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

Journal: Advanced Science

Article Title: Copper‐loaded Milk‐Protein Derived Microgel Preserves Cardiac Metabolic Homeostasis After Myocardial Infarction

doi: 10.1002/advs.202401527

Figure Lengend Snippet: The effects of CuCMG on cardiomyocytes, endothelial cells, and fibroblasts. A) Schematic diagram of OGD model building, dosing, and detection times. B) JC‐1 staining of H9C2 cells from Blank, Control, CMG, Cu, and CuCMG groups. n = 4. Scale bars = 100 µm. C) Statistical analysis of the JC‐1 staining. D) The ATP levels of H9C2 cells in different groups after OGD treatment. E) qRT‐PCR analysis of the expression levels of HIF1A , VEGFA , and FGF2 in HUVECs treated with CMG and CuCMG separately. F) qRT‐PCR analysis of the expression levels of Postn , Col1a1 , and Ccn2 in pre‐induced MCFs treated with CMG and CuCMG separately. one‐way ANOVA was used for statistical analyses. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. ns, no statistical significance.

Article Snippet: Isolation of myocardial fibroblasts (MCFs) from neonatal mice was performed following literature using a Neonatal Cardiac Fibroblast Isolation Kit (Miltenyi, Germany).

Techniques: Staining, Control, Quantitative RT-PCR, Expressing

Journal: Nature

Article Title: The extracellular matrix protein agrin promotes heart regeneration in mice

doi: 10.1038/nature22978

Figure Lengend Snippet: a, b, Representative fields of heart cultures stained with DAPI (blue), cTnT (green) and Ki67 (red). White arrows indicate Ki67+cTnT+ cells. CMs, cardiomyocytes. c, d, Percentage of proliferating cardiomyocytes from P1 (c) or P7 (d) hearts in response to P1 and P7 ECM particles. n = 2,069 cardiomyocytes from three samples (c); n = 2,221 cardiomyocytes from four samples (d). e, f, Percentage of proliferating cardiomyocytes (Ki67+cTnT+) in response to P1 and P7 ECM in P1 (e) or P7 (f) cultures, with or without the broad MMP inhibitor (Marimastat). n = 3,480 cardiomyocytes from three samples (e); n = 23,445 cardiomyocytes from four samples (f). g, h, Percentage of P1 (g) or P7 (h) proliferating cardiomyocytes in response to MMP9- or MMP12-cleaved ECM fragments. n = 11,820 cardiomyocytes from four samples (g); n = 15,509 cardiomyocytes from four samples (h). i, qPCR of Agrn mRNA in P1 and P7 hearts. n = 8 P1 and 3 P7 hearts. j, Quantification of western blots for agrin from P1, P7 and 12-week-old (12W) adult heart lysates. A.U., arbitrary units. n = 3 samples per group. k, Images of P1 and P7 heart sections stained for agrin (green) and DAPI (blue). n = 3 samples per group. l, qPCR analysis of cardiac populations (FB, fibroblasts; CM, cardiomyocytes; EC, endothelial cells). n = 4 cardiomyocyte, 4 non-cardiomyocyte, 4 fibroblast, 4 non-fibroblast, 7 endothelial cells and 7 non-endothelial cell samples. Scale bars, 40 μm (a) and 10 μm (k). Data are presented as mean ±s.e.m. *P < 0.05, **P< 0.01, ***P< 0.001; statistical significance was calculated using ANOVA followed by a Dunnett’s post hoc test relative to the control group (c–h) or a Tukey’s post hoc test (j), statistical significance was calculated using a one-tailed t-test (i, l).

Article Snippet: Separation to distinct cardiac cell populations was performed by using the Neonatal Cardiac Endothelial Cell Isolation kit (130-104-183, Miltenyi Biotec), Neonatal Cardiac Fibroblast Cell Isolation kit (130-101-372, Miltenyi Biotec) or by Neonatal Cardiomyocyte Isolation kit (130-100-825, Miltenyi Biotec), according to the manufacturer’s instructions.

Techniques: Staining, Western Blot, Control, One-tailed Test