Fig 1: FGFR1 mediates the inflammatory effects of S100B. (A) FGFR1 mRNA expression levels were increased or decreased in S100B-overexpressing and S100B-siRNA synovial fibroblasts, respectively, as measured by RT-qPCR. (B) FGFR1 protein expression levels were increased or decreased in S100B-overexpressing and S100B-siRNA synovial fibroblasts, respectively, as measured by western blotting. (C) Validation of the FGFR1 knockdown FGFR1, by RT-qPCR. (D) RT-qPCR and (E) western blotting were used to determine FGFR1 expression levels in LPS-simulated S100B overexpressed synovial fibroblasts. Increased FGFR1 expression induced by LPS and S100B overexpression was significantly attenuated by FGFR1 siRNA. (F) TNF-a expression levels were decreased following FGFR1 knockdown in LPS-simulated, S100B-overexpressing synovial fibroblasts. (G) IL-1ß expression levels were decreased following FGFR1 knockdown in LPS-simulated, S100B-overexpressing synovial fibroblasts. **P<0.01 vs. LPS + Empty vector or LPS + Scrambled siRNA; †P<0.05 vs. LPS + Empty vector; $P<0.05 vs. LPS + Scrambled siRNA; #P<0.05 vs. LPS + S100B + Scrambled siRNA. IL-1ß, interleukin 1ß; FGFR, fibroblast growth factor receptor; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA; TNF-a, tumor necrosis factor.
Fig 2: S100B expression levels are increased in patients with OA. (A) Immunohistochemical expression patterns and relative staining intensity quantification of the inflammatory mediators and growth factor in the cartilage tissue of patients with OA and healthy Controls. Correlation analysis between S100B and either (B) TNF-α or (C) IL-1β. IL, interleukin; OA, osteoarthritis; TNF, tumor necrosis factor. *P<0.05 vs. Control.
Fig 3: S100B regulates inflammatory cytokine expression levels in human synovial fibroblasts. (A) S100B overexpression was confirmed by RT-qPCR. (B) Increased TNF-α levels in S100B-overexpressing, LPS-stimulated fibroblast, as measured by ELISA. (C) Increased expression levels of IL-1β in S100B-overexpressing LPS-stimulated fibroblasts, as measured by ELISA. (D) S100B knockdown was confirmed by RT-qPCR. (E) TNF-α levels were reduced in LPS-stimulated fibroblasts transfected with S100B siRNA. (F) IL-1β levels were reduced in LPS-stimulated fibroblasts transfected with S100B siRNA. *P<0.05 vs. Empty vector or Scrambled siRNA controls; **P<0.01 vs. Empty vector; #P<0.05 vs. LPS group; †P<0.05 vs. LPS + Empty vector; ‡P<0.05 vs. LPS + scramble siRNA. IL, interleukin; LPS, lipopolysaccharide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; siRNA, small interfering RNA; TNF-α, tumor necrosis factor.
Fig 4: H&E staining and immunohistochemical analysis of the regenerated nerves. A1–D1) H&E staining in the autograft, PCL nerve conduit, HA‐Pam‐Mg hydrogel, and MeHA hydrogel group samples. A2–D2) Immunohistochemical analysis of NF200 staining in all four groups. A3–D3) Immunohistochemical analysis of S100 staining in all four groups. E) Diameter of the regenerated nerves (µm; n = 4). F) Percentage of NF200‐positive area (n = 4). G: percentage of S100‐positive area (n = 4). Data are expressed as mean ± standard deviation. Statistical analysis is evaluated by one‐way ANOVA and Tukey's post‐hoc test. *P < 0.05; **P < 0.01. H&E, hematoxylin and eosin; HA, hyaluronic acid; Pam, pamidronate; Mg magnesium; PCL, polycaprolactone.
Fig 5: Experimental Design. The cell culture protocol consists of three main steps: Step 1, Stem-cell reprogramming, requires 2 to 4mo to be completed. Blood samples were obtained from 3 PwMS and 3 age- and sex-matched controls, and used for isolation of PBMCs. The PBMCs were cultured and nucleofected with episomal vectors containing the Yamanaka factors required for cell reprogramming into iPSCs. To characterize the iPSCs, we used Oct3/4, SSEA-4 and SOX2 markers; Step 2, Neural commitment, lasts approximately 2mo. iPSCs were cultured in suspension as EBs, exposed to neural differentiation factors and then plated to form neural rosettes. Rosettes were manually picked, expanded and dissociated, giving rise to the NPCs. Here, we characterized NPCs using Nestin and SOX1 markers; Step 3, Astrocyte differentiation, takes about 1mo and consists of NPC expansion and culture in suspension as neurospheres, exposed to astrocyte differentiation factors. Neurospheres are then plated and give rise to astrocytes, which were the cells used for further experiments in this study. We used GFAP, Vimentin, S100-ß and AQP4 as astrocyte markers. The figure was generated using Biorender.
Supplier Page from MilliporeSigma for Anti-S100B antibody produced in rabbit