Figure 3
From: Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model
Little effect of NAS on autophagy or interaction with autophagy inducers. (a) A variety of structurally diverse NAS tested for their effects on autophagy at 1 and 10 µM, along with known autophagy inducer, Torin1 0.3 µM, and blocker, BafilomycinA1 0.5 µM. All treatments were for 24 h. Each point represents a well in a 96 well-plate (n = 4 replicates per condition from 3 separate experiments). Circles represent first experiment, squares second, and triangles third. Black asterisks represent significantly different conditions to the control based on results of separate one-way ANOVAs. 6 one-way ANOVAs (one per NAS) revealed an effect of ent-AlloP (F (2, 33) = 15.78, p < 0.0001). Dunnett’s multiple comparisons showed a difference between control and ent-AlloP 1 µM (*p = 0.0134) and ent-AlloP 10 µM (****p < 0.0001). However when accounting for multiple drug comparisons, two nested one-way ANOVAs (one for each concentration) revealed no differences from control for any NAS at a given concentration. Two unpaired t-tests, one of Torin1 and the other of BafilomycinA1, revealed significant effects of treatment (****p < 0.0001), including a Bonferroni correction for multiple comparisons. (b) Dose–response curves of Torin1, Torin1 + AlloP 10 μM, Torin1 + ent-AlloP 10 μM revealed a dose-dependent effect of Torin1 on autophagy induction and no statistical interaction of NAS with Torin1. An extra sum-of-squares F-test (F (6, 204) = 0.6823, p = 0.6641) revealed no difference between the best-fit values (Bottom, HillSlope, EC50) for each curve. The values of Torin1 0.3 µM are represented in (a) as a positive control since these treatments were conducted on the same plates. (c) Time course study of EBSS showed time-dependent autophagy. A two-way ANOVA revealed a difference between time (F (4, 165) = 33.92, p < 0.0001) and treatment (F (2, 165) = 5.712, p = 0.0040), with no significant interaction (F (8, 165) = 1.082, p = 0.3780). Dunnett’s multiple comparison showed a difference between EBSS and ent-AlloP at 3 h p = 0.0328 (purple asterisk), and EBSS and AlloP p = 0.0259 and ent-AlloP p = 0.0134 at 12 h (brown asterisk). In all panels of this figure, fluorescence was measured using a microplate reader, n = 4 per condition, 3 independent experiments.
