Figure 9.
ORP5 affects the localization of seipin in a Mito-MAM contact sites integrity dependent way. (A) Representative confocal images (single focal plane) of HeLa cells treated with siCtrl or siORP5 or siORP8 RNA oligos. The cells were then transfected with YFP-seipin (green) and Mito-BFP (blue). The LDs were stained LTox (purple). Arrowheads point to seipin enrichment at MAM–LD contact sites. Scale bar, 5 µm. (B) Analysis of the distribution of seipin enrichments in HeLa cells expressing seipin and treated with either siCtrl, siORP5, or siORP8 interfering RNAs. Data are shown as % mean ± SEM of cells with n = 64 cells in siCtrl, n = 44 cells in siORP5, and n = 32 cells in siORP8 (* = P < 0.05; ** = P < 0.01; **** = P < 0.0001; Wilcoxon-Mann-Whitney test). (C) Confocal images (single focal plane) of HeLa cells treated with siCtrl or siORP5 or siORP8 RNA oligos. The cells were then transfected with YFP-seipin (green), Mito-BFP (blue) and either RFP-Sec22b (red) or ORP5∆PH (red). The LDs were stained LTox (purple). Arrowhead points seipin enrichment at MAM–LD contact sites. (D) Analysis of the distribution of seipin enrichments to MAM-LD contact sites in HeLa cells treated with siCtrl or siORP5 RNA oligos and then co-transfected with seipin and RFP-Sec22b or siRNA-resistant ORP5∆PH. Cells were treated with OA (300 μM) for 2 h before analysis. Data are shown as % mean ± SEM of cells. n = 14 cells in siCtrl + Sec22b, n = 17 cells in siORP5 + Sec22b, n = 41 cells in siCtrl + ORP5∆PH rescue and n = 19 cells in siORP5 + ORP5∆PH rescue (* = P < 0.05; **** = P < 0.0001; Wilcoxon-Mann-Whitney test). (E) Quantification of the distribution of the immunogold particles (15 nm) staining seipin in HeLa cells treated with siCtrl or siORP5. Data are shown as % mean ± SEM of cell profiles with n = 79 (1,500 gold particles analyzed) in siCtrl, and n = 64 (1,800 gold particle) in siORP5. * = P < 0.001, ** = P < 0.0001, unpaired two-tailed t test. (F) Representative confocal images (single focal plane) of HeLa cells treated with siPTPIP51 or siCtrl RNAs. The cells were then transfected with YFP-Seipin (green) alone or in co-expression with ORP5∆PH rescue (red). The LDs were stained with LTox (purple). Scale bar, 5 µm. (G) Quantification of the distribution of seipin immunogold particles in HeLa cells treated with siCtrl or siPTPIP51 and expressing seipin alone or in co-expression with siRNA-resistant ORP5∆PH. Data are shown as % mean ± SEM of cells with n = 41 cells in siCtrl, n = 25 cells in siPTPIP51, and n = 23 cells in siPTPIP51 + ORP5∆PH (* = P < 0.05; ** = P < 0.01; *** = P < 0.001; Wilcoxon-Mann-Whitney test).

ORP5 affects the localization of seipin in a Mito-MAM contact sites integrity dependent way. (A) Representative confocal images (single focal plane) of HeLa cells treated with siCtrl or siORP5 or siORP8 RNA oligos. The cells were then transfected with YFP-seipin (green) and Mito-BFP (blue). The LDs were stained LTox (purple). Arrowheads point to seipin enrichment at MAM–LD contact sites. Scale bar, 5 µm. (B) Analysis of the distribution of seipin enrichments in HeLa cells expressing seipin and treated with either siCtrl, siORP5, or siORP8 interfering RNAs. Data are shown as % mean ± SEM of cells with n = 64 cells in siCtrl, n = 44 cells in siORP5, and n = 32 cells in siORP8 (* = P < 0.05; ** = P < 0.01; **** = P < 0.0001; Wilcoxon-Mann-Whitney test). (C) Confocal images (single focal plane) of HeLa cells treated with siCtrl or siORP5 or siORP8 RNA oligos. The cells were then transfected with YFP-seipin (green), Mito-BFP (blue) and either RFP-Sec22b (red) or ORP5∆PH (red). The LDs were stained LTox (purple). Arrowhead points seipin enrichment at MAM–LD contact sites. (D) Analysis of the distribution of seipin enrichments to MAM-LD contact sites in HeLa cells treated with siCtrl or siORP5 RNA oligos and then co-transfected with seipin and RFP-Sec22b or siRNA-resistant ORP5∆PH. Cells were treated with OA (300 μM) for 2 h before analysis. Data are shown as % mean ± SEM of cells. n = 14 cells in siCtrl + Sec22b, n = 17 cells in siORP5 + Sec22b, n = 41 cells in siCtrl + ORP5∆PH rescue and n = 19 cells in siORP5 + ORP5∆PH rescue (* = P < 0.05; **** = P < 0.0001; Wilcoxon-Mann-Whitney test). (E) Quantification of the distribution of the immunogold particles (15 nm) staining seipin in HeLa cells treated with siCtrl or siORP5. Data are shown as % mean ± SEM of cell profiles with n = 79 (1,500 gold particles analyzed) in siCtrl, and n = 64 (1,800 gold particle) in siORP5. * = P < 0.001, ** = P < 0.0001, unpaired two-tailed t test. (F) Representative confocal images (single focal plane) of HeLa cells treated with siPTPIP51 or siCtrl RNAs. The cells were then transfected with YFP-Seipin (green) alone or in co-expression with ORP5∆PH rescue (red). The LDs were stained with LTox (purple). Scale bar, 5 µm. (G) Quantification of the distribution of seipin immunogold particles in HeLa cells treated with siCtrl or siPTPIP51 and expressing seipin alone or in co-expression with siRNA-resistant ORP5∆PH. Data are shown as % mean ± SEM of cells with n = 41 cells in siCtrl, n = 25 cells in siPTPIP51, and n = 23 cells in siPTPIP51 + ORP5∆PH (* = P < 0.05; ** = P < 0.01; *** = P < 0.001; Wilcoxon-Mann-Whitney test).

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