Figure 4.

Abnormal autophagy in Sumf1^−/− chondrocytes. (A) Number of BrdU positive cells present in the proliferative zone of the growth plate. Values are the mean ± SD. Student’s test (*) P < 0.05. (B) EM analysis of newborn chondrochostal cartilage revealed the presence of autophagosomes in wild-type chondrocytes. (Boxed inset) Note the double membrane vesicles surrounding a portion of cytoplasm (arrows). (C) Chondrocyte from newborn Sumf1^−/− showing more autophagosomes (AV) surrounded by enlarged lysosomes (L). (D) Confocal microscopy analysis of Sumf1^−/−;GFP-LC3 and wt;GFP-LC3 growth plate. In GFP-LC3 chondrocytes (left) the GFP fluorescence was more diffused throughout the cytoplasm while in Sumf1^−/−;GFP-LC3 it was aggregated in cytoplasmatic dots (right). (E) Western blot analysis showing a 2.5-fold increase in LC3II level in newborn Sumf1^−/− chondrocytes. No difference was observed in osteoblasts. Values shown are means of triplicate experiments. (F) Abnormal autophagy in Sumf1^−/− chondrocytes during serum and nutrient starvation. Wild-type and Sumf1^−/− chondrocytes were starved for the indicated period of time, harvested, and subjected to LC3 immunoblotting. Sumf1^−/− chondrocytes and wild type stimulated with Baf presented an increased amount of LC3II compared with wild-type chondrocytes at all time points analyzed. (G) ATP amount is decreased in wild-type chondrocytes when autophagy is inhibited with Baf. Sumf1^−/− chondrocytes displayed a lower level of ATP compared with wild-type and Baf treatment did not affected significantly ATP concentration. (H) Wild-type and Sumf1^−/− chondrocytes were cultured in serum and glucose-free medium for 2 d. Wild-type chondrocytes were also treated with baf and 3-methyladenine, another inhibitor of autophagy. Cell viability was monitored after 12 h, 24 h, and 48 h. Error bars represent SEM. Student’s test (*) P < 0.05; (**)P < 0.01.