Fig. 3: PAF1 interacts with YAP1 and regulates YAP1/TEAD4-mediated transcription and SOX9.
From: PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer
A Immunofluorescence images. HPNE, HPDE, MiaPaCa2 (Mia2), and SUIT2 cells were stained with PAF1, YAP1, and DAPI. Scale bar, 50 µm. B Light microscope images: Isolated pancreatic acini from KrasG12D; Pdx-1 Cre (KC) mice were cultured in collage under 3D conditions (acinar explants embedded in collagen). Images were captured on days 1 and 4. Immunofluorescence images: Collagen discs with acinar explants were stained using immunofluorescence for PAF1, YAP1, and DBA (ductal-specific lectin). C–E Immunoprecipitation assay. PAF1 or YAP1 pulldown followed by immunoblotting with YAP1 or PAF1 or TAZ in indicated samples. F, G Quantitative real-time PCR (qRT-PCR) analysis of PAF1, SOX9, YAP1, and TEAD4 in PAF1 KD and scramble control PC cells. qRT-PCR data were normalized with the Actb gene. H Western blot analysis of PAF1, YAP1, and SOX9 expression in PAF1 KD and scramble control MiaPaCa2 cells. β-actin was used as a control. I ChiP-re-ChiP assay was performed using PAF1, YAP1, and control IgG antibodies in HPNE and MiaPaCa2 (Mia2) cells using primers that amplify SOX9 promoter, which contains TEAD binding motif. Immunoprecipitated and purified DNA was amplified using PCR followed by running in agarose gel. J qRT-PCR analysis of PAF1 in PAF1 KD and scramble control KC6141 cells. qRT-PCR data were normalized with the Actb gene. K–M 8xTEAD luciferase, and SOX9 promoter activity luciferase assays in indicated cells. The bar graphs show activities of 8xTEAD synthetic, and SOX9 promoters represented as the percentage of control (scramble). For all graphs, data are mean ± SD, n = 3. For all panels, significance was determined with a student’s t test. *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant, p > 0.05.
