Fig 6

IP₃R2 silencing decreases lysosomal pH and increases Ca²⁺ content in the lysosome.
(A) Representative images of confocal imaging in B16 cells loaded with LysoSensor Yellow/Blue DND-160 dye for 5 min after transfected with siNT or siIP₃R2, scale bar, 10 µm. (B) Bar graph showing the quantification of the intraluminal lysosomal pH in Yellow/Blue ratio, where ‘N’ denotes the number of cells imaged. (C) Representative images of confocal imaging in B16 cells loaded with OG-BAPTA-dextran (OBDx) after transfected with siNT or siIP₃R2, scale bar, 10 µm. (D) Bar graph showing the quantification of the green intensity, where ‘N’ denotes the number of cells imaged. (E) Representative traces of Fura2AM-based Ca²⁺ imaging in B16 cells stimulated with the BafilomycinA1 after 72 h of siRNA transfection. (F) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 1 µM BafilomycinA1, where ‘N’ denotes the total number of cells imaged. (G) Representative Ca²⁺ imaging trace using the TRPML1-G-GECO probe in B16 cells stimulated with the GPN after 72 h of siRNA transfection. (H) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 300 µM GPN, where ‘N’ denotes the total number of ROI in that trace. (I) Representative traces of Fura2AM-based Ca²⁺ imaging in B16 cells stimulated with the Tg after 72 h of siRNA transfection. (J) Bar graph showing the quantification of Ca²⁺ imaging traces stimulated with 2 µM Tg, where ‘N’ denotes the total number of cells imaged. (K) A schematic illustration demonstrating the mechanism of PLA by utilizing Primary antibody of LAMP1 and VAP-A. Created in BioRender. Motiani, R. (2026) https://BioRender.com/km2dti6. (L) Representative images of confocal imaging in B16 cells performing PLA between LAMP1 and VAP-A proteins after transfected with siNT or siIP₃R2 or treated with 100 nm Bafilomycin A1 or with Ab VAP-A only, scale bar, 20 µm. (M) Bar graph showing the quantification of the number of PLA spots per cell, where ‘N’ represents the number of images analyzed in the respective condition. (N) Representative TEM image showing B16 cells transfected with siNT or siIP₃R2, scale bar, 500 nm. (O) Bar graph shows the frequency of Mitochondria around ER within 50 nm range, where ‘N’ denotes the number of ER. (P) Bar graph shows the frequency of Lysosome around ER within 100 nm range, where ‘N’ denotes the number of ER. (Q) Bar graph shows the distance between ER and Mitochondria within 50 nm range, where ‘N’ denotes the number of distances measured between organelles. (R) Bar graph shows the distance between ER and Lysosomes within 100 nm range, where ‘N’ denotes the number of distances measured between organelles. (S) A schematic illustration demonstrating that IP₃R2 silencing decreases pH and increases lysosomal Ca²⁺ content. 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, D, F, H, J, O, P, Q, R and Dunnett’s multiple comparisons test was performed for panel M using GraphPad Prism software. Here, ‘ns’ means non-significant; ** p < 0.01, *** p < 0.001 and **** p < 0.0001.