Fig. 3: PDCD5 mediates TGF-β-dependent transcriptional activation of matricellular genes via formation of a PDCD5/β-catenin/Smad3 complex.
From: Club cell-specific role of programmed cell death 5 in pulmonary fibrosis
a C22 cells were treated with TGF-β1 (20 ng/ml) and harvested at indicated time points. Whole-cell lysates were analyzed by immunoblotting with indicated antibodies. Representative blots for three independent experiments are shown. b GSEA of RNA-seq signals of GO-defined ECM gene clusters in C22 cells. The normalized enrichment score (NES) is pictured in the left panel. A heat map of the ECM gene signature is pictured in the right panel. c Primary club cells were isolated from Pdcd5f/f and Ccsp-Pdcd5d/d mouse lungs, and AT2 cells were isolated from Pdcd5f/f and Spc-Pdcd5d/d mice after BLM injection. The levels of indicated genes were analyzed by qRT-PCR. (n = 3 mice/group); *p = 0.0293; **p = 0.0034; ***p < 0.0008; ****p < 0.0001; n.s. not significant. d C22 cells were transfected with Pdcd5 siRNAs for 36 h and then treated with TGF-β1 for 12 h. Nuclear extracts were immunoprecipitated with the indicated antibodies and immunoblotted with the indicated antibodies. Representative blots from three independent experiments are shown. e Primary club cells or AT2 cells from BLM-injected mice were analyzed by in situ PLA, using Smad3 and β-catenin antibodies. Scale bars = 20 µm. Number of signals was quantified using ImageJ software (n = 5/group); **p = 0.0074; ****p < 0.0001; n.s. not significant. f C22 cells were treated with Pdcd5 siRNA for 36 h and/or TGF-β1 for 12 h, immunofluorescence analysis was performed. Scale bars = 10 μm. Representative images from three independent experiments are shown. g C22 cells were treated with TGF-β1 for 24 h and a ChIP assay was performed using the indicated antibodies. Precipitated samples were analyzed by qRT-PCR, and results are presented as the percentage of input (n = 3 in each group); ****p < 0.0001. Data are shown as mean ± s.e.m. Statistical analysis was performed with one-way ANOVA with Tukey’s post hoc test (c, e, g). Source data are provided in the Source Data file.
