Fig. 6: TRβ modulates KLF2 and CEBPA to drive the expression of AT1 cell marker genes.

a Principal component analysis (PCA) of TFs in bulk RNA-Seq of mouse lung (same as Fig. 3c, n = 3). The lower and upper bounds of the boxplot correspond to the first and third quartiles (the 25th and 75th percentiles); whiskers represent minima/maxima or 1.5*IQR. b Histogram showing the contributions of TFs in PC.2 and PC.5. c, d Protein and mRNA levels (n = 3) of KLF2 and CEBPA in the mouse lung, as shown in Fig. 3a. e qPCR analysis of KLF2 and CEBPA expression in isolated AT2 cells from mice (n = 3). f, g Immunoblotting and qPCR analysis (n = 3) of KLF2 and CEBPA in A549 and MLE12 with a GC-1 gradient. h The mRNA level of Klf2 and Cebpa in primary AT2 cells after 10 nM GC-1 treatment for 6 h (n = 3). i–l Protein (i, k) and mRNA (j, l) tests of AT1 cell markers, KLF2, and CEBPA expression in A549 after KLF2 (i, j) and CEBPA (k, l) overexpression (n = 3). m Luciferase activity of KLF2, CEBPA, and AT1-marker promoters cloned in pGL3.0 after THRB transfection for 36 h with or without GC-1 10 nM for 12 h (n = 3). Values were normalized to the transfection vector. n, o Luciferase activity of indicated promoters after KLF2 (n) and CEBPA (o) transfection with 600 ng plasmid for 36 h (n = 3). p–r TRβ (p), KLF2 (q), and CEBPA (r) bind to the promoter regions (pro) of KLF2, CEBPA, and AT1 cell genes in ChIP q-PCR assays (n = 3). KRT5 promoter as negative control. Values were normalized to IgG. s Luciferase activity of AT1-maker promoters with KLF2 (300 ng) and CEBPA (300 ng) co-transfection for 36 h, as shown in (n, o) (n = 3). t IF image of KLF2 and CEBPA after co-transfection in A549. Scale bar, 10 μm. u CoIP of KLF2 and CEBPA overexpressed in A549. v Illustration of the regulatory processes of GC-1 on AT1 cell markers. The value of n indicates biologically independent samples. Similar results were repeated in two biologically independent experiments. The statistical tests used were one-way ANOVA (d, e, and g), two-tailed unpaired Student’s t test (h), and two-way ANOVA (j, l, m–s). Data are mean ± SEM.