Extended Data Fig. 8: Brg1 knockdown in iHA-Etv2 MEFs using shRNA lentiviral particles.
From: ETV2 functions as a pioneer factor to regulate and reprogram the endothelial lineage
(a) Schematic diagram of shRNA lentiviral knockdown of Brg1 in iHA-Etv2 MEFs. Briefly, MEFs were exposed to shRNA particles 72 hrs before reprogramming was started and cells were collected for analysis and sequencing at various time points throughout the reprogramming process (D1, D2 and D7). shRNA particles were added throughout the reprogramming process to ensure BRG1 expression was not increased. (b) Western blot analysis of BRG1 expression in iHA-Etv2 MEFs exposed to normal reprogramming media versus media with shRNA particles against Brg1 [representative blots (b) from 3 independent experiments with similar results]. (c-l) Compared to control, shRNA knockdown of (d) Brg1 (****P < 1×10-4) in the context of (c) Etv2 overexpression (****P < 1×10-4) leads to a significant decrease in the expression of (e) Flk1 (****P < 1×10-4, ***P = 0.0005), (f) Hopx (***P = 0.0004, **P = 0.0052), (g) Otor (***P = 0.0001, ***P = 0.0009), (h) Emcn (****P < 1×10-4), (i) Sox18 (****P < 1×10-4, ***P = 0.0003), (j) Lmo2 (****P < 1×10-4), (k) Mmp9 (****P < 1×10-4, ***P = 0.001) and (l) Lax1 (****P < 1×10-4, ***P = 0.0003) transcripts, which are upregulated at D7 following overexpression of ETV2 in iHA-Etv2 MEFs (n = 3 biological replicates; one-way ANOVA with multiple comparison). Data are presented as mean ± SEM.
