Fig 1: EHMT2 restrains Tcf4-mediated gene transcription by repressing chromatin bound ß-catenin through RUVBL2.(A) Western blot demonstrating expression of HIF1-a and ß-catenin in A549 lysates immunoprecipitated with RUVBL2 antibody. Cells were cultured under hypoxic conditions (1% O2) vs control (ambient O2). (B) Western blot demonstrating co-immunoprecipitation of RUVBL2 in A549 lysates co-immunoprecipitated with ß-catenin antibody. Cells were treated with EHMT2 inhibitor vs control. (C) Proximity ligation assay in EHMT2i-treated vs vehicle-treated tumor-propagating cells (TPCs) (red, RUVBL2-ß-catenin proximity ligation; blue, DAPI counterstain). Insets show a magnification of the red signal in nuclei of vehicle-treated TPCs (scale 10 µm). (D) Quantification of normalized nuclei with a positive signal (n=2; mean ± SEM, two-tailed paired t-test, **p<0.05). (E) Cytoplasmic (cyt), nuclear (nuc) and chromatin (chro) subcellular fractionation of EHMT2i-treated (as indicated) tumorspheres compared to control. Histone H4 and Tubulin are loading controls of chromatin and cytoplasmic fractions, respectively. (F) Chromatin immunoprecipitation using RUVBL2 antibody followed by qPCR (ChIP-qPCR) of areas flanking a Tcf4 binding sites in promoters of the alveolar type 2 (AT2) genes Etv5, Slc34a2 and Lamp3 (n=2 mean ± SEM; Etv5, Slc34a2, two-tailed paired t-test, *p<0.05). (G) Representative qPCR of Tcf4 binding motif within promoters of AT2 genes. Figure 5—source data 1.Western blot for Figure 5E showing G9a (EHMT2) expression in subcellular fractionation of EHMT2i-treated tumorspheres compared to control. Figure 5—source data 2.Western blot for Figure 5E showing TUBULIN expression in middle membrane (upper band) in subcellular fractionation of EHMT2i-treated tumorspheres compared to control. Figure 5—source data 3.Western blot for Figure 5E showing histone H4 expression in the bottom membrane and beta catenin expression (upper membrane) in subcellular fractionation of EHMT2i-treated tumorspheres compared to control.