Fig. 5.

Confirmation of increased autophagy and mineralization in EphrinB2-deficient Ocy454 osteocytes, and effect of autophagy on mineralization. a Efnb2 stable knockdown (short hairpin RNA (shRNA) 1 and shRNA 2) in Ocy454 cells differentiated for 7, 11, and 14 days, mean ± SD, 3–6 replicates, representative of three independent cultures; *p < 0.05 vs. vector by Student’s t test. b, c Autophagosomes (LC3 punctae) in Efnb2-deficient Ocy454 cells; bafilomycin (Baf) treatment (50 nM for 2 h) was used to block autophagosome degradation. Data shown are mean ± SD, five replicates, representative of three independent cultures; *p < 0.05; ***p < 0.001 as indicated by one-way analysis of variance (ANOVA). Scale bar = 10 μm d Fold change of LC3-II:I ratio in Efnb2 shRNA knockdown cells treated with chloroquine (CQ) for 4 h compared to basal levels of each shRNA construct. Data are represented as mean ± SEM, three replicates, representative of three independent cultures, *p < 0.05 vs. vector control by one-way ANOVA. e, f Alizarin Red stain for mineralization in Ocy454 cells with vector or Efnb2 shRNA 1 knockdown, grown under mineralizing conditions for 14 days. Data shown are mean ± SD, four replicates, representative of three independent cultures; ***p < 0.001 vs. vector by two-way ANOVA; f Alizarin Red-stained wells at day 14, showing three replicates. g Effect of rapamycin treatment (0.05 nM in dimethyl sulfoxide (DMSO) for 24 h) on Ocy454 cells grown under mineralizing conditions for 6 days. Data shown are mean ± SD, three replicates, representative of two independent cultures; *p < 0.05 vs. DMSO and p < 0.01 vs. untreated by one-way ANOVA