Fig. 5: MERCs regulate lipophagy to sustain OxPHOS.

a MOLM14 cells transfected with siRNA control (Ctrl) or mitofusin2 (Mtfn2) were processed for electron microscopy analysis. Electron microscopy pictures from one experiment are shown. Scale bar: 1 µm. b Histograms represent the % of mitochondria in contact with ER. Each dot corresponding to one cell. c Images of proximity ligation assay between VDAC1 and IP3R1 from MOLM14 transfected with Ctrl or Mtfn2 siRNAs (n = 3). Scale bar: 10 µm. Numbers represent the % of dots in cells transfected with siRNA Mtfn2 compared with cells transfected with siRNA Ctrl. d, e MOLM14 cells were transduced with Ctrl or VDAC1 shRNAs (n = 4) or transfected with Ctrl or Mtfn2 siRNAs (n = 6), and stained for LC3B and DAPI. Confocal sections (at least four independent experiments) are shown. Scale bar: 10 µm (d). Histograms represent the number of LC3B puncta per cell (e) (unpaired t-test). f, g MOLM14 cells transduced with Ctrl or VDAC1 shRNAs (n = 3) or transfected with Ctrl or Mtfn2 siRNAs (n = 6) were stained for Bodipy and DAPI. Scale bar: 10 µm, (f). Histograms show the number or the area of Bodipy dots per cell (g) (unpaired t-test). h Measurement of basal oxygen consumption rate (OCR) in MOLM14 cells transduced with Ctrl or VDAC1 shRNAs (n = 6) or transfected with Ctrl or Mtfn2 siRNAs (n = 6, unpaired t-test). i, j MOLM14 cells transfected with Ctrl or Beclin1 (Bec) or Mtfn2 siRNAs and incubated with RC₁₂, were stained for TOMM20 and confocal Z-stacks were acquired (three independent experiments). Scale bar: 10 µm (i). Fraction of RC₁₂ overlapping TOMM20 staining (j). siCtrl (n = 17), siBec (n = 18), and siMtfn2 (n = 13, unpaired t-test). k Measurement of OCR in MOLM14 cells transfected with Ctrl (n = 8), Bec (n = 7), or Mtfn2 siRNAs (n = 8) ± Oleate-BSA. l–o MOLM14 cells were transduced with the mitochondria–ER organelle linker (OMM-ER) and its control (OMM), treated ± metformin, and subjected to Western blot analysis for actin and LC3B (l, m) or stained for Bodipy and DAPI (n, o) (three independent experiments). Scale bar: 10 µm. Histograms represent the number of LC3B puncta (m) or the area of Bodipy dots per cell (o) unpaired t-test. Data are means ± s.e.m.