Fig 7

IP₃R2 knockdown augments TRPML1 activity and enhances melanophagy flux.
(A) Representative traces of Fura2AM-based Ca²⁺ imaging in B16 cells stimulated with the MLSA1 after 72 h of siRNA transfection. (B) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 20 µM MLSA1, where ‘N’ denotes the total number of cells in that trace. (C) Representative Ca²⁺ imaging trace using the TRPML1-G-GECO probe in B16 cells stimulated with the MLSA1 after 72 h of siRNA transfection. (D) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 20 µM MLSA1, where ‘N’ denotes the total number of ROI in that trace. (E) Representative traces of Fura2AM-based Ca²⁺ imaging in B16 cells stimulated with the GPN after 72 h of siRNA-mediated dual transfections of IP₃R2 and TRPML1. (F) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 300 µM GPN, where ‘N’ denotes the total number of cells in that trace. (G) Representative images of confocal imaging in B16 cells after co-transfected with siIP₃R2 and siTRPML1 along with mCherry-Tyrosinase-EGFP probe, scale bar, 10 µm. (H) Bar graph shows the quantification of the autolysosome intensity in the EGFP:mCherry ratio, where ‘N’ denotes the number of cells. (I) Representative trace of lysosomal Ca²⁺ imaging using the TRPML1-G-GECO probe in B16 cells transfected with siIP₃R2 demonstrating rescue with TRPML1-WT and TRPML1-M. (J) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 20 µM MLSA1, where ‘N’ denotes the total number of ROI in that trace. (K) Representative images of confocal imaging using the mCherry-Tyrosinase-EGFP construct in B16 cells transfected with siIP₃R2 demonstrating rescue with TRPML1-WT and TRPML1-M, scale bar, 10 µm. (L) Bar graph showing the quantification of the EGFP intensity (mCherry inherently present in the TRPML1 plasmid), where ‘N’ denotes the number of cells imaged. (M) Representative images of Assam wild type (ASWT) zebrafish at 48hpf treated with either MLSA1 or vehicle control. (N) Bar graph showing melanin content estimation of zebrafish embryo after 48hpf (N = 3 with 20 embryos in each “N”. Data presented are mean ± SEM. For statistical analysis, unpaired t test was performed for panels B, D, F, H, and Dunnett’s multiple comparisons test was performed for panels J, L using GraphPad Prism software. Here, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.