Figure 1
From: Pomegranate activates TFEB to promote autophagy-lysosomal fitness and mitophagy
PE upregulates autophagic compartments and autophagy activity in SY5Y cells. (a,b) Top: Immunoblots of LC3 (a) and LAMP1 (b) in SY5Y cells treated with vehicle control DMSO and increasing concentrations of PE at 50, 150 and 300 μg/ml for 24 h. Bottom: Quantification of LC3-II and LAMP1 levels, calculated as fold change against DMSO control. Full-length blots are presented in Supplementary Figure S6a,b. (c,d) Top: Immunofluorescence images of endogenous LC3 (c) and LAMP1 (d) puncta in SY5Y cells treated with DMSO, 150 or 300 μg/ml PE for 24 h. The white outlines highlight the cell shape. Bottom: Quantification of LC3 (c) and LAMP1 (d) puncta per cell area, calculated as fold change against DMSO control. (e) Left: Fluorescence images of yellow (autophagosomes) and red (autophagolysosomes) puncta in SY5Y cells transfected with tandem mRFP-GFP-LC3 reporter for 24 h, followed by 24 h incubation under basal serum containing (S+), serum starvation (S−) or treatment with 150 or 300 μg/ml PE conditions. The white dotted outlines highlight the cell nucleus. The yellow triangles indicate autophagosomes highlighted by both mRFP and GFP signals. Right: Quantification of yellow and red puncta per cell area, calculated as fold change against S+. The values inside the graph bars represent the total number of yellow or red puncta per cell area ± S.E.M under the respective conditions. (f) Left: Immunoblot of LC3 in SY5Y cells treated with DMSO, S− or 150 and 300 μg/ml PE for 24 h, followed by 4 h treatment with or without lysosomal inhibitors (NL: 20 mM ammonium chloride and 100 μM leupeptin). Right: Quantification of LC3-II flux, calculated as fold increase in LC3-II levels in the presence of NL over LC3-IIs level in the absence of NL. Full-length blots are presented in Supplementary Figure S6f. At least 30 cells from random fields were analyzed for each condition for all imaging experiments. Nuclei were stained with DAPI. All values are mean + S.E.M (n = 3–4). Differences against DMSO or S+ control are significant at *p<0.05 and **p < 0.01. Scale bar, 10 μm.
