Figure 3.

FoxO-activated autophagy mediators Gabarapl1, and Beclin-1 were activated in response to A2AR signaling. TC28⍺2 human chondrocytes were treated with 1 µM CGS (Gabarapl1-assessed cells were incubated with HCQ overnight to enhance autophagosome visualization). (A) RNA was isolated from cells and Gabarapl1 and Beclin-1 gene expression were assessed. (B) Autophagy proteins were visualized by IF in TC28⍺2 human chondrocytes specifically for Gabarapl1 (left) and Beclin-1 (right) shown as untreated (CTRL) or 1 h of CGS. Both proteins are demonstrated by green fluorescence. Development of punctate autophagy initiation complexes can be observed after 1 h. These are 40 × hpf views of representative cells repeated in 3 separate experiments. (C) Western blot for Gabarapl1 protein levels in total cellular lysates from TC28a2 cells treated ± CGS21680 (1.45 ± 0.10 vs. 1.0 ± 0.25, n = 3–4 per group, p < 0.05, Student’s T-test).