Fig. 3: ISO stimulates autophagic flux in vitro and in vivo.

A–D Human osteosarcoma U2OS cells stably expressing the tandem reporter construct GFP-LC3-ATG4-RFP-LC3ΔG (A) or the tandem reporter mCherry-GFP-p62 (B) were treated with torin 1 (300 nM) or isobacachalcone (ISO; 25 μM) with or without bafilomycin A1 (Baf A1, 100 nM) for 6 h. After fixation, GFP and RFP fluorescence was measured by automated image analysis, and the ratio of RFP to GFP was calculated (C, D). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. untreated control;^### P < 0.001 vs. without Baf A1; Tukey’s multiple comparisons test). E, F Rat adrenal gland PC12 cells stably expressing an inducible variant of Q74-GFP were treated with doxycycline (1 μg/mL) for 8 h for the induction of Q74 expression. Then the medium was changed, and ISO (10, 25, 50 μM) was added for 24 h. Torin 1 (300 nM) was used as a positive control. Representative images are shown in (E), and GFP-Q74 levels were quantitated in (F). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^**P < 0.01, ^***P < 0.001 vs. DMSO/Ctr, Student’s t test). G–M C57BL/6 mice received two intraperitoneal (i.p.) injections of 20 mg/kg/day ISO (n = 3 mice per condition, n = 2 experiments). Organs were collected, and representative immunoblots showing regulators and LC3I-to-LC3-II conversion in the heart (G–K) and in the liver (L–P). AKT, mTOR, and p70 abundance was evaluated, and parallel samples were probed with phosphoneoepitope-specific antibodies. β-actin (ACTB) or vinculin levels were monitored to ensure equal protein loading (H, J). Band intensities of pAKT and ACTB, pmTOR and Vinculin, pS6K and ACTB, as well as LC3-II and ACTB, were assessed, and their ratios were calculated (H–K, M–P). Data are means ± SD (n = 3; (^*P < 0.05, ^**P < 0.01 vs. DMSO/Ctr, Student’s t test).