Figure 4.

LRG1 promotes fibrosis in primary synovial cells. Western Blot (WB) and agarose gel images were taken by Chemidoc (Bio-Rad, Hercules, CA, USA), and densitometry analysis of all the images was carried out using Image Lab V6 (Bio-Rad). All graphs were generated and the statistical analysis carried out using GraphPad Prism (version 8.4.686). Data are presented as the mean ± standard deviation (SD). Paired Student’s t-test was applied for groups comparisons in (B) to show statistical significance, while one-way ANOVA was used for group comparisons in (C, E, F). (A) Western blot image showing LRG1 knockdown using siRNA (50 nM) in primary fibroblast-like cells isolated from OA synovium. The upper panel shows the LRG1 expression level with the transfection reagent for 48 h, while the lower panel shows GAPDH as the loading control in the respective sample. (B) Bar graph plotted using the densitometric values after the analysis of bands depicting the 60% downregulation of the LRG1 expression after knockdown using siRNA. (C) Bar graph showing the mRNA level of COMP after the knockdown of LRG1 in OA primary cells compared to the vehicle control group. (D) PCR products run on 1% agarose gel for visualization of the mRNA expression levels of IL-6 and COMP after treatment with siRNA. (E) Bar graph showing the mRNA level of LRG1 after knockdown. (F) Bar graph showing the mRNA level of the inflammatory marker IL-6 after the knockdown of LRG1 compared to the untreated groups. *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001. ns, non-significant; LRG1, leucine-rich alpha-2 glycoprotein; OA, osteoarthritis; COMP, cartilage oligomeric matrix protein; IL-6, interleukin 6; VC, vehicle group; HC, healthy control; GAPDG, glyceraldehyde-3-phosphate dehydrogenase.