Fig 5

IP₃R2 silencing reduces mitochondrial Ca²⁺ uptake and triggers melanophagy.
(A) Representative mitochondrial Ca²⁺ imaging trace using the Cepia2mt probe in B16 cells stimulated with the Histamine after 72 h of siRNA transfection. (B) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 50 µM histamine, where ‘N’ denotes the total number of regions of interest (ROI) in that trace. (C) Representative mitochondrial Ca²⁺ imaging trace using the Cepia2mt probe in B16 cells having stable IP₃R2 knockdown demonstrating rescue with IP₃R2 and IP₃R2-M, stimulated with Histamine. (D) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 50 µM histamine, where ‘N’ denotes the total number of ROI in that trace. (E) Representative images of confocal imaging in B16 cells after transfected with siNT or siIP₃R2 along with SPLICS Mt-ER Short P2A construct, scale bar, 10 µm. (F) Bar graph showing the quantification of the GFP intensity, where ‘N’ denotes the number of cells imaged. (G) Representative traces of CEPIA2mt-based Ca²⁺ imaging in B16 cells transfected with either siNT or siIP₃R2 alone or siIP₃R2 with MCU overexpression, stimulated with the Histamine. (H) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 50 µM Histamine, where ‘N’ denotes the total number of cells imaged. (I) Representative images of confocal imaging in B16 cells transfected with mCherry-Tyrosinase-EGFP probe. These cells were transfected with either siNT or siIP₃R2 alone or siIP₃R2 with MCU overexpression, scale bar, 5 µm. (J) Bar graph shows the quantification of the autolysosome intensity in the EGFP:mCherry ratio, where ‘N’ denotes the number of cells. (K) Bar graph shows quantification of the luminescent intensity measures changes in the ADP/ATP ratio (N = 12), where ‘N’ denotes the different wells imaged. (L) Representative image of immunoblot showing expression of p-AMPK and AMPK in B16 cells after siRNA silencing of IP₃R2. (M) Bar graph showing the densitometric analysis of the relative changes in the levels of p-AMPK to total AMPK (N = 3). (N) A schematic illustration demonstrating that IP₃R2 silencing augments melanophagy, by reducing mitochondrial Ca²⁺ uptake which subsequently triggers ULK1 complex. Created in BioRender. Motiani, R. (2026) https://BioRender.com/h5bus03. Data presented are mean ± SEM. For statistical analysis, an unpaired t test was performed for panels B, F, K, one-sample t test was performed for panel M, and Tukey’s multiple comparisons test was performed for panels D, H and J using GraphPad Prism software. Here, ‘ns’ means non-significant; * p < 0.05, ** p < 0.01, and **** p < 0.0001.