Figure 2.

Autophagy protein p62 analysis shows A2AR agonism increases autophagic flux. Of note, p62 protein is directly bound to cargo in the autophagosome leading to its degradation by lysosomal proteases. Hence, its degradation can be used as a positive marker of autophagy. (A) Western blot time course for p62 levels from TC28⍺2 cells all in starvation media with FBS reduced from 10 to 1% and treated ± 1 µM CGS with line graph trend below (+ CGS, blue line, -CGS, orange line). Densitometry of p62 was compared to that of actin and normalized to time 0 are plotted for the time points with time course analysis by time series mixed model ANOVA (time series CGS vs. CTRL 0.74 ± 0.17 vs. 1.0 ± 0.24, * p < 0.05, n = 3 per time point per group). Individual comparison of 3-h CGS vs. CTRL sample sets showed statistically significant percent decrease in p62 by 3 h compared to time 0 in treated vs. untreated (61% ± 3.3% vs. 87% ± 15%, p = 0.044, n = 3). (B) p62 mRNA levels were measured in vitro after CGS treatment. CGS vs. CTRL was significantly increased (1.44 ± 0.34 vs. 1.0 ± 0.082, p = 0.045, n = 4). (C) TC28⍺2 human chondrocytes were stained for p62 (red) by IF at 1 h after CGS treatment. CTRL, untreated; CGS, treated A2AR agonist; CGS + ZM, A2AR antagonist pre-treatment with A2AR agonist treatment. Representative cells at 40 × HPF from 4 repeated experiments per condition.