Fig 4

IP₃R2 silencing enhances melanophagy flux.
(A) A schematic illustration demonstrating the melanophagy flux upon IP₃R2 silencing by utilizing a mCherry-Tyrosinase-EGFP construct. Created in BioRender. Motiani, R. (2026) https://BioRender.com/m0tpcom. (B) Representative images of confocal imaging in B16 cells after transfected with siNT or siIP₃R2 along with mCherry-Tyrosinase-EGFP probe, scale bar, 10 µm. (C) Bar graph shows the quantification of the autolysosome intensity in the EGFP:mCherry ratio, where ‘N’ denotes the number of cells. (D) A schematic illustration demonstrating the melanophagy flux upon IP₃R2 silencing by utilizing Tyrosinase-mKeimaN1 construct. Created in BioRender. Motiani, R. (2026) https://BioRender.com/m0tpcom. (E) Representative images of confocal imaging in B16 cells after transfected with siNT or siIP₃R2 along with Tyrosinase-mKeimaN1 probe, scale bar, 10 µm. (F) Bar graph shows the quantification of the autolysosome intensity in the 543 nm:488 nm ratio, which is alternatively excited at 543 nm and 488 nm, where ‘N’ denotes the number of cells. (G) Representative images of confocal imaging in B16 cells having stable IP₃R2 knockdown transfected with mCherry-Tyrosinase-EGFP construct, scale bar, 10 µm. (H) Bar graph showing the quantification of the EGFP intensity (mCherry inherently present in the shRNA), where ‘N’ denotes the number of cells imaged. (I) Representative images of confocal imaging in B16 cells having stable IP₃R2 knockdown transfected with mCherry-Tyrosinase-EGFP construct, scale bar, 10 µm. (J) Bar graph showing the quantification of the EGFP intensity (mCherry inherently present in the shRNA), where ‘N’ denotes the number of cells imaged. (K) Representative images of confocal imaging in B16 cells transfected with mCherry-Tyrosinase-EGFP probe. These cells were also transfected with either siNT or siIP₃R2 and treated with 10 µM MG132 for 6 h, scale bar, 5 µm. (L) Bar graph shows the quantification of the autolysosome intensity in the EGFP:mCherry ratio, where ‘N’ denotes the number of cells. Data presented are mean ± SEM. For statistical analysis, an unpaired t test was performed for panels C, F, H, Tukey’s multiple comparisons test was performed for panels J and L using GraphPad Prism software. Here, ‘ns’ means non-significant, and **** p < 0.0001.