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Figure Lengend Snippet: (a-d) Human HaCaT keratinocytes were treated with recombinant OSM (25ng/ml) for 72h. (a) p63 mRNA expression was measured using RT-PCR. Data are expressed as mean ± SEM (n=4). * p <0.05 compared with vehicle. (b) p63 protein expression was measured using Western blot. β-ACTIN was used as a loading control. Representative blot from four independent experiments has been provided. Densitometry quantification of band intensity has been presented. Data are expressed as mean ± SEM (n = 4). * p <0.05 compared with vehicle. (c, d) p63 protein expression was measured using immunocytochemistry. Scale bar, 20μm. Data are expressed as mean ± SEM (n = 8-9). * p <0.05 compared with vehicle. (e-g) Full-thickness (skin and panniculus carnosum) dorsal wounds were created on the male C57bl/6 mice by using a 6-mm biopsy punch. The wounds were stented and treated with recombinant mouse OSM (1.25 μg.15μl −1 .wound − ). Control wounds received vehicle only. (e) p63 mRNA expression in LCM-captured wound-edge keratinocytes from d3 wounds of OSM-treated mice. Data are expressed as mean ± SEM (n = 5-7). * p <0.05 compared with vehicle-treated wounds. (f) Representative images of d3 wound-edge tissue sections from OSM-treated mice stained with p63 (red) and counterstained with DAPI (blue, nuclear). Scale bar, 50μm. (g) Quantification of p63 on d3 post-wounding. Data are expressed as mean ± SEM (n = 4). * p <0.05 compared with vehicle-treated wounds. (h) OSM in human wound fluid (see Supplementary Table 1 for subject demographics) was neutralized with OSM neutralizing antibody (1 μg/ml) for 4h and subjected to ELISA to check the levels of OSM. Data are expressed as mean ± SEM (n = 5). * p <0.05 compared with IgG-treated wound fluids. (i) HaCaT cells were treated with OSM-neutralized wound fluid (10%v/v; 72h) and p63 mRNA expression were measured using RT-PCR. Data are expressed as mean ± SEM (n = 3). * p <0.05 compared with human serum albumin (HSA) treated HaCaT cells. † p <0.05 compared with HaCaT cells exposed to IgG-treated wound fluids. (j) Human HaCaT keratinocytes were transfected with luciferase reporter vector pEZX-PG04 containing promoter for p63 upstream of secreted Gaussia luciferase (Gluc) and secreted alkaline phosphatase (SEAP, endogenous control), followed by treatment with OSM (25ng/ml, 72h). Luciferase activity measured as GLuc/SEAP. Data are expressed as mean ± SEM (n=4). * p <0.05 compared with vehicle. (k-n) Human HaCaT keratinocytes were transfected with si p63 or si Control . (k) p63 protein expression was measured following knockdown of p63 using siRNA for 72h. Data are expressed as mean ± SEM (n = 5). * p <0.05 compared with si Control . (1, m) Knockdown of p63 in keratinocytes using siRNA was followed by treatment with OSM (25ng/ml; 72h) after which cells were subjected to migration assay. Migration of cells was observed at 3h and 6h following removal of inserts. Scale bar, 100μm. Black and red dotted line represents migration under similar conditions in presence of vehicle alone at 3h and 6h respectively. Data are expressed as mean ± SEM (n = 4). * p <0.05 compared with si Control treated with OSM. (n) Cells were activated with OSM (25ng/ml) for 72h after knockdown of p63 in keratinocytes using siRNA. Cell proliferation was measured under low (0.2%) serum using CyQUANT. Black dotted line represents proliferation under similar conditions in presence of vehicle alone. Data are expressed as mean ± SEM (n = 5). * p <0.05 compared with si Control treated with OSM.
Article Snippet: The glycation of OSM was determined using Methylglyoxal (MG) Competitive ELISA (Cell Biolabs, San Diego CA) as per manufacturer’s instructions.
Techniques: Recombinant, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Immunocytochemistry, Staining, Enzyme-linked Immunosorbent Assay, Transfection, Luciferase, Plasmid Preparation, Activity Assay, Knockdown, Migration, CyQUANT Assay
