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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

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Figure Lengend Snippet: (a) Human HaCaT keratinocytes were treated with recombinant OSM (25ng/ml) for 72h. pSTAT3/STAT3 was measured in the cell lysate using pSTAT3 and STAT3 ELISA kit. Data are expressed as mean ± SEM (n=6). * p <0.05 compared with vehicle (b) Human HaCaT keratinocytes were transfected with si STAT3 or si Control . STAT3 was measured in the cell lysate using STAT3 ELISA kit. Data are normalized to total protein and expressed as mean ± SEM (n=4). * p <0.05 compared with si Control . (c-e) Human HaCaT keratinocytes were transfected with si STAT3 or si Control followed by treatment with recombinant OSM (25ng/ml; 72h). (c) p63 mRNA expression was measured using RT-PCR. Data are expressed as mean ± SEM (n=6). * p <0.05 compared with si Control . † p 0.05 compared with si Control treated with OSM. (d, e) p63 protein expression. was measured using immunocytochemistry. Scale bar, 20μm. Data are expressed as mean ± SEM (n=6). * p <0.05 compared with si Control . † 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: Expressing, Recombinant, Enzyme-linked Immunosorbent Assay, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Immunocytochemistry

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Figure Lengend Snippet: (a) Levels of OSM in wound fluid (WF) obtained from diabetic and non-diabetic chronic wounds (see Supplementary Table 2 for subject demographics). OSM levels were determined using ELISA and normalized to albumin. Data are mean ± SEM (n = 4). * p <0.05 compared with non-diabetic WF. (b) Full-thickness dorsal wounds were created using a 6-mm biopsy punch on dorsal side of diabetic (Leprdb, db/db) or corresponding non-diabetic controls (heterozygous, Leprdb/+, db/+) mice. The wounds were stented and left to heal by secondary intention. OSM levels in d5 wound-edge tissue was determined using ELISA. Data are mean ± SEM (n = 6). * p <0.05 compared to db/+. (c) Immunoprecipitation (IP) of OSM from d5 wound-edge tissue lysates of diabetic (db/db) or corresponding non-diabetic controls (db/+) mice. The IP was subjected to SDS–PAGE followed by immunoblotting (IB) with anti-AGE antibody. Input represents cell lysates 5% of the lysate not subjected to immunoprecipitation reaction. Data are mean ± SEM (n = 6). * p <0.05 compared to db/+. (d-f) OSM was incubated with Methyl Glyoxal (MGO, 12.5mM, 5d) in presence of aminoguanidine hydrochloride (AG, 25mM, 5d). (d) Glycation was measured using Methylglyoxal Competitive ELISA kit. Data are expressed as mean ± SEM (n = 3-4). * p <0.05 compared with OSM (sham). † p <0.05 compared with OSM treated with MGO without AG. (e, f) The glycated OSM was used to treat human HaCaT keratinocytes (equivalent of 25ng/ml of OSM; 3d). Cells were then subjected to migration assay. Migration of cells was observed at 3h and 6h following removal of inserts. Scale bar, 100μm. Data are expressed as mean ± SEM (n = 3-7). * p <0.05 compared with cells treated with OSM (sham). † p <0.05 compared with cells treated with MGO-treated OSM without AG. (g) p63 protein expression in db/db wound-edge tissues 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 mean ± SEM (n = 5). * p <0.05 compared to db/+. (h) p63 mRNA expression in db/db wound-edge tissues. Data are mean ± SEM (n = 5). * p <0.05 compared to db/+. (i, j) Representative images and quantification of d5 wound-edge tissue sections from db/db and db/+ mice stained with ITGB1 (red) and counterstained with DAPI (blue, nuclear). Scale bar, 20μm. Data are mean ± SEM (n = 3). * p <0.05 compared to db/+. (k) Itgb1 mRNA expression in db/db wound-edge tissues. Data are mean ± SEM (n = 6). * p <0.05 compared to db/+. (l) Representative images of K14-stained d7 wound tissues. Yellow dotted lines represent the non-re-epithelialized region. Scale bar, 500μm. (m) Wound re-epithelialization calculated on d7 post-wounding. Data are expressed as mean ± SEM (n = 4-6). * p <0.05 compared to db/+.

Article Snippet: The glycation of OSM was determined using Methylglyoxal (MG) Competitive ELISA (Cell Biolabs, San Diego CA) as per manufacturer’s instructions.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, SDS Page, Western Blot, Incubation, Competitive ELISA, Expressing, Control, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Rapid serological detection of smooth Brucella strain antibodies in bovine and sheep using a dynamic flow immunochromatographic test

doi: 10.3389/fcimb.2026.1804374

Figure Lengend Snippet: Diagnostic performance of the developed DFICT. (A-C) Receiver operating characteristic (ROC) analysis of DFICT (A) , iELISA (B) , and cELISA (C) for diagnosing brucellosis in bovine and sheep serum. (D-G) Correlation analysis between DFICT and iELISA in bovine (D) and sheep (E) serum, and between DFICT and cELISA in bovine (F) and sheep (G) serum.

Article Snippet: The Brucella antibody competitive ELISA (cELISA) kit was purchased from Eurofins Technologies Ingenasa (Spain).

Techniques: Diagnostic Assay