Fig 3

Fig 3 IP₃R2 silencing stabilize melanogenic proteins and triggers autophagy.
(A) Representative image of immunoblot showing expression of GP100 in B16 B16 cells transfected with siNT or siIP₃R2 along with 10 µM MG132 treatment for 6 h. (B) Bar graph showing the densitometry of the GP100 band normalized to β-Tubulin (N = 3). (C) Representative image of immunoblot showing expression of DCT in B16 cells transfected with siNT or siIP₃R2 along with 10 µM MG132 treatment for 6 h. (D) Bar graph showing the densitometry of the DCT band normalized to β-Tubulin (N = 3). (E) Representative image of immunoblot showing expression of IP₃R2 in B16 cells transfected with siNT or siIP₃R2 along with 10 µM MG132 treatment for 6 h. (F) Bar graph showing the densitometry of the IP₃R2 band normalized to β-Tubulin (N = 3). (G) Representative image of immunoblot showing expression of Calnexin in B16 cells transfected with siNT or siIP₃R2 along with 10 µM MG132 treatment for 6 h. (H) Bar graph showing the densitometry of the Calnexin band normalized to β-Tubulin (N = 4). (I) Representative image of immunoblot showing expression of LC3II and LC3I in B16 cells transfected with siNT or siIP₃R2 along with 10 µM MG132 treatment for 6 h. (J) Bar graph showing the densitometric analysis of the LC3II/LC3I ratio (N = 4). (K) Representative image of immunoblot showing expression of LC3II and LC3I in B16 cells after siRNA silencing of IP₃R2. (L) Bar graph showing the densitometric analysis of the LC3II/LC3I ratio (N = 3). (M) Representative images of confocal imaging in B16 cells after transfected with siNT or siIP₃R2 along with pMRX-IP-GFP-LC3-RFP probe, scale bar, 10 µm. (N) Bar graph shows the quantification of the autolysosome intensity in the GFP:RFP ratio, where ‘N’ denotes the number of cells. (O) Representative image of immunoblot showing expression of LC3II in B16 cells after siRNA transfection including 6 h of treatment with 100 nM of the lysosomal inhibitor Bafilomycin A1. (P) Bar graph showing the densitometric analysis of the LC3II/LC3I ratio (N = 3). Data presented are mean ± SEM. For statistical analysis, one-sample t test was performed for panel L, an unpaired t test was performed for panel N, and Tukey’s multiple comparisons test was performed for panel B, D, F, H, J, P using GraphPad Prism software. Here, ‘ns’ means non-significant; * p < 0.05, ** p < 0.01, and **** p < 0.0001.