Journal: Advanced Biology

Article Title: Fibro‐Adipogenic Progenitors From Skeletal Muscle Enhance Microvascular Angiogenesis in a 3D Model

doi: 10.1002/adbi.70162

Figure Lengend Snippet: Experimental Design and Characterization of FAPs and Freshly Isolated MVFs. (A) Schematic representing the study design (Created with BioRender.com). (B) Representative phase contrast image of a single microvascular fragment immediately after isolation. (C) Representative flow cytometry gating strategy and scatterplots of PDGFRα and SCA‐1 expression in FAPs after magnetic‐activated cell sorting.

Article Snippet: Briefly, the adherent cells were resuspended in 200 μL of DMEM + 2% FBS and incubated with antibodies (1 μL per 2.0 × 10 6 cells) against PDGFRα (ab203491, Abcam), SCA‐1 (AB4336, Millipore Sigma), CD31‐PE (130‐116‐538, Miltenyi Biotec), and CD45‐PE (130‐126‐312, Miltenyi Biotec) on ice for 30 min. For CD31/CD45 depletion, cells were incubated with 10 μL of Anti‐PE MicroBeads (130‐048‐801, Miltenyi Biotec) on ice for 30 min, washed twice in 1 mL of MACS buffer + 0.5% bovine serum albumin (BSA) via centrifugation (2000 rpm × 3 min at 4°C), and subjected to negative selection with LD Columns (130‐042‐901, Miltenyi Biotec) according to manufacturer protocols.

Techniques: Isolation, Phase Contrast, Flow Cytometry, Expressing, FACS, Co-Cultures, Confocal Microscopy

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 used to specifically detect the proteins of interest were as follows: SIK2 (6919s, CST), GAPDH (G9545, Sigma-Aldrich), α-SMA (A5228, Sigma-Aldrich), Collagen I (AB765P, Sigma-Aldrich), Collagen III (AB7778, Abcam), CD31 (3528s, CST), Phospho-NF-κB P65 (Ser 536 ) (3033s, CST), NF-κB P65 (8242s, CST), Phospho-P38 MAPK (Thr 180 /Tyr 182 ) (4511s, CST), P38 MAPK (8690s, CST), α-Tubulin (66031-1, Proteintech), Histone H3 (68345-1, Proteintech), NOS2 (13120s, CST), FLAG-Tag (14793s, CST), HA-Tag (3724s, CST), TRAF1 (4710s, CST), TRAF2 (4724s, CST), TRAF4 (18527s, CST), TRAF6 (67591s, CST), His-Tag (12698s, CST), and Myc-Tag (2276s, CST).

Techniques: Analysis, Staining, Immunostaining, Tissue