Fig. 5.

Modulating PACS2–TRPV1 axis with CPS rescues ER–mitochondrial tethering and PACS2 protein in cells overexpressing SPC^Δexon4. A MEL188 cells were stably transfected with either SPC^WT or SPCΔ^exon4 plasmids or left un-transfected, followed by immunoblots for the given proteins. B and C Quantification of PACS2 (B) and CHOP (C) protein levels are shown. Relative protein amounts were normalized to GAPDH and their mean value in untransfected cells was set as one. D Representative fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in SPC^WT or SPCΔ^exon4 overexpressing cells, scale bar = 60 µm. E Fluorescence intensity was quantified using ImageJ, and its intensity in untransfected cells was set to one. F Cells stably expressing SPC^WTor SPCΔ^exon4 or control cells were treated with DMSO or CPS, followed by immunoblotting of PACS2 and GAPDH. G Quantification of PACS2 protein expression is shown. Relative protein amounts were normalized to GAPDH and its mean value in control cells was set as one. H Fluorescence microscopy images following proximity ligation assay with antibodies against calnexin and VDAC1 in control cells or in cells overexpressing SPC^WT or SPCΔ^exon4, followed by DMSO or CPS treatments at the indicated doses, scale bar = 60 µm. I Fluorescence intensity was quantified using ImageJ, and its intensity in untreated control cells was set as one. ‘n’ of three independent experiments were performed and statistical significance is indicated as: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001