Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Representative parasternal long-axis views and M-mode images. ( B ) Echocardiographic analysis of EF, FS, LVESV, LVEDV, LVESD, and LVEDD on days 0, 7, and 14 after MI or sham operation in Sik2 F/F and Sik2 F/F ; LysM Cre mice ( n = 8). ( C ) Masson’s trichrome staining for infarct size measurements in Sik2 F/F and Sik2 F/F ; LysM Cre mice at day 14 after MI or sham operation ( n = 8). ( D ) Post-MI survival analysis. n = 42 in Sik2 F/F MI; n = 38 in Sik2 F/F ; LysM Cre MI. ( E ) Masson’s trichrome and Picrosirius red (PSR) staining of heart transverse sections to detect collagen within the scar of Sik2 F/F and Sik2 F/F ; LysM Cre hearts at day 14 post-MI (scale bar, 100 μm). Collagen density measured as the total collagen area divided by scar area examined ( n = 7). ( F ) Immunostaining analyses of α-SMA (green), Collagen I (green) and DAPI (blue) in the heart tissue at day 7 post-MI ( n = 8) (scale bar, 40 μm). ROI indicates the region of interest. ( G ) Gating strategy for endothelial cells (ECs) and CFs in the infarcted heart. ( H ) Statistics of ECs and CFs in the infarcted heart at day 7 post-MI from Sik2 F/F and Sik2 F/F ; LysM Cre mice ( n = 6). ( I ) Representative fibroblasts immunostaining of α-SMA (green), Collagen I (red), and DAPI (blue). Scale bar, 20 μm.

Article Snippet: Primary antibodies against the following targets were used in these experiments: SIK2 (6919s, 1:200; CST, Danvers, MA, USA), Ly6G (551459, 1:200; BD Biosciences, Franklin Lakes, NJ, USA), CD68 (ab53444, 1:200; Abcam, Cambridge, UK), TNNI3 (SAB2502170, 1:200; Sigma-Aldrich, St. Louis, MO, USA), α-SMA (A5228, 1:700; Sigma-Aldrich), Collagen I (NBP1-30054, 1:200; Novus Biologicals, Littleton, CO, USA), CD31 (ab222783, 1:200; Abcam), NOS2 (13120s, 1:200; CST), and Arginase-1 (93668s, 1:200; CST).

Techniques: Analysis, Staining, Immunostaining, Tissue

Journal: Science Advances

Article Title: SIK2 antagonizes postinfarction cardiac repair by driving macrophage proinflammatory and impairing efferocytosis

doi: 10.1126/sciadv.adw6624

Figure Lengend Snippet: ( A ) Representative TUNEL/TNNI3/DAPI staining of cardiomyocytes from Sik2 F/F and Sik2 F/F ; LysM Cre mice on day 3 post-MI. White arrows indicate TUNEL + (green) cardiomyocytes (red) and DAPI (blue) (scale bar, 40 μm). Percentage of TUNEL + cardiomyocytes ( n = 6). ( B ) Immunostaining shows colocalization of CD68 + macrophages with TNNI3 + cardiomyocytes (yellow). Scale bar, 40 μm. Macrophages positive for TNNI3 are scored as internalizing cardiomyocyte-derived proteins ( n = 6). ( C ) Heatmap of efferocytosis-related DEGs in cardiac macrophages on day 3 post-MI. ( D ) WT or SIK2-knockout cardiac macrophages were incubated with Calcein-AM + apoptotic cardiomyocytes (AC:Mφ = 2:1 or 5:1) for 1 hour. Efferocytosis was quantified as the total percentage of macrophages positive for Calcein-AM + ACs ( n = 4 for 2:1; n = 6 for 5:1). ( E ) Calcein-AM + ACs were incubated with WT or SIK2-knockout macrophages (5:1) for 1 hour. Efferocytosis was analyzed by fluorescence microscopy for Calcein-AM mean fluorescence intensity (MFI) in macrophage ( n = 7). ( F ) Flow cytometry analysis of efferocytosis in PMs for 2 hours. ( G ) PMs from WT and SIK2-cKO mice were treated with CFSE + RBCs (RBC:Mφ = 50:1 or 100:1) for 2 hours. Phagocytic macrophages were quantified as the total percentage of macrophages positive for CFSE + RBCs ( n = 5). ( H and I ) qPCR analysis of Cd36 , Cx3cr1 , Il-10 , Tgfb1 , and Vegfa mRNA levels in macrophages cocultured with RBCs for 8 hours ( n = 4). ( J ) qPCR analysis of α-SMA and Col1a1 mRNA levels in control and ARN3236 (3 μmol)–treated CFs with or without Tgfb1 (20 ng/ml) ( n = 6). ( K ) Immunostaining staining of α-SMA (green), Collagen I (red), and DAPI (blue) in WT mouse CFs treated with macrophage-conditioned media (ARN3236-treated or control) after efferocytosis. Scale bar, 20 μm. ( L ) qPCR analysis of α-SMA and Col1a1 mRNA levels in WT mouse CFs cultured with macrophage-conditioned media (ARN3236-treated or control) after efferocytosis ( n = 6).

Article Snippet: Primary antibodies against the following targets were used in these experiments: SIK2 (6919s, 1:200; CST, Danvers, MA, USA), Ly6G (551459, 1:200; BD Biosciences, Franklin Lakes, NJ, USA), CD68 (ab53444, 1:200; Abcam, Cambridge, UK), TNNI3 (SAB2502170, 1:200; Sigma-Aldrich, St. Louis, MO, USA), α-SMA (A5228, 1:700; Sigma-Aldrich), Collagen I (NBP1-30054, 1:200; Novus Biologicals, Littleton, CO, USA), CD31 (ab222783, 1:200; Abcam), NOS2 (13120s, 1:200; CST), and Arginase-1 (93668s, 1:200; CST).

Techniques: TUNEL Assay, Staining, Immunostaining, Derivative Assay, Knock-Out, Incubation, Fluorescence, Microscopy, Flow Cytometry, Analysis, Control, Cell Culture

Journal: Cardiovascular Therapeutics

Article Title: CircIdh2 Attenuates Endothelial‐to‐Mesenchymal Transition and Myocardial Fibrosis and Exhibits Translational Potential

doi: 10.1155/cdr/2005876

Figure Lengend Snippet: AAV‐mediated delivery of circIdh2 or circIdh2‐aa attenuated myocardial fibrosis and EndMT‐related marker changes in vivo. (A) HE staining of myocardial infarction tissues from the sham, model, circIdh2‐AAV, and circIdh2‐aa‐AAV groups. (B) Masson staining of MI tissues. (C) IF detection and quantification of fibrotic markers (Collagen I and TGF‐ β ) in myocardial tissues. Scale bars, 100 μ m. (D) Western blot analysis of EndMT‐related proteins (VE‐cadherin, vimentin, and N‐cadherin). Data are mean ± SD; n = 3 rats per group; one‐way ANOVA; ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: The sections were then incubated overnight at 4°C with primary antibodies against Collagen I (1:200, #66761‐1‐Ig, Proteintech) and TGF‐ β 1 (1:200, #81746‐2‐RR, Proteintech).

Techniques: Marker, In Vivo, H&E Stain, Staining, Western Blot, Analysis