Fig. 3. αV integrins mediate mechanical stress-induced TGFβ activation in chondrocytes.

a Double immunofluorescence staining of αVβ6 (red) and pSmad2/3 (green) in AC of human knee joints. HC healthy control, OA-M OA specimen with minimal cartilage degeneration, OA-S OA specimen with severe cartilage degeneration. b Quantitative analysis of αVβ6 and pSmad2/3 double staining in a. n = 10 biologically independent specimens, *P < 0.05 in comparison to αVβ6 and pSmad2/3 double-positive cells. Data are presented as mean values + /− SEM. Data were analyzed using one-way ANOVA LSD post hoc test. PS+: pSmad2/3+, AV+: αVβ6+. c Immunofluorescence staining of αVβ6 (red) and pSmad2/3 (green) in mouse AC harvested 1 month after ACL-T or sham surgery. Nuclei were labeled with DAPI (blue). d–f Quantitative analysis of the percentage of pSmad2/3⁺ cells (d) and αVβ6⁺ cells (e) in total chondrocytes and the ratio of αVβ6⁺ cells in pSmad2/3⁺ cells (f). n = 3 biologically independent animals, data are presented as mean values + /− SEM. Data were analyzed by two-tailed unpaired t test. g Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line. The cells were cultured in the free well or subjected to shear stress (6.58 dynes/cm²) for 48 h. The gene expression of αV integrin or α5 was knocked down using siRNA. Source data are provided as a Source data file. h ELISA of TGFβ in the conditional medium of the SV40 chondrocyte culture. The cells were cultured in the free well or subjected to shear stress (6.58 dynes/cm²) for 48 h. n = 3 independent experiments, data are presented as mean values + /− SEM. Data were analyzed using one-way ANOVA LSD post hoc test. Ctr or si-alphaV: cells treated with siRNA of control or αV integrin, respectively. i Immunofluorescence staining of pSmad2/3 and Smad2 in the SV40 cells with or without shear stress. Ctr or si-alphaV cells treated with siRNA of control or αV integrin, respectively. j Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line subjected to shear stress at different speeds. k Western blot of αV integrin and pSmad2 protein in SV40 chondrocyte cell line. Shear shear stress, hTGFβ recombinant human TGFβ1 (2 ng/ml), SB SB505124, TβRI selective inhibitor (1 µM). All the in vitro experiments were repeated three times independently. Source data are provided as a Source data file.