Figure 3

PE does not influence ERK1/2, AKT, mTOR and calcineurin signaling, but regulates TFEB nuclear shuffling in a manner dependent on cytosolic Ca²⁺ levels. (a–c) Left: Immunoblots of phosphorylated (P) and total (T) forms of ERK1/2 (a), AKT (b), mTOR and p70/S6K (c) in SY5Y cells treated with DMSO, 150 or 300 μg/ml PE for 16 h. Right: Quantification of the various phosphorylated proteins against their respective total proteins, expressed as fold change relative to DMSO control. Full-length blots are presented in Supplementary Figure S8a–c. (d) Top: PCR analysis of calcineurin mRNA expression levels in SY5Y cells after 24 h treatment with DMSO or 300 μg/ml PE. Graph shows quantification of calcineurin mRNA levels, expressed as fold change against DMSO control. Bottom: Immunoblot of calcineurin in SY5Y cells treated with DMSO or 300 μg/ml PE for 16 h. Graph shows quantification of calcineurin levels, calculated as fold change relative to DMSO control. Full-length blots are presented in Supplementary Figure S8d. (e) Top: Fluorescence images depicting spatial localization of GFP-TFEB in GFP-TFEB SY5Y stable cells treated with DMSO, starved (S−) in the absence or presence of 10 µM Ca²⁺ chelator BAPTA-AM, or treated with 300 μg/ml PE in the absence or presence of BAPTA-AM for 24 h. Bottom: Quantification of percentage TFEB nuclear localization. Nuclei were stained with DAPI. At least 100 cells from random fields were analyzed for each condition. All values are mean + S.E.M (n = 3–7). Differences against DMSO are significant at *p<0.05, **p<0.01 and ***p<0.005 and ^#p < 0.05. Scale bar, 10 μm.