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: American Heart Hournal Plus: Cardiology Research and Practice

Article Title: Oncostatin M mediates cardioprotection via angiogenesis in ischemic heart disease

doi: 10.1016/j.ahjo.2023.100331

Figure Lengend Snippet: Recombinant OSM (10 ng/ml for 24 h) upregulated VEGF concentration in the culture supernatant. Experiments were repeated 3 times.

Article Snippet: The cultured HCAEC were treated with 10 ng/ml of recombinant OSM (Cell Signaling Technology #5367) for 24 h. Culture supernatant was collected and centrifuged at 1500 g for 5 min at 4 °C.

Techniques: Recombinant, Concentration Assay

Journal: Immunity & Ageing : I & A

Article Title: Impact of Klotho genotype on lactylation in Alzheimer’s disease and mechanistic insights

doi: 10.1186/s12979-026-00563-x

Figure Lengend Snippet: Klotho genotypes differentially regulate the OSM/JAK1/STAT3 pathway in BV − 2 cells. A mRNA expression levels of OSM, JAK1, and STAT3 measured by qRT − PCR. B Protein levels of OSM, JAK1, STAT3, and phosphorylated STAT3 (p−STAT3) detected by Western blot. Data are presented as mean ± SD ( n = 3). Statistical significance: ** p < 0.01; *** p < 0.001

Article Snippet: Thereafter, we blocked the membranes with 5% skim milk for 1 h. Post-blocking, we incubated those membranes with primary antibodies targeting Aβ (A17911, Abclonal, Wuhan, China), Klotho (bs-2925R, Bioss, Beijing, China), L-lactyl (PTM-1401, PTM, Hangzhou, China), H4K12la (PTM-1401RM, PTM), OSM (A8705, Abclonal), JAK1 (A18323, Abclonal), STAT3 (A16975, Abclonal), p-STAT3 (AP0070, Abclonal), or GAPDH (A19056, Abclonal) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Immunity & Ageing : I & A

Article Title: Impact of Klotho genotype on lactylation in Alzheimer’s disease and mechanistic insights

doi: 10.1186/s12979-026-00563-x

Figure Lengend Snippet: Klotho genotypes modulate OSM/JAK1/STAT3 signaling and lactylation in primary microglia. A mRNA expression levels of OSM, JAK1, STAT3 and Klotho detected by qRT − PCR. B Protein levels of OSM, JAK1, STAT3, p−STAT3, L−Lactyl and H4k12la detected by Western blot. Data are presented as mean ± SD ( n = 3). Statistical significance: * p < 0.05; *** p < 0.001; # p < 0.05; ## p < 0.01; ### p < 0.001; & p < 0.05; && p < 0.01; &&& p < 0.01; ^^ p < 0.01; ^^^ p < 0.001

Article Snippet: Thereafter, we blocked the membranes with 5% skim milk for 1 h. Post-blocking, we incubated those membranes with primary antibodies targeting Aβ (A17911, Abclonal, Wuhan, China), Klotho (bs-2925R, Bioss, Beijing, China), L-lactyl (PTM-1401, PTM, Hangzhou, China), H4K12la (PTM-1401RM, PTM), OSM (A8705, Abclonal), JAK1 (A18323, Abclonal), STAT3 (A16975, Abclonal), p-STAT3 (AP0070, Abclonal), or GAPDH (A19056, Abclonal) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Immunity & Ageing : I & A

Article Title: Impact of Klotho genotype on lactylation in Alzheimer’s disease and mechanistic insights

doi: 10.1186/s12979-026-00563-x

Figure Lengend Snippet: Klotho genotypes influence OSM/JAK1/STAT3 signaling and lactylation in BV − 2 cells. A mRNA expression levels of OSM, JAK1, STAT3 and Klotho detected by qRT − PCR. B Protein levels of OSM, JAK1, STAT3, p−STAT3, L−Lactyl and H4k12la detected by Western blot. Data are presented as mean ± SD ( n = 3). Statistical significance: * p < 0.05; *** p < 0.001; ### p < 0.001; & p < 0.05; && p < 0.01; &&& p < 0.01; ^ p < 0.05; ^^ p < 0.01; ^^^ p < 0.001

Article Snippet: Thereafter, we blocked the membranes with 5% skim milk for 1 h. Post-blocking, we incubated those membranes with primary antibodies targeting Aβ (A17911, Abclonal, Wuhan, China), Klotho (bs-2925R, Bioss, Beijing, China), L-lactyl (PTM-1401, PTM, Hangzhou, China), H4K12la (PTM-1401RM, PTM), OSM (A8705, Abclonal), JAK1 (A18323, Abclonal), STAT3 (A16975, Abclonal), p-STAT3 (AP0070, Abclonal), or GAPDH (A19056, Abclonal) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Immunity & Ageing : I & A

Article Title: Impact of Klotho genotype on lactylation in Alzheimer’s disease and mechanistic insights

doi: 10.1186/s12979-026-00563-x

Figure Lengend Snippet: Molecular effects of OSM knockdown in APP/PS1 mouse brain. A mRNA expression levels of Klotho, OSM, JAK1, and STAT3 measured by qRT − PCR. B Protein levels of OSM, AK1, STAT3, p−STAT3, L−Lactyl and H4k12la detected by Western blot. C Concentrations of TNF − α, IL−1β, iNOS, and IL − 10 assessed by ELISA. C Expression levels of were detected by ELISA. Data are presented as mean ± SD ( n = 3). Statistical significance: * p < 0.05; *** p < 0.001; ### p < 0.001; && p < 0.01; &&& p < 0.001; ^ p < 0.05; ^^ p < 0.01; ^^^ p < 0.001

Article Snippet: Thereafter, we blocked the membranes with 5% skim milk for 1 h. Post-blocking, we incubated those membranes with primary antibodies targeting Aβ (A17911, Abclonal, Wuhan, China), Klotho (bs-2925R, Bioss, Beijing, China), L-lactyl (PTM-1401, PTM, Hangzhou, China), H4K12la (PTM-1401RM, PTM), OSM (A8705, Abclonal), JAK1 (A18323, Abclonal), STAT3 (A16975, Abclonal), p-STAT3 (AP0070, Abclonal), or GAPDH (A19056, Abclonal) overnight at 4°C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 2. Activity of selected aptamer clones in a HEPG2 re- porter gene cell line. OSM signaling via the OSM receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Aptamer clones ADR58 (L), 120 (E), and 152 (M) have affinities of ,10 nM for OSM; ADR147 (f) is a negative control, and ADR66 (Œ) is an aptamer with an affinity .1000 nM for OSM. Aptamers were preincubated with 2 ng/ml OSM (which is approximately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The experiment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Activity Assay, Clone Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 6. Effect of aptamers on LIF- and OSM-induced interleu- kin 6 release from KB cells. Shown are ADR58 (f) and ADR147 control (Œ). Aptamers were preincubated with either 1 ng/ml LIF (a) or 1 ng/ml OSM (b). The mixture was incubated with KB cells for 24 h. Interleukin 6 levels in the cell culture medium were then measured by ELISA. The experiment was carried out in triplicate. Error bars repre- sent S.D. The concentrations of LIF and OSM used were at the approx- imate ED50 for this assay. In the absence of aptamer, the IL-6 release induced by 1 ng/ml LIF was 1113 6 7.5 pg/ml, and the release induced by OSM was 4702 6 67 pg/ml. From cells incubated with medium alone, the IL-6 release was 49 6 9 pg/ml.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 7. Effect of aptamer on IL-6 signaling in the HEPG2 re- porter gene cell line. IL-6 signaling via the IL-6 receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Shown are ADR58 (f) and ADR147 control (Œ). Aptamer and control RNA were preincubated with 4 ng/ml IL-6 (which is approx- imately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of IL-6/aptamer mixture to the HEPG2 reporter cell line. In the absence of aptamer, cells incu- bated with IL-6 gave an A of 0.3, and in cells incubated with medium alone, the A was 0.06. ADR58 inhibition of OSM-induced secreted alkaline phosphatase (M) was included as a positive control. The exper- iment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Inhibition, Positive Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 9. Functional activity of truncated ADR58. a, effect of trun- cated ADR58 on OSM signaling via the OSM receptor in the HEPG2 reporter cell line, measured by production of secreted alkaline phospha- tase (f). Aptamers were preincubated with 2 ng/ml OSM, and produc- tion of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The A from cells incubated with OSM in the absence of aptamer was 0.443, and that from cells incubated with medium alone was 0.035. b, activity of trun- cated ADR58 in OSM-gp130 ELISA (f). The ability of truncated ADR58 to block the interaction of microtiter plate-immobilized OSM with gp130 is shown. gp130-IgG-Fc fusion binding was quantitated with an alkaline phosphatase-linked anti-IgG antibody. The experiment was carried out in triplicate. Error bars represent S.D. At the concentration of gp130-IgG-Fc used, the OSM was approximately at the ED50. The A values are corrected for nonspecific binding (where either OSM or gp130-IgG-Fc is excluded; A 5 0.06). The A in the presence of OSM and gp130-IgG-Fc but without aptamer was 1.29 6 0.3.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Concentration Assay