LC3-II steady-state level, APG biogenesis and APG degradation after treatment with Nef and/or ART for 7 days. Primary human astrocytes were treated daily for 7 days with extracellular Nef and/or ART, without and with chloroquine (CQ) for the last 2 or 4 h, then LC3 Western blotting was performed. Steady-state level, biogenesis, flux and net flux for LC3-II were calculated as described in Methods. (A,G,M) Representative LC3-II Western blots after A. Nef, G. ART, or M. Nef+ART treatment. B-E, H-K, and N-Q show individual experiments (dots) and the means of the experiments (bars). (B,H,N) LC3-II steady-state level in control and B. Nef, H. ART, or N. Nef+ART treated astrocytes. (C,I,O) APG biogenesis in control and C. Nef, I. ART, or O. Nef+ART treated cells. (D,J,P) Rate of APG degradation (Flux) in control and D. Nef, J. ART, P. Nef+ART treated astrocytes. (E,K,Q) Amount of APG degradation (net flux) in control and E. Nef, K. ART, or Q. Nef+ART treated cells. (F,L,R) show the fold changes of the same autophagy parameters after treatment with F. Nef, L. ART, and R. Nef+ART over controls of individual experiments (dots) and the means of the fold changes of the experiments (bars). Controls were set to 1, represented by the dashed lines. Error bars depict SD. n = 7–11; *, p < 0.05; **, p < 0.005 by the Wilcoxon Matched-Pairs Signed Rank test or the Wilcoxon Signed Rank test.