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. 2015 Sep;53(3):391–399. doi: 10.1165/rcmb.2014-0150OC

Figure 5.

Figure 5.

PP2A/HDAC4 axis regulates type I collagen expression via miR-29. (A and B) HDAC4 was knocked down by HDAC4 shRNA in IPF fibroblasts in which PP2Ac was overexpressed using an adenoviral vector (Ad-PP2Ac/HDAC4-shRNA). Controls consisted of IPF fibroblasts in which PP2A was overexpressed and treated with scrambled shRNA (Ad-PP2Ac/Scr-shRNA), cells infected with empty vector (control for PP2A) and treated with HDAC4-shRNA (Ad-EV/HDAC4-shRNA), and cells infected with empty vector and treated with scrambled shRNA (Ad-EV/Scr-shRNA). qRT-PCR was done to assess mRNA levels of Col1A1 (A) and miR-29c (B). (C and D) Control fibroblasts in which HDAC4 had been knocked down were infected with a lentiviral vector containing a miR-29c construct to overexpress miR-29 (HDAC4-shRNA/miR-29 overexpression [OE]). Controls consisted of control fibroblasts in which HDAC4 had been knocked down and then treated with empty vector (HDAC4-shRNA/EV), control fibroblasts treated with scrambled shRNA in which miR-29c had been overexpressed (Scr-shRNA/miR-29 OE), and control fibroblasts treated with scrambled shRNA and empty vector (Scr-shRNA/EV). The cells were seeded on polymerized type I collagen matrices for 4 hours. (C) Left panel: To confirm HDAC4 knockdown, HDAC4 expression was quantified by Western blot analysis. Shown is the ratio of HDAC4 to GAPDH. Right panel: To confirm miR-29 overexpression, miR-29c expression was quantified by qRT-PCR. Shown is the ratio of miR-29 to RNU. EV, empty vector. (D) Type I collagen expression was assessed by Western blot analysis. GAPDH is shown as a loading control (top panel). Collagen I protein expression was quantified by densitometric analysis (bottom panel).