Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 1. Oncostatin M (OSM)–induced expression of CCL13. A and B, Normal dermal fibro- blasts (nDF), normal lung fibroblasts (nLF), normal cervical fibroblasts (nCF), and normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM for the indicated periods of time. A, Levels of CCL2, CCL13, and CCL11 mRNA were determined by RNase protection assay. Results are representative of 3 independent experiments. B, The amounts of CCL13 and CCL2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 3). P 0.05 versus control. C, Primary human blood neutrophils were stimulated with 10 ng/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) for 3 hours. The amounts of OSM in culture supernatants were determined by ELISA. Values are the mean SD (n 3). P 0.05 versus control. D, Culture supernatants (SNs) from untreated neutrophils or neutrophils stimulated with GM-CSF for 3 hours were transferred onto normal SFs. The contribution of OSM was evaluated by additional application of blocking antibodies to OSM or control antibodies (ctrl. Ab). The amounts of CCL13 produced in normal SF culture supernatants were then determined by ELISA. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus antibody control.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Rnase Protection Assay, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Produced

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 2. Significance of OSM and other key cytokines involved in rheumatoid arthritis (RA) pathogenesis for CCL13 expression. A, Normal SFs were treated for 2 hours with 10 ng/ml of OSM, 200 units/ml of interleukin-6 (IL-6)/0.5 g soluble IL-6 receptor (sIL-6R), 10 ng/ml of IL-1, or 10 ng/ml of tumor necrosis factor (TNF). CCL13 mRNA levels were quantified, normalized to L32 levels, and compared with untreated cells. Values are the mean SD (n 4). P 0.05 versus specific control. B, Normal SFs were stimulated as described in A for 2 hours or 12 hours. Values are the mean SD (n 4). P 0.05 versus time-specific control. C, Normal SFs were treated for 3 hours with OSM, alone or in combination with IL-6/sIL-6R, IL-1, or TNF (at the concentrations described in A). Values are the mean SD (n 4). P 0.05 versus control. D, RASFs were stimulated with cytokines as described in A for 3 hours. Values are the mean SD (n 4). P 0.05 versus normal SF control; d P 0.05 versus control. E, OSM and CCL13 protein in the supernatant of unstimulated fibroblasts was determined by ELISA. Values are the mean SD (n 3). P 0.05 versus normal SF. F and G, Both normal SF and RASF supernatants were treated with OSM and either control or OSM-neutralization antibodies. Normal SFs and RASFs were restimulated with OSM-depleted supernatant for 3 hours. Values are the mean SD (n 3). and # P 0.05 versus control; d P 0.05 versus stimulated control. H, Supernatants of untreated RASFs were treated with control antibody, CCL13 antibody, or both CCL13 and OSM-neutralizing antibodies. Depleted supernatants were added to untreated normal SFs. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus control supernatant; # P 0.05 versus CCL13-depleted supernatant. w/o without; OASF osteoarthritis SF (see Figure 1 for other definitions).

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 4. Comparison of oncostatin M (OSM)–, interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R)–, IL-1–, and tumor necrosis factor (TNF)–induced signal transduction, showing a potential role of phosphorylated (pY) STAT-5 in CCL13 induction. A, Normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM, 200 units/ml of IL-6/0.5 g of sIL-6R, 10 ng/ml of IL-1, or 10 ng/ml of TNF. Lysates were subjected to Western blot analysis, using antibodies specific for the indicated proteins. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status or with loading control antibodies. B, Normal SFs were stimulated with OSM as indicated. Western blots of lysates were stained with the antibodies indicated. C, Normal SFs, normal cervical fibroblasts (nCF), normal lung fibroblasts (nLF), and normal dermal fibroblasts (nDF) were stimulated with 10 ng/ml of OSM. Cell lysates were analyzed as described in B. D, Normal SFs were transfected with control or STAT-5 small interfering RNA (siSTAT5). Forty-eight hours later, cells were stimulated with 10 ng/ml of OSM for 3 hours. CCL13 and CCL2 protein in normal SF culture supernatants was measured by enzyme-linked immunosorbent assay. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus stimulated siRNA control; w/o without.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Comparison, Transduction, Western Blot, Activation Assay, Control, Staining, Transfection, Small Interfering RNA, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 5. Role of OSM-induced p38 activation in the stabilization of CCL13 mRNA through inhibition of tristetraprolin (TTP). A and C, Normal SFs and rheumatoid arthritis SFs (RASFs), respectively, were pretreated with 10 M AG490, 10 M SB202190, 10 M U0126, 10 M SP600125, 1 M JAK inhibitor I (JI-1), or the solvent DMSO, and then exposed to 10 ng/ml of OSM for 3 hours. Subsequently, CCL13 protein was quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 4). and # P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. B, Western blots from treated normal SFs were analyzed using the indicated antibodies. D, Normal SFs were stimulated with 10 ng/ml of OSM for 30 minutes. Then, cells were washed and cultivated for an additional 20 minutes in OSM-free medium containing actinomycin D (4 M). SB202190 (10 M) was added to the medium before cells were restimulated with OSM for the indicated periods of time. CCL13 and GAPDH mRNA levels were analyzed by reverse transcription–polymerase chain reaction. One of 3 representative independent experiments is shown. E, Normal SFs were transfected with TTP or control siRNA. Forty-eight hours later, cells were pretreated with 10 M SB202190 or DMSO for 20 minutes and then stimulated with 10 ng/ml of OSM for 3 hours. CCL13 was measured by ELISA. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. See Figure 4 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Activation Assay, Inhibition, Solvent, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Transfection

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 6. Contribution of STAT-5 and ERK-1/2 to CCL13 expres- sion. RASFs were transfected with STAT-5 or control small interfering RNA (siRNA). Forty-eight hours later, cells were preincubated with DMSO or U0126 for 30 minutes, prior to stimulation with 10 ng/ml of OSM for 3 hours. CCL13 protein was measured in culture superna- tants of RASFs, by ELISA. Values are the mean SD (n 4). P 0.05 versus unstimulated control; # P 0.05 versus OSM- stimulated DMSO control; d P 0.05 versus stimulated siRNA control. Inset, Knockdown efficiency was controlled by Western blot analysis of lysates, using a specific antiserum against tyrosine- phosphorylated STAT-5 (pY-STAT5). After stripping, the blots were reprobed with antiserum recognizing STAT-3. See Figure 5 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Transfection, Control, Small Interfering RNA, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Stripping Membranes

Journal: Communications Biology

Article Title: Oncostatin M induces epigenetic reprogramming in renal cell carcinoma-associated endothelial cells

doi: 10.1038/s42003-025-08907-x

Figure Lengend Snippet: a Left: HMEC-1 cells, a human microvascular EC cell line, were treated with recombinant human OSM (hOSM) at 10 ng/ml for 48 h. Total mRNAs of OSM-treated HMEC-1 and the untreated counterpart (CT) were harvested and analyzed by mRNA sequencing. Right: Heatmap of normalized gene-expression levels (TPM, transcripts per million; Supplementary Data ) shows distinct gene expression patterns between untreated control (CT) and OSM-treated samples. Each column represents one individual sample. b Representative genes upregulated in OSM-treated ECs also identified previously as OSM-responsive genes in Vhlh KO kidney . c Biological process enrichment analysis shows enriched pathways in the OSM-treated EC transcriptome that underlie the phenotypes of ECs observed in vivo. NES: normalized enrichment score. d Gene set enrichment analysis (GSEA) shows Histone modification as the most significantly induced. e GSEA identifies “Histone modification” and “Histone 3 acetylation” as significantly enriched in OSM-treated vs CT groups. f Western blotting of different acetylation sites of histone 3 (H3K14ac, H3K9ac, H3K18ac, and H3K27ac) of OSM-treated ECs and their untreated counterparts was performed to validate the acetylation of histone H3 shown in ( e ). Statistical testing was performed by a two-tailed paired Student’s t -test. ns no significance; ** P ≤ 0.01. Error bars show the standard deviation. Only acetylation at K14 was significantly induced by OSM. Uncropped blots are shown in Fig. . The source data points for quantification are provided in Supplementary Data .

Article Snippet: In the neutralizing OSM experiment, tumor-bearing mice were treated with 100 μg anti-human OSM antibodies (R&D System, MAB295-500) or IgG control (BioXCell, BE0085) once a week (for a total of 4 weeks) by I.V. injection.

Techniques: Recombinant, Sequencing, Gene Expression, Control, In Vivo, Modification, Western Blot, Two Tailed Test, Standard Deviation

Journal: Communications Biology

Article Title: Oncostatin M induces epigenetic reprogramming in renal cell carcinoma-associated endothelial cells

doi: 10.1038/s42003-025-08907-x

Figure Lengend Snippet: a Heatmap shows the expression of histone acetylation/deacetylation enzyme genes found in hOSM-treated (OSM) and untreated (CT) HMEC-1 transcriptomes. The order is arranged according to Log fold change (FC). 7/9 upregulated genes encode acetyltransferases (in red), while 8/9 downregulated genes encode deacetylases (in blue). Each column represents one individual sample. b The volcano plot based on -Log 10 ( P value) and Log (FC) of HAT and HDAC genes shown in ( a ). Top three significantly upregulated genes are indicated. c Western blotting of selected HAT and HDAC genes to validate the mRNA results in ( a ). Uncropped blots are shown in Fig. . Statistical testing was performed by a two-tailed paired Student’s t -test. ns no significance; ** P ≤ 0.01; * P < 0.05. Error bars show the standard deviation. d Double staining for KAT6B (green) and CD31 (red) in human ccRCC tissue. Colocalization of KAT6B and CD31 is found in peritumor (Peri) and ccRCC tissues but not in normal adjacent tissue (NAT). Quantification of KAT6B intensity is shown on the right. NAT ( n = 17), Peri ( n = 9), ccRCC ( n = 14). Statistical testing was performed by one-way ANOVA with Tukey’s post hoc test. *** P ≤ 0.001. Error bars show the standard deviation. Scale bars are 20 μm. KAT6B intensity was quantified by CellProfiler (version 4.2.5) per the supplier’s instructions. e Kidney tissues from WT or Vhlh KO mice were harvested, homogenized, and selected for primary ECs (ADD-7 − CD45 - CD31 + ). Primary ECs from WT (WT-EC) or Vhlh KO (Vhlh KO -EC) tissue were then cultured on a gelatin-coated culture dish. These primary ECs were propagated for three passages over 6 days. f Proteins of each passage of primary ECs from WT or Vhlh KO were collected for Western blotting for detection of Kat6b, H3K14ac, Icam-1, and Snail1. Uncropped blots are shown in Fig. . Statistical testing was performed by a two-tailed paired Student’s t -test. *** P ≤ 0.001; ** P ≤ 0.01; * P ≤ 0.05. Error bars show the standard deviation. Upregulation of OSM-induced markers are sustained for three passages over 6 days. g The primary ECs from WT and Vhlh KO as shown in ( e ) were cultured for three passages over 6 days and observed under a light microscope. The mesenchymal morphology of Vhlh KO -ECs is maintained. The source data points for quantification are provided in Supplementary Data .

Article Snippet: In the neutralizing OSM experiment, tumor-bearing mice were treated with 100 μg anti-human OSM antibodies (R&D System, MAB295-500) or IgG control (BioXCell, BE0085) once a week (for a total of 4 weeks) by I.V. injection.

Techniques: Expressing, Western Blot, Two Tailed Test, Standard Deviation, Double Staining, Cell Culture, Light Microscopy

Journal: Communications Biology

Article Title: Oncostatin M induces epigenetic reprogramming in renal cell carcinoma-associated endothelial cells

doi: 10.1038/s42003-025-08907-x

Figure Lengend Snippet: a Volcano plot of genes with increased or decreased H3K14ac-containing chromatins in response to hOSM treatment determined by CUT&Tag-Seq of hOSM-treated compared with untreated HMEC-1 cells. Upregulation is defined by Log 2 (FC) ≥1 and P value ≤0.05. Downregulation is defined by Log 2 (FC) ≤−1 and P value ≤0.05. Non-significant genes are defined by P value ≥0.05. b Venn diagram shows the overlapped genes that are significantly altered in mRNA sequencing of hOSM-stimulated vs untreated datasets and CUT&Tag-Seq of H3K14ac-containing genes in hOSM-treated vs untreated datasets. c The Gene set enrichment analysis of commonly upregulated genes identified in ( b ). The identified enriched biological processes are the same as in Fig. . d Peak plots of representative genes that are known OSM targets and are involved in angiogenesis (VEGFA), inflammatory response (IL-6), cell-cell adhesion (ICAM-1), regulation of mesenchymal cell shape (SNAIL1), and hypoxia/glycolysis (HIF-1α) signaling pathways from CUT&Tag-Seq of hOSM-stimulated (red) or untreated (blue) ECs. All show increased presence of H3K14ac upon hOSM stimulation.

Article Snippet: In the neutralizing OSM experiment, tumor-bearing mice were treated with 100 μg anti-human OSM antibodies (R&D System, MAB295-500) or IgG control (BioXCell, BE0085) once a week (for a total of 4 weeks) by I.V. injection.

Techniques: Sequencing, Protein-Protein interactions

Journal: Communications Biology

Article Title: Oncostatin M induces epigenetic reprogramming in renal cell carcinoma-associated endothelial cells

doi: 10.1038/s42003-025-08907-x

Figure Lengend Snippet: a HMEC-1 cells were treated or untreated with recombinant human OSM and with histone acetyltransferase inhibitors (CBP300, a EP300 inhibitor; or PF9363, a KAT6 inhibitor). Total proteins from CT (0 ng hOSM), OSM (10 ng/ml hOSM), CBP300 (10 ng/ml hOSM + 10 μM CBP300), or PF9363 (10 ng/ml hOSM + 30 μM PF9363) were isolated after 48 h. The concentrations of the inhibitors followed the published protocols and were selected empirically. Western blotting was performed for the expression of H3K14ac, H3K9ac, and HIF-1α. Uncropped blots are shown in Fig. . b EC cord formation assay of CT, OSM, CBP300, and PF9363 experimental groups. Prior to seeding onto Matrigel (growth-factor-reduced), samples were labeled with CellTracker Green CMFDA (1 μM). Images were obtained after 6 h using fluorescence microscopy. Scale bars are 200 μm. c Quantification of the experiment in ( b ) by measuring total network length and number of nodes (branching points). OSM significantly induces cord formation compared with the CT group. Treating hOSM-stimulated ECs with KAT6 inhibitor PF9363 reduces the cord formation, while EP300 inhibitor CBP300 has no effect. Each data point represents an independent experiment. d Heatmap of HMEC-1 cells from CT, OSM, PF9363, and CBP300 groups by unsupervised hierarchical clustering based on normalized gene expression levels (TPM, transcripts per million; Supplementary Data ). Heatmap shows that the OSM and CT groups are located in different branches of clusters. EP300 inhibitor (CBP300) has a limited effect on the overall gene expression profile altered by OSM. In contrast, CT and OSM + PF9363 show similarity in hierarchical grouping. In particular, two clusters (Clusters 1 and 5; bracketed) upregulated by hOSM are reverted to the CT levels by KAT6 inhibitor PF9363. Each column represents one individual sample. e Gene set enrichment analysis of HMEC-1 genes in Clusters 1 and 5 of the heatmap shown in ( d ). These clusters compare the PF9363-rescued gene sets (PF9363) with the corresponding clusters in the OSM treatment group and the control (untreated with OSM). These genes represent those reverted by treatment of PF9363 (OSM+inhibitor PF9362) versus the OSM treatment group, to a level similar to the control (CT, untreated with OSM or PF9362). They show gene set enrichment in biological processes similar to those identified as common pathways in hOSM-treated HMEC-1 cells (Fig. ). f Western blotting of proteins from HMEC-1 cells with (shKAT6B-51 and -63) or without (Scr) KAT6B knockdown, treated with (OSM) or without (CT) hOSM, for representative OSM downstream targets. hOSM can induce expression of KAT6B, and the knockdown efficiency is confirmed by the lack of KAT6B protein expression in shKAT6B lines. Correspondingly, H3K14ac expression is induced by hOSM, and the induction is ameliorated in KAT6B knockdown. Conversely, KAT6B knockdown has no effect on the expression of KAT6A and H3K9ac with or without hOSM treatment. Importantly, hOSM can induce the expression of ICAM-1 and SNAIL1, and loss of KAT6B reduces the extent of induction. Uncropped blots are shown in Fig. . This result also validates the specificity of anti-KAT6B and anti-H3K14ac antibodies, as no signal was detected in KAT6B knockdown samples. g The schematic showing how cancer cell invasion through the EC monolayer was evaluated (left panel). The invasive cancer cells (RFP positive cells) across the EC monolayer was observed using fluorescence microscopy. Quantification of invasive cell numbers is shown in the bar graph. Each data point represents one independent experiment. KAT6B knockdown reduces the extent of ccRCC cell invasion. Scale bars are 1 mm. h Cord formation assay to test the angiogenic ability of HMEC-1 treated with (OSM) or without (CT) hOSM, and with or without KAT6B knockdown. HMEC-1 were grown on Matrigel (growth-factor-reduced). Quantification of network length and number of nodes is shown on the right. Each data point represents one independent experiment. hOSM can induce cord formation, and KAT6B knockdown can ameliorate the induction. Scale bars are 200 μm. Statistical testing of Western blotting quantification was performed by a two-tailed paired Student’s t -test. ns no significance; * P ≤ 0.05; ** P ≤ 0.01. Other statistical tests were performed by one-way ANOVA (Tukey post hoc test). *** P ≤ 0.001. Error bars show the standard deviation. The source data points for quantification are provided in Supplementary Data .

Article Snippet: In the neutralizing OSM experiment, tumor-bearing mice were treated with 100 μg anti-human OSM antibodies (R&D System, MAB295-500) or IgG control (BioXCell, BE0085) once a week (for a total of 4 weeks) by I.V. injection.

Techniques: Recombinant, Isolation, Western Blot, Expressing, Tube Formation Assay, Labeling, Fluorescence, Microscopy, Gene Expression, Control, Knockdown, Two Tailed Test, Standard Deviation

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 3. Normal human epidermal keratinocytes (NKs) secrete oncostatin M (OSM) upon keratinocyte growth factor (KGF) stimulation inducing fibroblast activation. (a) The OSM messenger RNA (mRNA) expression analyzed in NKs after 24hours treatment with recombinant KGF compared with untreated NKs and kFCM and sFCM treatment compared with incubation with nFCM. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments with n ¼ 3 for each sample analyzed with analysis of variance (ANOVA) þ Kruskal–Wallis test. (b) OSM protein measured by ELISA secreted by NKs upon direct treatment with recombinant KGF (n ¼ 6) compared with untreated NKs (n ¼ 7) and upon incubation with kFCM and sFCM compared with nFCM (each n ¼ 5). The graph represents mean values of two independent experiments analyzed with ANOVAþ Tukey test. (c) Collagen type I-a1 (ColIa1) and (d) fibroblast activation protein (FAP) mRNA expression in normal fibroblasts (NFs), KFs, and scleroderma fibroblasts (SFs) with and without OSM treatment. (e) Fibroblast migration after treatment with recombinant OSM (10ng ml 1). (f) OSM mRNA expression upon KGF-treatment in NKs transfected with control small interfering RNA (CTRL siRNA) and siRNA against KGFR (each n ¼ 3). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (g) The OSM mRNA expression in keloid tissue (n ¼ 4) and scleroderma tissue (n ¼ 4) compared with normal skin (n ¼ 5). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVA þ Kruskal–Wallis test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Migration, Transfection, Control, Small Interfering RNA, MANN-WHITNEY

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 4. STAT3 is activated in fibroblasts upon keratinocyte growth factor (KGF)–induced oncostatin M (OSM) secretion. Western blot analyis was performed for STAT3 and phosphorylated STAT3 (pSTAT3) using b-actin as a loading control. (a) STAT3 is constitutively expressed in normal fibroblasts (NFs) but phosphorylation only occurs upon direct OSM treatment or treatment with OSM-containing keratinocyte-conditioned media (KCMs) as confirmed by the OSM ELISA in Figure 3c. (b) Direct KGF treatment had no unspecific effect on STAT3 phosphorylation. NFs showed decreased STAT3 phosphorylation when treated with KCM:kFCM:siKGF compared with KCM:kFCM:siCTRL, but increased phosphorylation after treatment KCM:nFCM:KGF compared with KCM:nFCM:mock. (c) pSTAT3 and STAT3 expression status in NFs, keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (d) pSTAT3 and STAT3 expression in NFs treated with rOSM and KCM in the presence or absence of OSM-neutralizing antibodies ( þ antiOSM).

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Western Blot, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 5. STAT3 mediates fibroblast activation. (a) Collagen type I-a1 (ColIa1) expression and (b) fibroblast migration cannot be induced upon oncostatin M (OSM) stimulation when pretreated with the STAT3 inhibitor S3I-201. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (c) The expression of the STAT3 target gene urokinase-type plasminogen activator (uPA) is induced upon direct OSM treatment and upon incubation with KCM (keratinocyte-conditioned medium):kFCM, KCM:sFCM, and KCM:rKGF. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with analysis of variance (ANOVA)þ Kruskal–Wallis test and Mann–Whitney test, respectively. (d) uPA mRNA expression in normal fibroblasts (NFs), keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (e) The induction of fibroblast migration measured upon OSM treatment and incubation with KCM:kFCM or KCM:sFCM is completely suppressed when the uPA expression is decreased by means of small interfering RNA transfection. The graphs in d and e represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (f) The uPA expression is significantly higher in keloid and scleroderma tissue compared with normal skin. The graphs represent mean values of four independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVAþ Kruskal–Wallis test. (g) OSM-induced STAT3-mediated uPA expression can be suppressed by pretreatment with S3I-201. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Migration, MANN-WHITNEY, Incubation, Recombinant, Small Interfering RNA, Transfection