Journal: Biotechnology Reports

Article Title: Xenogeneic-free platform for the isolation and scalable expansion of human bladder smooth muscle cells

doi: 10.1016/j.btre.2025.e00878

Figure Lengend Snippet: Isolation and characterization of bladder smooth muscle cells under xeno(geneic)-free culture conditions. A) Samples were fragmented into explants of approximately 1 mm diameter, and 10 explants per well were plated onto 6-well plates (Corning®). Phase contrast imaging of cells migrating and proliferating from explants (first image: donor 63 yo at day 2 post-processing; second image: donor 58 at day 6 post-processing; third and fourth images: donor 70 yo at day 6 post-processing); Scale bar: 100 µm. B) Representative images of fluorescence microscopy for the detection of typical SMC markers (red); left panel: donor 58 yo, cell passage 2; center panel: donor 80 yo, cell passage 5; right panel: donor 70 yo, Passage 3; Nuclei counterstained with DAPI (blue); Scale bar: 100 µm. C) Flow cytometry panel for SMC identity markers (positive and negative) for all four donors. αSMA: α-Smooth muscle actin; MYH11: Myosin heavy chain 11; SM22: Smooth Muscle Protein 22-α; SMC: Smooth muscle cells.

Article Snippet: Primary antibodies anti-human α-Smooth muscle actin (αSMA) (Invitrogen), Caldesmon, Desmin (Santa Cruz Biotechnology), Smooth Muscle Protein 22-α (SM22α) (Novus Biological), Myosin heavy chain 11 or smooth muscle myosin heavy chain (MYH11) (Abcam), CD44, CD90 and CD29 (Becton Dickinson, BD), were diluted in 1% (v/v) BSA and incubated for 2 h at RT.

Techniques: Isolation, Imaging, Fluorescence, Microscopy, Flow Cytometry

Journal: Biotechnology Reports

Article Title: Xenogeneic-free platform for the isolation and scalable expansion of human bladder smooth muscle cells

doi: 10.1016/j.btre.2025.e00878

Figure Lengend Snippet: Assessment of smooth muscle cell identity post-spinner flask culture. A) Live/dead staining assessed by flow cytometry immediately upon cell retrieval at the end of the spinner flask cultures for each donor. B) Flow cytometry for the depicted cell identity markers (positive and negative), C) Representative immunofluorescence images for SMC markers (red) and nuclei counterstained with DAPI (blue) on cells retrieved from microcarriers and adhered to static cultures; Upper panel: donor 58 yo; Middle panel: donor 80 yo; Bottom panel: donor 58 yo (for CD29 and CD44) and donor 80 yo (for CD90); Scale bar: 100 μm. αSMA: α-Smooth muscle actin; MYH11: Myosin heavy chain 11; SM22: Smooth Muscle Protein 22-α, SMC: smooth muscle cells.

Article Snippet: Primary antibodies anti-human α-Smooth muscle actin (αSMA) (Invitrogen), Caldesmon, Desmin (Santa Cruz Biotechnology), Smooth Muscle Protein 22-α (SM22α) (Novus Biological), Myosin heavy chain 11 or smooth muscle myosin heavy chain (MYH11) (Abcam), CD44, CD90 and CD29 (Becton Dickinson, BD), were diluted in 1% (v/v) BSA and incubated for 2 h at RT.

Techniques: Staining, Flow Cytometry, Cell Retrieval, Immunofluorescence

Journal: British Journal of Pharmacology

Article Title: Inhibition of polycomb repressor complex 2 ameliorates neointimal hyperplasia by suppressing trimethylation of H3K27 in vascular smooth muscle cells

doi: 10.1111/bph.14754

Figure Lengend Snippet: Trimethylation of H3K27 was induced in PDGF‐BB treated vascular smooth muscle cells (VSMCs) and neointima of rat wire‐injured carotid arteries. (a) VSMCs were treated with PDGF‐BB (40 ng·ml−1) for 36 hr after serum starvation. Histone methylation was detected by NANO‐HPLC/MS. The change in methylation is shown as a heat map. (b,c) VSMCs were treated with 10% FBS (b) or PDGF‐BB (40 ng·ml−1) (c) for 36 hr after being starved for 48 hr: western blot analysis of protein levels of H3K27me3, total H3, and GAPDH; data are mean ± SEM, n = 5. (d,e) Rats underwent carotid artery wire injury (n = 5 rats in each group). At 14 days after surgery, arteries were collected. (d) Representative H&E staining and immunostaining of H3K27me3, α‐SMA, and DAPI (left panel, scale bar = 50 μm); quantification of mean integrated OD of H3K27me3 in α‐SMA positive area (right panel). (e) Western blot analysis of protein levels of H3K27me3, total H3, α‐SMA, SM‐22, PCNA, and α‐tubulin. Data are mean ± SEM. *P < .05. Ctrl, control

Article Snippet: Proteins from VSMCs or rat carotid arteries were resolved by 10% or 12% SDS‐PAGE and transferred to PVDF membranes, which were blocked with 5% non‐fat milk for 2 hr at room temperature, then incubated with primary antibodies for H3K27me3 (1:5,000, cat#: 07449), EZH1 (1:2,500, cat#: ABE281), and EZH2 (1:1,000, cat#: 07689, RRID:AB_417397; all from Millipore); total H3 (1:3,000, cat#: 4499, RRID:AB_10544537) and PCNA (1:1,000, cat#: 2586, RRID:AB_2160343; both from CST); α‐SMA (1:8,000, cat#: A2547; Sigma Aldrich); smooth muscle protein 22‐α (SM‐22α; 1:500, cat#: SC‐53932, RRID:AB_1129519) and α‐tubulin (1:1,000, cat#: SC‐8035, RRID:AB_628408; both from Santa Cruz Biotechnology, CA, USA); and GAPDH (1:10,000, cat#: 600041, RRID:AB_2107436; Proteintech) at 4°C overnight, then secondary antibodies (goat anti‐mouse IgG, 1:5,000, cat#: 4741806, RRID:AB_2307348; goat anti‐rabbit IgG, 1:5,000, cat#: 0741506, RRID:AB_2721169; KPL, Gaithersburg, MD, USA) were applied at room temperature for 1 hr.

Techniques: Methylation, Western Blot, Staining, Immunostaining, Control