Journal: JVS-Vascular Science

Article Title: Canonical transient receptor potential 6 channel deficiency promotes smooth muscle cells dedifferentiation and increased proliferation after arterial injury

doi: 10.1016/j.jvssci.2020.07.002

Figure Lengend Snippet: Canonical transient receptor potential 6 deficient ( TRPC6 -/- ) SMC are phenotypically modulated in culture and in situ. A-C , Lysates were prepared from cultured wild-type (WT) and TRPC6 -/- VSMC (passages 6-8), resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and immunoblotted for SM22 and MYH11. Actin served as the loading control. A , Representative immunoblot. B and C , Relative MYH11 and SM22 protein expression quantified by densitometry (n = 3 immunoblots). D , Immunohistochemistry (IHC) performed on common carotid artery (CCA) cross-sections of 8- to 10-week-old male WT and TRPC6 -/- mice. ( Top ) IHC for SM22 and ( Bottom ) MYH11. E , SM22 protein levels were assessed at passages 1, 4, and 7 in WT and TRPC6 -/- VSMC by immunoblot analysis and quantified by densitometry. F and G , Lysates were prepared from cultured WT and TRPC6 -/- VSMC (passages 6-8) and immunoblotted for beta-actin and GAPDH. F , Representative western blot. G , The ratio of beta actin to GAPDH protein was quantified by densitometry (n = 3). H and I , Relative MYH11 and SM22 mRNA levels were assessed by quantitative real-time PCR in cultured WT and TRPC6 -/- VSMC (passages 6-8; n = 4).

Article Snippet: Primary antibodies included SM22 (Genetex, Irvine, Calif; cat. GTX101608, 1:100), MYH11 (Santa Cruz Biotechnology, Santa Cruz, Calif; cat. sc-6956, 1:100), α-smooth muscle actin (α-SMA; Cell Signaling Technology, Danvers, Mass; cat. D4K9N, 1:100), and Ki67 (Genetex, cat. GTX16667, 1:100), Von-Willebrand factor (Abcam, Cambridge, UK; cat. ab6994, 1:100).

Techniques: In Situ, Cell Culture, Polyacrylamide Gel Electrophoresis, Control, Western Blot, Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction

Journal: JVS-Vascular Science

Article Title: Canonical transient receptor potential 6 channel deficiency promotes smooth muscle cells dedifferentiation and increased proliferation after arterial injury

doi: 10.1016/j.jvssci.2020.07.002

Figure Lengend Snippet: Acute, transient knockdown of canonical transient receptor potential 6 (TRPC6) promotes loss of the contractile phenotype in MOVAS cells. A , Wild-type ( WT ) and TRPC6 -/- CCA from 10-week-old male mice were explanted and homogenized in lysis buffer (n = 3 mice and 6 CCA per genotype). Lysates were resolved by SDS page and immunoblotted against TRPC3 and GAPDH. B-F , Immortalized mouse aortic smooth muscle cell line (MOVAS) cells were transiently transfected with 50 nmol/L control small interfering RNA ( NsiRNA ) or TRPC6 siRNA. Transfected cells were harvested at 48 hours and immunoblotted for TRPC6, TRPC3, and contractile biomarkers. B , Representative immunoblot of total TRPC6 and TRPC3 protein. C , Relative TRPC6 protein expression in NsiRNA and TRPC6 siRNA transfected cells was quantified by densitometry (n = 3). D , Representative immunoblot of contractile biomarkers. E and F , Relative protein expression of MYH11 and SM22 in NsiRNA and TRPC6 siRNA transfected cells quantified by densitometry (n = 3).

Article Snippet: Primary antibodies included SM22 (Genetex, Irvine, Calif; cat. GTX101608, 1:100), MYH11 (Santa Cruz Biotechnology, Santa Cruz, Calif; cat. sc-6956, 1:100), α-smooth muscle actin (α-SMA; Cell Signaling Technology, Danvers, Mass; cat. D4K9N, 1:100), and Ki67 (Genetex, cat. GTX16667, 1:100), Von-Willebrand factor (Abcam, Cambridge, UK; cat. ab6994, 1:100).

Techniques: Knockdown, Lysis, SDS Page, Transfection, Control, Small Interfering RNA, Western Blot, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Phenotypical and functional characteristics of mesenchymal stem cells from bone marrow: comparison of culture using different media supplemented with human platelet lysate or fetal bovine serum

doi: 10.1186/scrt97

Figure Lengend Snippet: Western blot analysis showing differentiation markers of osteogenic, vascular smooth muscle, and adipogenic lineages expressed by BM-derived MSCs cultured in different expansion media . The media are M1 (10% FBS + FGF2), M2 (10% FBS + 5% HPL), M3 (10% HPL), and M4 (5% HPL). The experiments were performed in triplicate, and one representative blot is shown here. Beta-actin expression was used as positive control. Osteogenic proteins studied are calcium-sensing receptor (CaSR) and parathormone receptor (PTHR). Adipogenic protein studied is Leptin. Vascular smooth muscle proteins studied are smooth muscle 22alpha (SM22α) and alpha-smooth muscle actin (ASMA). D0, day 0 (before induction); D14, day 14 (after induction).

Article Snippet: Membranes were blocked with 5% skim milk for an hour and left to react with anti-human β-actin (clone AC-15, mouse IgG1; Sigma-Aldrich) as a positive control, anti-human leptin (monoclonal; R&D Systems, Inc., Minneapolis, MN, USA, reference number 398), anti-human calcium-sensing receptor (CaSR) (monoclonal; AbCam, Cambridge, UK, reference number ab 3513), anti-human parathormone receptor (PTHR) (mouse monoclonal anti-human; Novocastra, part of Leica Microsystems, Wetzlar, Germany), anti-human ASMA (clone 1A4, mouse anti-human, -mouse, -rat; Sigma-Aldrich), and anti-human transgelin (SM22α) (mouse monoclonal anti-human; Novocastra).

Techniques: Western Blot, Derivative Assay, Cell Culture, Expressing, Positive Control

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: MG132 pretreatment blocks the reduced phenotypic transition of HA-VSMCs induced by crenolanib. HA-VSMCs were pretreated with crenolanib (50 nM) for 48 h or MG132 (5 μM) for 1 h and then exposed to 10 ng/mL PDGF-BB treatment for 24 h. Untreated cells were used as control. ( A ) Cell proliferation was checked by EdU assay (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to the total number of cells. **, p < 0.01 vs. the control group; ##, p < 0.01 vs. the group treated with PDGF-BB; $$, p < 0.01 vs. the PDGF-BB and crenolanib-treated group ( n = 3). ( B ) Cell migration was checked by wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm). Histograms showing the quantification of the wound healing (migrating cells from the scratched boundary) and transwell assay (crystal violet stained cells migrating to the lower chamber) results. **, p < 0.01 vs. the control group; #, p < 0.05 and ##, p < 0.01 vs. the PDGF-BB-treated group; $, p < 0.05 vs. the PDGF-BB and crenolanib-treated group ( n = 3). These results show that MG132 pretreatment promoted the reduced cell proliferation and migration mediated by crenolanib. ( C ) Western blot analysis of MYH-11, SM22, and smoothelin. MG132 reduced the increased smoothelin expression induced by crenolanib. Histograms showing the ratios of target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group; $, p < 0.05 vs. the PDGF-BB and crenolanib-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Control, EdU Assay, Migration, Transwell Assay, Staining, Western Blot, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: RhoGDI1 suppression inhibits PDGF-BB-induced phenotypic transition in HA-VSMCs. Cells were transfected with RhoGDI1 siRNA for 48 h and then treated with 10 ng/mL of PDGF-BB for another 24 h. Untreated cells were used as control. ( A ) Confirmation of RhoGDI1 knockdown by western blotting. ( B ) EdU assay showed that RhoGDI1 suppression inhibited PDGF-BB-induced cell proliferation (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to total cells. **, p < 0.01 vs. the control group; ##, p < 0.01 vs. the group treated with PDGF-BB ( n = 3). ( C ) Wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm) showed that RhoGDI1 knockdown reduced cell migration induced by PDGF-BB. The quantification method refers to . **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ( D ) Western blot analysis of MYH-11, SM22, and smoothelin showed that RhoGDI1 suppression promoted the expression of smoothelin in PDGF-BB-treated cells. Histograms showing the ratios of target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Transfection, Control, Knockdown, Western Blot, EdU Assay, Migration, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: Cdc42 inhibition reduces PDGF-BB-induced phenotypic transition in HA-VSMCs. HA-VSMCs were pretreated with ZCL278 (50 μM) for 30 min followed by treatment with 10 ng/mL of PDGF-BB for 24 h. Untreated cells were used as control. ( A ) Cell proliferation was analyzed by EdU assay (scale bar = 100 μm). Histogram showing the ratio of EdU-positive cells (red) to total cells. **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ( B ) Cell migration was analyzed by wound-healing (scale bar = 200 μm) and transwell assays (scale bar = 100 μm). The quantification method refers to . **, p < 0.01 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3). ZCL278 pretreatment significantly decreased cell proliferation and migration induced by PDGF-BB. ( C ) Western blot analysis of MYH-11, SM22, and smoothelin. ZCL278 pretreatment increased the expression of smoothelin in PDGF-BB-treated cells. Histograms showing the ratios of the target proteins to β-tubulin. *, p < 0.05 vs. the control group; #, p < 0.05 vs. the PDGF-BB-treated group ( n = 3).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Inhibition, Control, EdU Assay, Migration, Western Blot, Expressing

Journal: Biomedicines

Article Title: RhoGDI1-Cdc42 Signaling Is Required for PDGF-BB-Induced Phenotypic Transformation of Vascular Smooth Muscle Cells and Neointima Formation

doi: 10.3390/biomedicines9091169

Figure Lengend Snippet: Both RhoGDI1 and Cdc42 suppression inhibit neointima formation and improve the expression of contractile proteins in a rat carotid injury model. For shRNA-mediated knockdown, the carotid artery was injected with approximately 0.2 mL of virus solution (titer: 1 × 10 10 pfu) after balloon injury operation. ( A ) H&E of arteries 14 days after balloon injury. Rats without balloon injury were used as the sham operation group. Histogram showing the area ratio of intima to media. Both RhoGDI1 and Cdc42 knockdown reduced intima/media area ratio. **, p < 0.01 vs. the sham operation group; ##, p < 0.01 vs. the injury model group ( n = 10). ( B ) Immunofluorescence staining for contractile proteins such as MYH-11, SM22, smoothelin (green), ki-67 (green), and α-SMA (green) and CD31 (red) as double staining (merge); nuclei were stained with DAPI (blue). Histograms showing the fluorescence intensity of the staining. RhoGDI1 and Cdc42 knockdown reduced the expression of ki-67 and co-localization of α-SMA with CD31 and increased the expression of the three contractile proteins. *, p < 0.05 vs. the sham operation group; #, p < 0.05 vs. the injury model group ( n = 10). ( C ) Western blots showing the expression of MYH-11, SM22, and smoothelin. Histograms showing the ratios of the contractile proteins to β-tubulin. The results are consistent with those from immunofluorescence analysis. *, p < 0.05 vs. the sham operation group; #, p < 0.05 and ##, p < 0.01 vs. the injury model group ( n = 10).

Article Snippet: Anti-RhoGDI1 (A1214), –Ubiquitin (A3207), –smoothelin (# 6745), –MYH11 (# 10827), -ki-67 (A11390), and -SM22α (# A6760) antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Expressing, shRNA, Knockdown, Injection, Virus, Immunofluorescence, Staining, Double Staining, Fluorescence, Western Blot