Figure 10. ZEB1 deletion epigenetically represses TGF-β signaling in LLC tumor ECs.

(A) Comparison of indicated gene expression in the in vitro–cultured control (i.e., Ad–β-gal–infected) versus ZEB1-deleted (i.e., Ad-Cre–infected) LLC tumor ECs (n = 3 independent experiments). (B) Immunoblot analysis of control and ZEB1-deleted tumor ECs as described in A. (C) Luciferase reporter analysis of control and ZEB1-deleted LLC-ECs that were transfected with Tgfb1, Tgfb2, and Tgfb3 promoter reporter constructs (n = 3 independent experiments). Right panel: immunoblot analysis of ZEB1 expression in control and ZEB1-deleted LLC-ECs. (D) ChIP-PCR for confirming the loading of ZEB1 on the proximal but not distal promoters of Tgfb1, Tgfb2, and Tgfb3 in LLC-ECs (n = 3 independent experiments). Around 5 × 10⁶ LLC-ECs were used for each ChIP-PCR experiment. (E) ChIP-PCR for analyzing the enrichments of H3K4Ac, H3K14Ac, H3K18Ac, and H3K27me3 on Tgfb1 promoter in control and ZEB1-deleted LLC-ECs (n = 3 independent experiments). (F) IP analysis confirming association of endogenous ZEB1 with CBP or p300 in LLC-ECs (n = 3 independent experiments). (G) Sequential ChIP-PCR for analyzing the co-occupancy of ZEB1 and CBP on the promoters of Tgfb1, Tgfb2, and Tgfb3 (n = 3 independent experiments). (H) Luciferase reporter assays for analyzing Tgfb1 promoter activity in HEK293T cells that were cotransfected with the indicated constructs (n = 3 independent experiments). Right panel: immunoblot analysis confirming ectopic expression of HA-tagged ZEB1, p300, and CBP in HEK293T cells. Asterisks and arrows mark nonspecific bands and specific bands, respectively, with the expected molecular weights. All data are represented as mean ± SD. *P < 0.05; **P < 0.01. Differences were tested using unpaired 2-sided Student’s t test (A and C) and 1-way ANOVA with Tukey’s post hoc test (H).