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: Breast Cancer Research : BCR

Article Title: OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

doi: 10.1186/s13058-018-0971-5

Figure Lengend Snippet: Oncostatin M (OSM) is highly expressed in ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC). a To detect the presence of OSM in breast cancer tissue, histological microarrays from 72 patients with breast cancer were stained with human OSM antibody by IHC. Twelve patients had in situ DCIS, 54 patients had nonmetastatic IDC, and 16 patients had IDC with metastasis to lymph nodes (see Additional file ). The results showed that normal adjacent tissue expresses little OSM but that OSM is highly expressed in DCIS and IDC. Secondary antibody alone did not produce any background signals. b Intensity quantification of OSM stained tissues. Mean staining intensity for DCIS (2.00) and IDC (1.66) tissues was significantly higher than that of normal adjacent tissue (1.33) and metastatic tissue (1.24). There was no statistically significant difference between normal and metastatic tissue. Multiple cores from the same patients were averaged. Data are expressed as mean ± SD. * p < 0.05 by one-way analysis of variance with Tukey’s multiple comparisons test

Article Snippet: The serum was then diluted 1:3 in PBS and used in an hOSM ELISA (catalogue number DY295; R&D Systems, Minneapolis MN, USA), which was performed in accordance with the manufacturer’s instructions.

Techniques: In Situ, Staining

Journal: Breast Cancer Research : BCR

Article Title: OSM potentiates preintravasation events, increases CTC counts, and promotes breast cancer metastasis to the lung

doi: 10.1186/s13058-018-0971-5

Figure Lengend Snippet: Oncostatin M (OSM) produced by tetracycline (TET)-inducible MDA-MB-231 (MDA TO/OSM ) cell MDA TO/OSM ) tumors increase metastasis and decrease survival. a MDA TO/OSM human breast cancer cells were treated with (+TET) or without TET (−TET), and the resultant conditioned media (CM) from the treated cells were applied to parental MDA-MB-231, MDA-MB-231-LUC, and T47D cells. Left : The activity of OSM accumulated in the CM was compared with commercially obtained recombinant human OSM (rhOSM) (25 ng/ml). There was no significant difference between OSM produced by MDA TO/OSM versus rhOSM versus its ability to induce pSTAT3. Middle : Western blot analysis depicting that CM produced by MDA TO/OSM cells stimulated with TET contain OSM. Right : Enzyme-linked immunosorbent assay (ELISA) analysis showed that CM from TET-treated MDA TO/OSM cells contain 10.1 ng/ml of hOSM. b Left : Animals with MDA TO/OSM tumors were given drinking water with or without TET, and whole blood was collected at the experimental endpoint. After allowing the blood to clot and serum was separated by centrifugation, the resultant serum OSM levels were measured by ELISA. Animals with MDA TO/OSM tumors with drinking water containing TET had 67-fold higher serum OSM levels. Center : Platelet counts were higher in +TET MDA TO/OSM tumor-bearing mice than in −TET mice. Right : +TET MDA TO/OSM tumor-bearing mice had lower body weight than −TET mice. c Animals with MDA TO/OSM tumors were given drinking water containing TET for 1 week, and their lung metastasis levels were assessed by ex vivo bioluminescence imaging. Left : Representative ex vivo bioluminescence image. Right : Average radiance analysis of the ex vivo bioluminescence imaging in photons per second per square centimeter per square radian (p/s/cm 2 /sr). Animals with MDA TO/OSM tumors +TET had a fivefold higher bioluminescent radiance than −TET mice (−TET, n = 3; +TET, n = 6). Data are expressed as mean ± SEM. d Kaplan-Meier survival curve for mice with MDA TO/OSM tumors ± TET. Mice that did not receive TET survived, on average, 11 days longer (−TET, n = 9; +TET, n = 10). *** p < 0.001 by log-rank test. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 by two-tailed t test or one-way analysis of variance with Tukey’s posttest where appropriate

Article Snippet: The serum was then diluted 1:3 in PBS and used in an hOSM ELISA (catalogue number DY295; R&D Systems, Minneapolis MN, USA), which was performed in accordance with the manufacturer’s instructions.

Techniques: Produced, Activity Assay, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Centrifugation, Ex Vivo, Imaging, Two Tailed Test