Fig 1: iOPN promoted STAT1 deubiquitination. a 293T cells were transfected with Myc-iOPN expression plasmid together with expression plasmid of Flag-STAT1 or Vector-STAT1, followed by IP with anti-Myc antibody and western blot with anti-Flag antibody or adverse. Input, western blot of whole-cell lysate with the indicated antibodies. b MSCs were treated with or without TNF-α plus IFN-γ (10 ng/mL) for 24 h, followed by IP with anti-STAT1 antibody and western blot with anti-OPN antibody, IP with anti-OPN antibody and western blot with anti-STAT1 antibody. Input, western blot of whole-cell lysate with the indicated antibodies. c Fluorescent images of 293T cells transfected with Myc-iOPN together with Flag-STAT1. Nuclei, Myc-iOPN and Flag-STAT1 were labeled with DAPI (blue), antibody to Myc tag (green) and antibody to Flag tag (red), respectively. d MSCs were stimulated with TNF-α plus IFN-γ (10 ng/mL) for different periods of time, followed by immunoprecipitation with anti-STAT1 antibody and then blotted with anti-ubiquitin antibody. Next, lysates were immunoblotted with different antibodies, followed by band visualization. e Vector-MSCs and iOPN-MSCs or WT-MSCs and OPN−/−-MSCs were treated with or without TNF-α plus IFN-γ at the indicated concentration for 24 h, followed by immunoprecipitation with anti-STAT1 antibody and then blotted with the anti-Ubiquitin antibody. The lysates were immunoblotted with different antibodies. f 293T cells transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Flag antibody and then blotted with anti-HA antibody. Input, western blot of whole-cell lysate with the indicated antibodies. The data are representative of three biological replicates. All Full-length blots are presented in Additional file 1: Fig. 6a,b and d–f
Fig 2: iOPN was downregulated by IFN-γ-induced activation of STAT1 in MSCs. a and b Murine MSCs were treated with IFN-γ or TNF-α at the indicated concentration for 24 h. mRNA and protein of MSCs were collected. OPN expression was determined at the mRNA and protein levels by quantitative real-time PCR and immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 7a. c Neutralizing antibodies against IFN-γ, TNF-α or both were added to the medium of MSCs stimulated with IFN-γ or TNF-α. OPN expression was determined by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 7c. d–e The STAT1 inhibitor fludarabine (Flu, 2 μM) was added to the culture medium of MSCs with TNF-α plus IFN-γ (10 ng/mL) for 24 h. The expression of OPN and phosphorylation of STAT1 at Tyr701 were determined by immunoblotting analysis (d). Full-length blots are presented in Additional file 1: Fig. 7d. The mRNA expression of OPN was determined by quantitative real-time PCR (e). f iOPN luciferase activity in 293 T cells transfected with luciferase reporter and indicated expression vectors. g Schematic representation showing a conserved STAT1-binding motif located in the promoter region of OPN genes between − 2,895 and − 2,910 bp. h Enrichment of p-STAT1 at the promoter of OPN was analyzed by ChIP-PCR. i Working model of the regulation of iOPN on the immunosuppressive capacity of MSCs. The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. ns = no significance
Fig 3: iOPN upregulated iNOS expression to promote the immunosuppressive capacity of MSCs. a–h Vector-MSCs and iOPN-MSCs or WT-MSCs, OPN−/−-MSCs and iOPN-OPN−/−-MSCs were treated with TNF-α plus IFN-γ as indicated for 24 h. Protein, mRNA and cells were collected. a and b Expression of Opn and Nos2 was measured by quantitative real-time PCR. c and d Expression of Opn and iNOS was measured by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 4c and d. f Murine OPN−/−-MSCs were transfected with pcDNA3.1-Vector or pcDNA3.1 containing iOPN, and OPN expression was measured by immunoblotting analysis. Full-length blots are presented in Additional file 1: Fig. 4f. e, g The expression of iNOS in MSCs was determined by flow cytometry. h The concentration of nitric oxide (NO) in the culture supernatant was determined by Griess assay. i iOPN-MSCs were pretreated with the iNOS inhibitor LNMMA for 12 h. Irradiated MSCs were cocultured with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD4+ T cells and CD8+ T cells were collected for proliferation analysis by flow cytometry at the end of coculture. j and k Mice were intravenously injected with ConA or fed DSS, untreated, Vector-MSCs, or iOPN-MSCs were transfused every 2 days. LNMMA was intraperitoneally injected into the iOPN-MSC-treated group (n = 5 per group). Serum levels of ALT and AST were measured (j). Representative colonic specimens were observed, and the colonic length was measured (k). The results are representative of three to six independent experiments. Values are shown as the mean ± SEM and statistical significance is indicated as *P < 0.05, **P < 0.01 and ***P < 0.001
Fig 4: OPN could improve the migration, proliferation, and angiogenesis of HUVECs as well as the activation of SOCE, eNOS, and NO release. (A) Stimulation by purified OPN, setting three concentrations graded 0.5, 0.7, and 1.0 μg/mL at three incubated time periods of 12, 24, and 48 h, showed dose- and time-dependent influence on the scratch closure of human umbilical vein endothelial cells (HUVEC) after 24 h. The photographs were shown at a magnification of 40 ×. The statistical analysis indicated the cellular migration function. A CCK-8 assay was used to evaluate the proliferation function of cells. n = 5. (B) The interference of OPN was verified by a qPCR experiment. n = 6. (C) Migration and proliferation functional changes of OPN-siRNA-treated HUVECs. The magnification is 40 ×. n = 5. (D) On the basis of the division of groups in the experiment of migration, another group of OPN-knockdown cells stimulated by OPN was added. Then, the function of HUVECs' angiogenesis in every group was observed. The magnification is 40 ×. n = 5. (E) Measurement of eNOS (n = 6) and NO (n = 5) in each group by ELISA. (F) The expression level of OPN, Orai1, STIM1, eNOS, and p-eNOS in each group was detected by western blotting. The right chart shows the gray scale statistics including the ratio of p-eNOS and eNOS. n = 3. (G) The fluorescence changes of intracellular and extracellular calcium ions in five groups of cells were recorded using a calcium imaging system. F1 is the fluorescence intensity after calcium was added, and F0 is the baseline fluorescence intensity. All the data in the bar graph is presented as mean ± S.E.M. *p < 0.05, **p < 0.01 and ***p < 0.001.
Fig 5: The schematical figure shows the pathophysiological changes and molecular mechanisms of OPN-activated Orai1/STIM1-mediated SOCE during the TTT process. First, VEGF-A, OPN, and NO could be detected in the para-osseous tissues around the bone transport area. By acquiring the Orai1/STIM1-high-expressed tissue in the paraffin blocks using the LCM method and then undergoing protein profile analysis, the key protein OPN was found. Second, in vitro, after being stimulated by OPN, the migration, proliferation, and angiogenesis of HUVECs were observed to be enhanced. The activation of Orai1/STIM1 might increase the activity of eNOS and its effect on releasing NO, which could diastole the vascular smooth muscle to increase local blood flow. Therefore, OPN could improve the function of VECs to increase the local volume of blood flow and ultimately benefit the local vessels' growth and proliferation. Third, in vivo functional experimental verified that OPN activates SOCE and promotes the healing, formation of granulation tissue, local collagen deposition, and vascular formation on the skin wound surface of diabetic mice.
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