Fig 8

Fig 8 TRPML1-mediated lysosomal Ca²⁺ release regulates melanophagy via TFEB nuclear translocation.
(A) Representative images of confocal imaging in B16 cells demonstrates TFEB nuclear translocation stimulated with 20 µM MLSA1 for 1 h after transfecting with siNT or siIP₃R2 along with pEGFP-N1-TFEB construct, scale bar, 10 µm. (B) Bar graph showing the quantification of the GFP intensity from the cytoplasm and nucleus in the nucleus/cytoplasmic ratio following the addition of 20 µM MLSA1 for 1 h, where ‘N’ denotes the number of cells imaged. (C) Representative image of immunoblot showing expression of TFEB in B16 cells from the cytoplasm and nucleus following subcellular fractionation after siRNA silencing of IP₃R2. (D) Bar graph showing the densitometric analysis of the relative changes in the levels of TFEB by quantitating nucleus/cytoplasmic ratio (N = 3). (E) A schematic illustration demonstrating the transcriptional regulation of the TFEB on expression of genes RCHY1 and OPTN. Created in BioRender. Motiani, R. (2026) https://BioRender.com/wu3kyes. (F) qRT-PCR analysis showing increase in RCHY1 mRNA expression after 72 h of siRNA-mediated IP₃R2 silencing. (N = 3). (G) qRT-PCR analysis showing increase in OPTN mRNA expression after 72 h of siRNA-mediated IP₃R2 silencing. (N = 3). (H) Representative images of confocal imaging in B16 cells transfected with siRNA along with mCherry-Tyrosinase-EGFP probe, scale bar, 5 µm. (I) Bar graph shows the quantification of the autolysosome intensity in the GFP:RFP ratio, where ‘N’ denotes the number of cells. (J) Representative images of confocal imaging in B16 cells transfected with siRNA along with mCherry-Tyrosinase-EGFP probe, scale bar, 5 µm. (K) Bar graph shows the quantification of the autolysosome intensity in the GFP:RFP ratio, where ‘N’ denotes the number of cells. (L) A schematic illustration demonstrating that IP₃R2 silencing augments melanophagy, by releasing Lysosomal Ca²⁺ via TRPML1 which subsequently translocated TFEB into the nucleus. Created in BioRender. Motiani, R. (2026) https://BioRender.com/h5bus03. Data presented are mean ± SEM. For statistical analysis, one-sample t test was performed for panels B, D, F, G, and Tukey’s multiple comparisons test was performed for panels I and K, using GraphPad Prism software. Here, * p < 0.05, *** p < 0.001 and **** p < 0.0001.