Fig 1: TRIM27 is essential for ZNF165 transcriptional activity and tumor growth in vivo.(A) Cartoon maps showing the domains of ZNF446 and TRIM27. The KRAB-binding domain of TRIM27 is highlighted for reference. (B) Forty-eight hours after transfection with indicated cDNA, HEK293T lysates were subjected to immunoprecipitation with HA antibody. Immunoblotting was performed using indicated antibodies. Data are representative of three independent experiments. (C) Proximity ligation assays (PLAs) performed using V5 and TRIM27 antibodies in SUM159 cells stably expressing ZNF165-V5. Parental SUM159 cells were used as a negative control with the V5/TRIM27 antibody combination. Scale bar, 10 µm. The mean PLA signal (number of foci per nucleus) is quantified (right), where each data point represents the mean signal calculated within one image. P-values were calculated using an unpaired, two-tailed Student’s t-test. Five images were used per condition and data are representative of four independent assays. (D) WHIM12 cells were transfected with siRNA for 48 hr and qPCR was used to quantify relative expression (log2 fold change) of shared ZNF165-SMAD3 target genes upon depletion of ZNF165 (x-axis) or TRIM27 (y-axis). The Pearson correlation coefficient is indicated by r. Data are representative of three independent experiments. (E) SUM159 cells stably expressing indicated cDNA were transfected with siRNA targeting TRIM27 for 48 hr. Relative RRAD expression was measured using qPCR and the data were normalized to the CTRL sample (grey). Error bars represent mean + SEM. P-values were calculated using an unpaired, two-tailed Mann-Whitney test. Significance is indicated by asterisks, where *=p < 0.05. Data are representative of four independent experiments. (F) PLAs performed using antibodies against endogenous TRIM27 and SMAD3 in SUM159 cells, where either antibody alone was used as a negative control. Cells were pre-treated with 20 µM SB-431542 or DMSO for 15 min, followed by stimulation with 5 ng mL-1 TGFß for 30 min. Scale bar, 10 µm. The mean PLA signal (number of foci per nucleus) is quantified (right), where each data point represents the mean signal calculated within one image. P-value was calculated using an unpaired, two-tailed Student’s t-test. Ten images were used per condition and data are representative of three independent assays. (G) As in (F) except antibodies against endogenous TRIM27 and SMAD4 were used. (H) Tumor volumes from mice orthotopically injected with SUM159T-Luciferase cells stably expressing shRNAs against TRIM27 (n = 10) or a non-targeting control (n = 9). P-value was calculated using an unpaired, two-tailed Student’s t-test. (I) Representative images of bioluminescence (BLI) measurements taken for mice from each group. BLI data was quantified (right) and the p-value was calculated using an unpaired, two-tailed Student’s t-test.