recombinant mouse tgf β1  (R&D Systems)

Journal: Wound Repair and Regeneration

Article Title: Dendritic Cell–Associated MARCKSL1 Regulates Fibroblast Differentiation During Wound Healing

doi: 10.1111/wrr.70143

Figure Lengend Snippet: Induction of MARCKSL1 expression in mouse DCs by wounding stimulation. (A) Immunofluorescence staining of DCs following TGF‐β1 stimulation. Red: MARCKSL1; Green: CD11c; Blue: DAPI. Scale bar = 10 μm. (B) Western blot analysis of MARCKSL1 and p‐MARCKS expression in TGF‐β1‐treated DCs. * p < 0.05. (C) RT‐qPCR analysis of MARCKSL1 mRNA expression in TGF‐β1‐stimulated DCs. * p < 0.05. Each experiment was independently repeated three times (biological replicates). Within each experiment, three technical replicates were performed per condition and averaged prior to statistical analysis. Each data point represents one independent biological replicate. Data are shown as mean ± SEM. DAPI, 4′,6‐diamidino‐2‐phenylindole; DCs, dendritic cells; MARCKSL1, myristoylated alanine‐rich C‐kinase substrate‐like 1; TGF‐β1, transforming growth factor beta 1; CD11c, cluster of differentiation 11c.

Article Snippet: Cells were subsequently stimulated with 5 ng/mL TGF‐β1 (7666‐MB‐005, R&D Systems, Minneapolis, MN, USA) for 24 h. Following stimulation, DCs were washed thrice with phosphate‐buffered saline (PBS) to remove residual cytokines and resuspended in fresh medium.

Techniques: Expressing, Immunofluorescence, Staining, Western Blot, Quantitative RT-PCR

Journal: Wound Repair and Regeneration

Article Title: Dendritic Cell–Associated MARCKSL1 Regulates Fibroblast Differentiation During Wound Healing

doi: 10.1111/wrr.70143

Figure Lengend Snippet: MARCKSL1‐positive DCs promote fibroblast activation. (A) Immunofluorescence staining of co‐cultures of TGF‐β1‐stimulated DCs and fibroblasts. Green: Phalloidin; Red: CD11c; Blue: α‐SMA; White: MARCKSL1; Cyan: DAPI. Scale bar = 10 μm. (B) Western blot analysis of α‐SMA expression in fibroblasts after co‐culture with TGF‐β1‐stimulated DCs. * p < 0.05. (C) RT‐qPCR analysis of Acta2 (α‐SMA) expression in fibroblasts after co‐culture with TGF‐β1‐stimulated DCs. * p < 0.05. (D) Validation of MARCKSL1 knockdown in DCs transfected with pLKO.1‐shMARCKSL1 under TGF‐β1 stimulation. * p < 0.05. (E) Immunofluorescence staining of fibroblast co‐cultures with MARCKSL1‐shRNA‐treated DCs. Green: Phalloidin; Red: CD11c; Blue: α‐SMA; White: MARCKSL1; Cyan: DAPI. Scale bar = 10 μm. (F) Western blot analysis of α‐SMA expression in fibroblasts after co‐culture with MARCKSL1‐shRNA‐treated DCs. * p < 0.05. (G) RT‐qPCR analysis of Acta2 expression in fibroblasts co‐cultured with MARCKSL1‐shRNA‐treated DCs. * p < 0.05. Each experiment was independently repeated three times (biological replicates). Within each experiment, three technical replicates were performed per condition and averaged prior to statistical analysis. Each data point represents one independent biological replicate. Data are shown as mean ± SEM. CD11c, cluster of differentiation 11c; DAPI, 4′,6‐diamidino‐2‐phenylindole; DCs, dendritic cells; MARCKSL1, myristoylated alanine‐rich C‐kinase substrate‐like 1; RT‐qPCR, real‐time quantitative polymerase chain reaction; TGF‐β1, transforming growth factor beta 1; α‐SMA, α‐smooth muscle actin.

Article Snippet: Cells were subsequently stimulated with 5 ng/mL TGF‐β1 (7666‐MB‐005, R&D Systems, Minneapolis, MN, USA) for 24 h. Following stimulation, DCs were washed thrice with phosphate‐buffered saline (PBS) to remove residual cytokines and resuspended in fresh medium.

Techniques: Activation Assay, Immunofluorescence, Staining, Western Blot, Expressing, Co-Culture Assay, Quantitative RT-PCR, Biomarker Discovery, Knockdown, Transfection, shRNA, Cell Culture, Real-time Polymerase Chain Reaction