprecipitation was performed using an anti‐Flag antibody. G, Purified WT GST‐STAT3 or GST‐STAT3 T622A protein was incubated with purified WT His‐MST2 proteins for an in vitro kinase assay

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Western Blot, Purification, Incubation, In Vitro, Kinase Assay, GST Pulldown Assay, Mutagenesis, Expressing, Immunoprecipitation, Blocking Assay, shRNA

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: MST2‐dependent STAT3 T622 phosphorylation impairs STAT3 dimerization and activation. C‐H, Immunoblotting analyses were performed using indicated antibodies. A, A schematic of the SH2 domain in STAT3 protein sequence. B, Human STAT3 protein structure (PDB code: 6TLC) shows the spatial location of the T622 site. The SH2 domain is boxed and enlarged. T622 site is shown in yellow. C, DU145 cells with expression of HA‐STAT3 and Flag‐STAT3 at high (~80%) or low (~10%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. D, DU145 cells with expression of HA‐STAT3, Flag‐STAT3 or MST2 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. E, DU145 cells with expression of WT HA‐STAT3, WT Flag‐STAT3, or indicated mutants at high (~80%) density were treated with 50 ng/ml IL6 for 30 min. Immunoprecipitation was performed using an anti‐Flag antibody. F, Purified GST‐STAT3, Flag‐STAT3, or indicated mutant protein was incubated with purified His‐MST2 protein for an in vitro kinase assay and then incubated with His‐JAK2 protein for another in vitro kinase assay. Immunoprecipitation was performed using an anti‐Flag antibody. G, Purified Flag‐STAT3 or indicated mutant protein was incubated with purified His‐MST2 protein for an in vitro kinase assay and then incubated with His‐JAK2 protein for another in vitro kinase assay. Size exclusion chromatography assay was performed. H, Endogenous STAT3‐depleted DU145 cells were stably expressed with WT Flag‐STAT3 and Flag‐STAT3 T622A. STAT3 shRNA targets noncoding region. I‐K, Endogenous STAT3‐depleted DU145 cells with expression of WT Flag‐STAT3 or indicated mutant at high (~80%) density were treated with 50 ng/ml IL6 for 30 min (I‐J) or 2 h (K). DNA‐binding affinity (I) and transcription activity (J) of STAT3 were measured. The level of IL6 mRNA was measured by RT‐PCR (K). L, M, DU145 cells with expression of MST2 shRNA at high (~80%) density were treated with 50 ng/ml IL6 for 30 min (L) or 2 h (M). DNA‐binding affinity of STAT3 was measured (L). The level of IL6 mRNA was measured by RT‐PCR (M)

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Activation Assay, Western Blot, Sequencing, Expressing, Immunoprecipitation, shRNA, Purification, Mutagenesis, Incubation, In Vitro, Kinase Assay, Size-exclusion Chromatography, Stable Transfection, Binding Assay, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Hippo pathway monomerizes STAT3 to regulate prostate cancer growth

doi: 10.1111/cas.15463

Figure Lengend Snippet: STAT3 T622 phosphorylation promotes prostate cancer growth and predicts patient outcome. A, B, Endogenous STAT3‐depleted DU145 cells with stable expression of WT Flag‐STAT3 or indicated mutant were subcutaneously injected in mice. The images of two representative xenografts from each group are shown (A). The volume of mice tumor xenografts ( n = 7) was measured at indicated time points after injection (B). Data represent the mean and SD. *** p < 0.001. C, The specificity of the anti‐STAT3 pT622 antibody was measured by immunohistochemistry (IHC) staining using phosphorylated or nonphosphorylated blocking peptide. Scale bar, 80 μm. D, E, IHC staining using indicated antibodies was performed using mouse tumor samples (D, scale bar, 80 μm) or human prostate cancer samples (E, scale bar, 50 μm). F, The IHC staining scores were analyzed by linear regression. G, Kaplan‐Meier plots of the overall survival time of prostate cancer patients ( n = 55) with high or low STAT3 T622 phosphorylation or MST2 expression were generated. P value was calculated using the log‐rank test

Article Snippet: STAT3 transcription activity assay was performed by using a STAT3 reporter kit obtained from BPS Bioscience (#79730), following the manufacturer's instructions.

Techniques: Expressing, Mutagenesis, Injection, Immunohistochemistry, Blocking Assay, Generated