Figure S2.
SAMM50 knockdown affects oxidative phosphorylation. (A and B) Lysates from WT and SAMM50 CRISPR KD HeLa cells were analyzed for mitochondrial protein expression by immunoblotting (A) and quantified (B). Values are mean ± SD from three different experiments. *, P < 0.01; †, NS; one-way ANOVA. (NIPS1:NIPSNAP1, NIPS2:NIPSNAP2). (C and D) WT and SAMM50 KD HeLa cells were immunostained with antibody to mtDNA and nuclei stained with DAPI. Mitochondrial DNA (mtDNA) nucleoids were visualized by fluorescence confocal microscopy (C) and staining intensity quantified (D) from 100 cells per sample using the ImageJ software. Values are mean ± SD. †, NS; one-way ANOVA. Scale bars, 10 µm. (E) WT and SAMM50 KD HeLa cells lysates were analyzed for presence of cleaved caspase 3 by immunoblotting. Lysate from WT HeLa cells treated with 100 μM etoposide for 24 h is used as a control. (F) Relative ATP levels from WT and SAMM50 KD cells were measured using the ATP determination kit from three independent experiments. Values are mean ± SD. *, P < 0.01; one-way ANOVA. (G) Direct measurement of OCR was done using a Seahorse XFp flux analyzer. Basal respiration was measured for 30 min. Oligomycin was injected at 30 min, blocking ATP production due to oxidative phosphorylation. FCCP was injected at 60 min, followed by complex I and III inhibitors at 90 min, showing differences in maximal mitochondrial capacity. (H) Direct measurements of ECAR was done using a Seahorse XFp flux analyzer. (I) Glycolytic rates in WT and SAMM50 KD HeLa cells were measured using a Seahorse XFp flux analyzer. Graphs show one representative from three independent experiments. Values are ± SD from three replicates. †, NS. (J) Lysates from HeLa cells treated with either control (CTL) siRNA or two different siRNA to MTX1 and MTX2 for 6 d (three pulses of 48 h each) were immunoblotted with the indicated antibodies. (K and L) Lysates from HeLa cells treated with control siRNA or two different siRNAs to TOMM40 for 4 d (two pulses of 48 h each) were immunoblotted with indicated antibodies (K) and quantified (L). Values are mean ± SD from three different experiments. ***, P < 0.001; **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA.

SAMM50 knockdown affects oxidative phosphorylation. (A and B) Lysates from WT and SAMM50 CRISPR KD HeLa cells were analyzed for mitochondrial protein expression by immunoblotting (A) and quantified (B). Values are mean ± SD from three different experiments. *, P < 0.01; †, NS; one-way ANOVA. (NIPS1:NIPSNAP1, NIPS2:NIPSNAP2). (C and D) WT and SAMM50 KD HeLa cells were immunostained with antibody to mtDNA and nuclei stained with DAPI. Mitochondrial DNA (mtDNA) nucleoids were visualized by fluorescence confocal microscopy (C) and staining intensity quantified (D) from 100 cells per sample using the ImageJ software. Values are mean ± SD. †, NS; one-way ANOVA. Scale bars, 10 µm. (E) WT and SAMM50 KD HeLa cells lysates were analyzed for presence of cleaved caspase 3 by immunoblotting. Lysate from WT HeLa cells treated with 100 μM etoposide for 24 h is used as a control. (F) Relative ATP levels from WT and SAMM50 KD cells were measured using the ATP determination kit from three independent experiments. Values are mean ± SD. *, P < 0.01; one-way ANOVA. (G) Direct measurement of OCR was done using a Seahorse XFp flux analyzer. Basal respiration was measured for 30 min. Oligomycin was injected at 30 min, blocking ATP production due to oxidative phosphorylation. FCCP was injected at 60 min, followed by complex I and III inhibitors at 90 min, showing differences in maximal mitochondrial capacity. (H) Direct measurements of ECAR was done using a Seahorse XFp flux analyzer. (I) Glycolytic rates in WT and SAMM50 KD HeLa cells were measured using a Seahorse XFp flux analyzer. Graphs show one representative from three independent experiments. Values are ± SD from three replicates. †, NS. (J) Lysates from HeLa cells treated with either control (CTL) siRNA or two different siRNA to MTX1 and MTX2 for 6 d (three pulses of 48 h each) were immunoblotted with the indicated antibodies. (K and L) Lysates from HeLa cells treated with control siRNA or two different siRNAs to TOMM40 for 4 d (two pulses of 48 h each) were immunoblotted with indicated antibodies (K) and quantified (L). Values are mean ± SD from three different experiments. ***, P < 0.001; **, P < 0.005; *, P < 0.01; †, NS; one-way ANOVA.

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