Supplementary Figure 5: PRDM15-mediated regulation of the WNT/β-catenin pathway.
From: PRDM15 safeguards naive pluripotency by transcriptionally regulating WNT and MAPK–ERK signaling
(a) Box plot of β-catenin (left) and CDH1 (right) quantification in Prdm15fl/fl, Prdm15Δ/Δ and Prdm15Δ/Δ ESCs upon rescue with recombinant WNT3a or CHIR. Integrated density was measured for each channel, using ImageJ, and normalized to integrated density for DAPI (n = 6). (b) Immunofluorescence staining for β-catenin (β-CAT) and TUBULIN (TUBA) in Prdm15fl/fl, Prdm15Δ/Δ ESCs. (c) Western blot analysis of total levels of β-CAT, plakoglobin and CDH1 in Prdm15fl/fl and Prdm15Δ/Δ ESCs; uncropped images are in Supplementary Data. (d) Immunofluorescence staining for β-catenin (β-CAT) and plakoglobin (JUP) in Prdm15fl/fl, Prdm15Δ/Δ ESCs. (e) Immunofluorescence staining for TBX3 (green) and NANOG (red) in Prdm15fl/fl and Prdm15Δ/Δ ESCs; note the restoration of TBX3+NANOG+ in Prdm15Δ/Δ cells upon addition of recombinant WNT3a or CHIR. (f) Immunofluorescence staining for β-CAT and PRDM15 in Prdm15fl/fl, Prdm15Δ/Δ and Prdm15Δ/Δ ESCs expressing exogenous Prdm15. (g) qPCR analysis of canonical WNT target activation in Prdm15fl/fl and Prdm15Δ/Δ ESCs, 8 h after stimulation with recombinant WNT3a (10 ng). Data from three experiments are shown; center values, mean; error bars, s.d. Student’s t test (two-sided) was used. Representative images are shown in b–f (n = 10).
