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Figure Lengend Snippet: Antibodies.

Article Snippet: Western blot (mouse sample) , Novus Biologicals , NB100-2220.

Techniques: Western Blot

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