Fig 1: The injury of smooth muscle caused by specific knockout of Kindlin-2 leads to embryonic lethality. (A) Gross appearance of E12.5-15.5 embryos with different genotype. Embryos were fixed with paraformaldehyde. cKO (Kindlin-2fl/fl;SM22a-cre+) embryos are lethal at E14.5 (arrows). (B) Whole embryo sections were stained with hematoxylin and eosin (HE). Representative images and morphological abnormality are presented. The WT (Kindlin-2fl/fl;SM22a-cre-, Up panel) and cKO mice (Kindlin-2fl/fl;SM22a-cre+,Lower panel) were presented. H: heart; L: liver; GI: gastrointestinal tract; P: pancreas. Scale bar, 1mm. (C) HE staining of embryonic arteries from E12.5-E15.5. Scale bar, 50µm (D) Western blot analysis of whole embryo lysis from E12.4-E15.5, test for Myh11, a-SMA, CNN expression levels respectively. (E) Quantitation of (D) and two other independent experiments. Data were presented as the mean ± SEM. *p < 0.05 by student's t-test. See Figure S1, S2, Table S1, S2 and S3 for additional information.
Fig 2: PA increased miR-22 expression in VSMCs. (A) MiRNAs levels in VSMCs treated with PA. (B) The mRNA levels of miR-22, miR-23b, and miR-125b in VSMCs after transfecting with their specific miRNA mimics. (C) The protein expression of SM22a, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMCs treated with miR-22, miR-23b or miR-125b mimics. (D) Migration ability of VSMCs transfecting with miR-122 mimics determined by transwell assay. Scale bar=100 µm. (E, F) The cell proliferation of VSMCs treated with miR-22 mimics detected by EdU assay. (G, H) Apoptosis of VSMCs treated with miR-22 mimics determined by flow cytometry. (I) The protein expression of bax, bcl-2, caspase-3, and cleaved-caspase-3 in VSMCs treated with miR-22 mimics. n = 3. *P < 0.05, **P < 0.01.
Fig 3: Subset of wild-type double-positive cells expresses smooth muscle genes. (A,B) All wild-type uninjured and 3 dpci Tg(BAC-runx1P2:Citrine;kdrl:Hsa.HRASmCherry) double-positive cells visualised in an UMAP plot. (A) Expression of collagen 1a1b specifically in 3 dpci double-positive cells. (B) Cell clustering within the double-positive population identifies six different cell clusters. (C) Heatmap showing that both clusters 4 and 5 express high levels of collagens, whereas cluster 4 specifically expresses smooth muscle genes. (D) myh11a and tagln are expressed in cluster 4 after injury. Arrowhead indicates myh11a expression in cell cluster 4. (E-F) Immunohistochemistry for Citrine, mCherry and Myh11. Arrowheads indicate expression of Myh11 in Tg(BAC-runx1P2:Citrine;kdrl:Hsa.HRAS-mCherry) double-positive cells at 3 (E,E') and 7 (F) dpci. en, endocardium; v, ventricle; w, wound. Scale bars: 100 µm.
Fig 4: PA treatment enhanced miR-22 mimic impacts on aborted the inhibitive effect of EVI1 on VSMC phenotype switch. (A) Expression of miR-22 and EVIL1 in VMSCs treated with EVI1 or/and miR-22 overexpression followed by PA treatment. (B) Cell proliferation was detected by EdU in VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (C) Flow cytometry to detect the apoptosis of VSMCs with EVI1/miR-22 overexpression followed by PA treatment. (D) Western blot to detect the apoptosis associated markers in VSMCs. (E, F). Cell migration was detected by transwell assay in EVI1/miR-22-overexpressed VSMCs followed by PA treatment. Scale bar = 100 µm. (G) Protein levels of SM22a, calponin, SMMHC, vimentin, collagen I, and osteopontin in VSMC in VSMCs with EVI1/miR-22 overexpression followed by PA treatment detected by western blot. n = 3. *P < 0.05, **P < 0.01.
Fig 5: PA inhibited VSMC switch to synthetic phenotype. VSMC morphologies after PA treatments (0, 100, 200 or 400 µM) for 3d. (A) Live/dead cell assay of PA-treated VSMCs. (B and C). EdU assay to detect the proliferation on VSMCs treated with PA. (D and E). Flow cytometry to detect the apoptosis of PA-treated VSMCs. (F) Western blot determined the expression of apoptosis associated markers. (G) Transwell assay of PA-treated (200 µM) VSMCs. Scale bar = 100 µm. (H) Expression levels of SM22a, calponin, SMMHC, vimentin, collagen I, and osteopontin in PA-treated (200 µM for 3 d) VSMCs. n = 3. *P < 0.05, **P < 0.01.
Supplier Page from Abcam for Anti-smooth muscle Myosin heavy chain 11 antibody