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. 2022 Mar 24;11(7):e023401. doi: 10.1161/JAHA.121.023401

Figure 5. Overexpression of ChGn‐2 augments the cardioprotective effects in mouse cardiac fibroblasts (MCFs).

Figure 5

A, Immunoblotting for ChGn‐2 (chondroitin sulfate N‐acetylgalactosaminyltransferase‐2) in wild‐type (WT) mouse cardiac fibroblasts (MCFs) infected with retroviruses carrying either green fluorescent protein (GFP) or ChGn‐2 (ChGn‐2 overexpression). Molecular weight (MW) for protein ladders are shown. ChGn‐2 protein levels were quantified and normalized to GAPDH expression (n=3 for each group). B, Immunoblotting for caspase‐3, cleaved caspase‐3 (c.caspase 3), and GAPDH in H9C2 cells treated with conditioned medium (CM) derived from WT MCFs in either control (C‐CM) or stretch (S‐CM) condition in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. WT MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. Apoptosis was assessed by the cleaved caspase‐3 expression levels compared with caspase 3 (n=6 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in GFP MCF C‐CM + doxorubicin group. C, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining in in H9C2 cells treated with CM derived from WT MCFs in either C‐CM or S‐CM in the presence of doxorubicin. WT MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. TUNEL‐positive apoptotic cells were quantified (n=6 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. D, Immunoblotting for ChGn‐2 in ChGn‐2‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression). Molecular weight (MW) for protein ladders are shown. E, Immunoblotting for caspase‐3, cleaved caspase‐3, and GAPDH in H9C2 cells treated with CM derived from ChGn‐2‐/‐ MCFs in either C‐CM or S‐CM in the presence of doxorubicin. Molecular weight (MW) for protein ladders are shown. ChGn‐2‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 overexpression) were used. Apoptosis was assessed by the cleaved caspase‐3 expression levels (n=9 for each group). Quantitative data show cleaved caspase 3/total caspase 3 value relative to that in GFP ChGn‐2‐/‐ MCF C‐CM + doxorubicin group. F, TUNEL staining in H9C2 cells treated with CM derived from ChGn‐2‐/‐ MCFs in either C‐CM or S‐CM in the presence of doxorubicin. ChGn‐2‐/‐ MCFs infected with retroviruses carrying either GFP or ChGn‐2 (ChGn‐2 OE) were used. TUNEL‐positive apoptotic cells were quantified (n=6 for each group); bars: 100 μm. TUNEL, green; and Hoechst, blue. Doxorubicin was used at 1 μmol/L. Data represent median and interquartile range. *P<0.05, **P<0.01, and ***P<0.001. Statistical analyses were performed using Wilcoxon rank‐sum test (A) or Kruskal‒Wallis test followed by Wilcoxon rank‐sum test (B, C, E, and F). C‐CM indicates control conditioned medium; ChABC, chondroitinase ABC; ChGn‐2, chondroitin sulfate N‐acetylgalactosaminyltransferase‐2; GFP, green fluorescent protein; MCFs, mouse cardiac fibroblasts; S‐CM, stretch conditioned medium; and WT, wild‐type.