Fig. 3
From: RGS5 maintaining vascular homeostasis is altered by the tumor microenvironment
RGS5 protects against inflammatory response of VSMCs and ameliorates vascular remodeling. (A) qRT-PCR analysis of RGS5, inflammatory markers (MCP-1 and VCAM-1) and macrophage-like markers (C3, CD74 and Lyz2) mRNA levels in mouse VSMCs with RGS5 overexpression followed TNF-α treatment for 24 h. (B) Western blot analysis and quantification of protein expression of RGS5, inflammatory markers (MCP-1 and VCAM-1) and macrophage-like markers (C3, CD74 and Lyz2) in mouse VSMCs infected with lenti-RGS5 followed TNF-α treatment for 48 h. (C) Left, Representative immunofluorescence images of CD74 (green) stained in mouse VSMCs infected with lenti-RGS5 followed by TNF-α treatment for 72 h. Nucleuses stained by DAPI were indicated in blue. Scale bar = 75 μm. (D) Phagocytosis of neutral red of VSMCs with RGS5 overexpression followed TNF-α treatment for 7 days. (E) Phagocytosis of fluorescent microsphere of VSMCs with RGS5 overexpression followed TNF-α treatment for 7 days. (F) Western blot analysis and quantification of RGS5, CD74, GSDMD, GSDMD-N, Caspase-1 and cleaved Caspase-1 in Rgs5-KO VSMCs infected with lenti-RGS5 followed TNF-α treatment for 48 h. (G) The fluorescent intensity quantification of calcein-AM-labeled RAW264.7 cell adhesion to Rgs5-KO VSMCs infected with lenti-RGS5 followed TNF-α treatment. (H) Western blot analysis RGS5 and macrophage-like markers (C3, CD74 and Lyz2) in lysates of carotid arteries harvested from 10-week-old male WT mice intravenous infected with AAV-Con and AAV-Flag-RGS5 following by carotid artery ligation for 28 days. (I) Representative cross sections of immunofluorescence images of CD74 and α-SMA in sham-operated and ligated carotid arteries at day 28 post surgery of 10-week-old male WT mice intravenous infected with AAV-Con and AAV-RGS5. Scale bar = 75 µm. (J) Left: Representative cross sections of hematoxylin & eosin-stained (H&E) sham-operated and ligated carotid arteries at day 28 post surgery of 10-week-old male WT mice intravenous infected with AAV-Con and AAV-RGS5. Scale bar = 100 µm. Right: Quantitative analysis of the intima area, intima to media ratio, artery lumen diameter, artery lumen area, external elastic lamina (EEL) circumference and media area in histological sections from ligated carotid arteries of 10-week-old male WT mice at day 28 post surgery of 10-week-old male WT mice intravenous infected with AAV-Con and AAV-RGS5. The data were presented as the mean ± SD