Fig. 1: Isobacachalcone (ISO) is a candidate caloric restriction mimetic (CRM).

A Human neuroglioma H4 cells stably expressing GFP-LC3 were treated with a selection of chalcones from the TargetMol library of flavonoids at the indicated concentrations. We compared the selected agents at different concentrations with the standard autophagy inducer torin 1 (300 nM), and identified conditions with significantly increased GFP-LC3 puncta formation (1.25 times of the vehicle control (DMSO)) and viability of at least 80% with respect to DMSO, as potent autophagy activation. B, C H4 cells stably expressing GFP-LC3 were treated with isobacachalcone (ISO) (10, 25, and 50 μM) for 6 h. Then the cells were fixed and imaged to assess the formation of GFP-LC3 puncta (C). Torin 1 (300 nM) was used as a prototypical autophagy inducer. Representative images are shown in (B). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^**P < 0.01; ^***P < 0.001 vs. DMSO/Ctr, Student’s t test). D, E U2OS cells were treated as described above, followed by the incubation with specific antibodies to block acetylated tubulin. Thereafter, immunofluorescence was conducted with antibodies against acetylated lysine residues and appropriate AlexaFluor-conjugated secondary antibodies. Representative images of lysine acetylation are shown in (D), and the decrease of acetylation in the cytoplasm was measured in (E). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^**P < 0.01 vs. DMSO/Ctr, Student’s t test). F, H U2OS cells transfected with a plasmid expressing p62 protein fused with an HA tag (HA-p62) were treated with ISO (25 μM) in the presence or absence of bafilomycin A1 (Baf A1, 100 nM) for 6 h. SDS–PAGE and immunoblot were performed, band intensities of HA-p62 and β-actin (ATCB) were assessed, and the ratio (HA/ATCB) was calculated (H). In parallel samples, band intensities of LC3-II and ATCB were assessed, and their ratio (LC3-II/ATCB) was calculated (G). Data are means ± SD of three independent experiments (^*P < 0.05, ^**P < 0.01 vs. untreated control; ^##P < 0.01, ^###P < 0.001 vs. without Baf A1; Tukey’s multiple comparisons test). I, K Human osteosarcoma U2OS cell stably expressing GFP-LC3 either wild-type (WT) or ATG5 knockout (I) were treated with ISO (25 μM) or torin 1 (300 nM) for 6 h. The cells were fixed, imaged, and GFP-LC3 dots were quantified (K). Scale bar equals 10 μm. Data are means ± SD of quadruplicates (^***P < 0.001 vs. untreated control; ^##P < 0.01, ^###P < 0.001 vs. WT; Tukey’s multiple comparisons test).