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. 2022 Jun 17;12:10275. doi: 10.1038/s41598-022-14491-5

Figure 4.

Figure 4

Nintedanib increases expression of smooth muscle marker proteins in high-passage CSMC. (A) Immunofluorescent images of high passage CSMC showing labeling for smooth muscle marker proteins SM22 (red) or SMA (green) with nuclei stained with Hoechst (blue). Control (upper) or NIN-treated CSMC (1 µM, 48 h; lower) were imaged identically. Right, higher magnification of SMA staining for each condition. Scale bars, 30 µm (left images) and 20 µm (right images). (B) Image analysis of staining intensity for SM22 and SMA in CSMC treated with NIN (1 µM, 48 h). Average line density normalized to black background (n = 4 cell lines, 10–30 cells per cell line, *p < 0.05). (C,D) Western blotting for SMA and SM22 protein expression in CSMC treated with NIN (1 µM). (C) Representative images selected from independent western blots (see Supplementary Fig. 1 for further detail) and (D) outcome of image analysis. Equal cell numbers were loaded per lane (n = 5 cell lines, *p < 0.05). IOD, integrated optical density; GAPDH, loading control. *p < 0.05. (E) Quantitative PCR analysis of mRNA expression for SM22 and SMA in control vs NIN-treated CSMC (1 µM). All experiments done in triplicates and normalized to HPRT.