Figure 5.
HA-CD44 interaction mediated contractility through ROCK pathway. Human PDL cells were cultured on PDMS-coated glass coverslips. Representative fluorescent images of a fixed and stained (a) control, (b) HA-treated, (c) Y27-treated, (d) Y27+ HA-treated, (e) ML7-treated, (f) ML7+ HA-treated human PDL cells (red: vinculin; green: actin; blue: nucleus). (g) Y27 (P = 2.27e-3) and ML7 (P = 3.57e-3) treatments caused a reduction in stress fiber density relative to control PDL cells; however, treating the cells with exogenous HA caused an increase in stress fiber density for controls (P = 1.72e-3) and ML7-treated PDL cells (P = 0.019), but not in Y27-treated cells. (h) Regarding the focal adhesion assays, Y27 (population size of experimental repetition: n1 = 7, n2 = 5, n3 = 13; P = 9.63e-4) and ML7 (n1 = 9, n2 = 8, n3 = 11; P = 2.67e-3) treatments caused focal adhesions to decrease in size relative to control human PDL cells (n1 = 15, n2 = 13, n3 = 14); however, treating the cells with exogenous HA caused an increase in focal adhesion size for controls (n1 = 29, n2 = 20, n3 = 17; P = 2.71e-3) and ML7-treated PDL cells (n1 = 14, n2 = 11, n3 = 8; P = 0.039), but not Y27-treated cells (n1 = 7, n2 = 6, n3 = 10). (i) After stimulating with HA (n1 = 5, n2 = 12, n3 = 2; P = 4.44e-4), we found a significant increase in traction forces compared to untreated controls (n1 = 7, n2 = 8, n3 = 4). Conversely, we found that after treating cells with Y27 (n1 = 6, n2 = 11, n3 = 4; P = 0.020) and ML7 (n1 = 9, n2 = 3, n3 = 7; P = 7.66e-3), we found a significant loss in traction forces. After adding exogenous HA to Y27-treated (n1 = 6, n2 = 8, n3 = 7) or ML7-treated (n1 = 9, n2 = 7, n3 = 7) PDL cells, we observed a significant increase in traction force only in ML7-treated cells (P = 0.028) and not Y27-treated cells. Data shown as average ± SEM from three experimental replicates. Asterisk indicates P < 0.05 (one-way ANOVA with a Bonferroni’s and Fischer post-hoc adjustment).