Figure 2. Mutant TRPV4 had little effect on chondrogenic matrix production.

(A) Wildtype (WT), V620I, and T89I day-28 pellets exhibit similar matrix production shown by staining for sulfated glycosaminoglycans (sGAGs) with Safranin-O and hematoxylin and labeling with immunohistochemistry (IHC) for (B) COL2A1 (C), COL1A1 (D), and COL10A1. Scale bar = 500 µm. Representative images from 3 to 4 differentiations. (E) The sGAG/DNA ratio increased in all three lines from day 14 to 28 of chondrogenesis. There were no differences between lines at each time point. Mean ± standard error of the mean (SEM). n = 11–16 from 3 to 4 independent differentiation experiments. ****p < 0.0001 Statistical significance determined by an ordinary two-way analysis of variance (ANOVA) with Tukey’s post hoc test. (F) There were no differences in the elastic modulus of the matrix at day 28. Mean ± SEM. n = 11–14 from 3 experiments. Statistical significance determined by an ordinary two-way ANOVA with Tukey’s post hoc test.

Figure 2—figure supplement 1. Minor differences in V620I matrix were observed at day 42 of chondrogenesis.

(A) The sulfated glycosaminoglycan (sGAG)/DNA ratio increased in all three lines from day 14 to 28 of chondrogenesis. V620I also had an increase in the sGAG/DNA ratio from day 28 to 42. There were no differences between lines at each time point. Mean ± standard error of the mean (SEM). n = 11–16 from 3 to 4 differentiation experiments. ****p < 0.0001, ^#p < 0.05 to day-28 V620I. Statistical significance determined by an ordinary two-way analysis of variance (ANOVA) with Tukey’s post hoc test. (B) There were no differences in the elastic modulus of the matrix at day 28. Day-42 V620I had a significantly lower elastic modulus than wildtype (WT) and T89I. Mean ± SEM. n = 11–14 from 3 experiments. *p < 0.05, ***p < 0.001 Statistical significance determined by an ordinary two-way ANOVA with Tukey’s post hoc test.