Cells were synchronized by culture for 48h under serum starvation, then cultured under serum stimulation and assessed at 72h.
(A) Representative immunoblots of and quantification of ALDH1A3 protein normalized to GAPDH in donor PASMC with nontargeting (Con) and BMPR2 siRNA.
(B) Expression of ALDH1A3 and NFYA relative to β-actin in PAH PASMC with nontargeting (Con) and β-catenin siRNA detected by qPCR.
(C) β-catenin and IgG ChIP-qPCR of ALDH1A3 and NFYA relative to input in PAH PASMC.
(D) mRNA expression, by qPCR, of NFYA relative to β-actin in PAH PASMC treated with Con or ALDH1A3 siRNA.
(E) Protein expression by immunoblots of NFYA relative to GAPDH in three PAH PASMC treated with Con or ALDH1A3 siRNA.
(F) Representative LEF1 binding site of NFYA and H3K27ac histone mark in PAH PASMC treated with Con vs. ALDH1A3 siRNA visualized in the IGV genome browser.
(G) mRNA expression by qPCR of NFYA relative to β-actin in PAH PASMC treated with Con vs. KAT2B siRNA. (H) Model summarizing all data. n=3. *p<0.05**p<0.01, by paired Student t test.