Journal: eLife

Article Title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

doi: 10.7554/eLife.94794

Figure Lengend Snippet: ( A ) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from old, wt, and Arg2 -/- splenic cells (96 hr incubation) (n=3 mice in each group). The values shown are mean ± SD. mRNA expression levels of ( B ) Fib , ( C ) Col3a1, and ( D ) Tgfb1 in wt fibroblasts treated with conditioned media (CM) from old wt and Arg2 -/- splenic cells (96 hr incubation) were analyzed by qRT-PCR. Gapdh served as the reference. (n=4 mice per group); ( E ) Hydroxyproline content in fibroblasts treated with CM from old wt splenic cells (96 hr incubation). ILRa (50 ng/ml) is used to prevent IL-1β binding to its receptor (n=4 independent experiments). ( F ) Immunoblotting analysis of ARG2 and vimentin in human cardiac fibroblasts (HCFs) upon Arg2 gene overexpression. GAPDH served as protein loading control; ( G ) qRT-PCR analysis of mRNA expression levels Arg2 in HCF cells; ( H ) The plot graph shows the quantification of the vimentin signals on immunoblots shown in panel F. (n=3 independent experiments); ( I ) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells. Gapdh served as the reference. (n=6 independent experiments); ( J ) Representative confocal images of human cardiac fibroblasts (HCF) upon transfection with rAd-CMV-Con/ Arg2 for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. ( K ) Mitosox signal quantification (n=3 independent experiments); ( L ) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells treated as indicated. Gapdh served as the reference. (n=5 independent experiments). ( M ) Immunoblotting analysis of ARG2 and HIF-1α in human cardiac fibroblasts (HCFs) upon 1% hypoxia incubation for 48 h. Tubulin served as a protein loading control; ( N ) Representative confocal images of HCFs under normoxia (21% O 2 ) or hypoxia (1% O 2 ) for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. ( O ) Representative confocal images of HCFs upon transfection with shRNA for Arg2 gene silencing under normoxia or hypoxia. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). DAPI (blue) stains cell nuclei. Scale bar = 25 µm. ( P ) MitoSOX signal quantification of images shown in panel O (n=3 independent experiments). Data are expressed as fold change to respective control group. *p≤0.05, **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups. MØ, splenic macrophage; Con, control.

Article Snippet: Human cardiac fibroblasts (HCF) from adult human heart tissue were purchased from Innoprot ( P10452 ) and cultured in the proprietary fibroblast culture medium (P60108-2, Innoprot) composed as follows: 500 ml of fibroblast basal medium, 25 ml of FBS, 5 ml of fibroblast growth supplement-2, and 5 ml of penicillin/streptomycin in the Poly-L-Lysine (PLL) coated flasks and dishes.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Binding Assay, Western Blot, Over Expression, Control, Transfection, Staining, shRNA