Figure 5. ARL6IP1 deficiency reduces... | Rockefeller University Press

Figure 5.

$ARL6IP1 deficiency reduces physical and functional coupling of ER–mitochondria. (A) Representative image of immunofluorescence staining of MitoTracker Deep Red and Sec61β in ARL6IP1 KO or WT MEFs at three passages (600×; scale bar: 20 μm). (B) Protein levels of organelle markers in cytoplasmic and crude mitochondria (MT) fraction isolated from ARL6IP1 KO or WT MEFs at three passages. (C) Subcellular fractions were isolated via Percoll density gradient ultracentrifugation from WT MEFs at three passages and analyzed using western blotting. (D and E) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Seahorse XF24 Extracellular Flux Analyzer in ARL6IP1 WT and KO MEFs at two passages (n = 5/group). (F and G) Quantification of basal and maximal OCR rate using Wave Controller 2.6 (Agilent Seahorse XFe24 Analyzers). Data represent mean ± SD of n = 5/group and two independent experiments. (H) Representative mitophagy image in CCCP-treated ARL6IP1 KO or WT MEFs at three passages using Mitophagy detection kit (left, 600×; scale bar: 20 μm). Quantification of autophagosome/lysosome fusion by puncta numbers in ARL6IP1 KO or WT MEFs (right). Data represent mean values of ≥10 images assessed per experiment ± SD, calculated in GraphPad Prism 7.0. **P < 0.01 (I) Colocalization in fluorescent images by Rhod-2 AM and MitoTracker Green in ARL6IP1 KO or WT MEFs. Rhod-2 AM is used to indicate mitochondrial Ca2+ levels (left, 600×; scale bar: 20 μm). Quantification of immunofluorescence staining. Data represent mean ± SD of ≥10 images/group and are calculated in GraphPad Prism 7.0 (right). (J) Intracellular acetyl-CoA, α-ketoglutarate, and ATP synthesis levels were measured in ARL6IP1 WT and KO MEFs at three passages. Data represent mean ± SD of triplicates per group in three independent experiments. (K) Mitochondrial total and free cholesterol levels were quantified using cholesterol assay kit in ARL6IP1 WT and KO MEFs and represent three independent experiments. Data represent mean values of triplicates ± SD. (L) Representative TEM images of ER–mitochondria tethering in ARL6IP1 KO or WT MEFs. ER–mitochondria contact sites were indicated by arrowheads (left, 52,000×; scale bar: 200 nm; ER, endoplasmic reticulum; Mito, mitochondria; MAM, mitochondria-associated ER membranes). Quantification of ER–mitochondria contact length represents mean ± SD of ≥10 images/group and is calculated in GraphPad Prism 7.0 (right). *P < 0.05; **P < 0.01. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F5.$

ARL6IP1 deficiency reduces physical and functional coupling of ER–mitochondria. (A) Representative image of immunofluorescence staining of MitoTracker Deep Red and Sec61β in ARL6IP1 KO or WT MEFs at three passages (600×; scale bar: 20 μm). (B) Protein levels of organelle markers in cytoplasmic and crude mitochondria (MT) fraction isolated from ARL6IP1 KO or WT MEFs at three passages. (C) Subcellular fractions were isolated via Percoll density gradient ultracentrifugation from WT MEFs at three passages and analyzed using western blotting. (D and E) Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Seahorse XF24 Extracellular Flux Analyzer in ARL6IP1 WT and KO MEFs at two passages (n = 5/group). (F and G) Quantification of basal and maximal OCR rate using Wave Controller 2.6 (Agilent Seahorse XFe24 Analyzers). Data represent mean ± SD of n = 5/group and two independent experiments. (H) Representative mitophagy image in CCCP-treated ARL6IP1 KO or WT MEFs at three passages using Mitophagy detection kit (left, 600×; scale bar: 20 μm). Quantification of autophagosome/lysosome fusion by puncta numbers in ARL6IP1 KO or WT MEFs (right). Data represent mean values of ≥10 images assessed per experiment ± SD, calculated in GraphPad Prism 7.0. **P < 0.01 (I) Colocalization in fluorescent images by Rhod-2 AM and MitoTracker Green in ARL6IP1 KO or WT MEFs. Rhod-2 AM is used to indicate mitochondrial Ca²⁺ levels (left, 600×; scale bar: 20 μm). Quantification of immunofluorescence staining. Data represent mean ± SD of ≥10 images/group and are calculated in GraphPad Prism 7.0 (right). (J) Intracellular acetyl-CoA, α-ketoglutarate, and ATP synthesis levels were measured in ARL6IP1 WT and KO MEFs at three passages. Data represent mean ± SD of triplicates per group in three independent experiments. (K) Mitochondrial total and free cholesterol levels were quantified using cholesterol assay kit in ARL6IP1 WT and KO MEFs and represent three independent experiments. Data represent mean values of triplicates ± SD. (L) Representative TEM images of ER–mitochondria tethering in ARL6IP1 KO or WT MEFs. ER–mitochondria contact sites were indicated by arrowheads (left, 52,000×; scale bar: 200 nm; ER, endoplasmic reticulum; Mito, mitochondria; MAM, mitochondria-associated ER membranes). Quantification of ER–mitochondria contact length represents mean ± SD of ≥10 images/group and is calculated in GraphPad Prism 7.0 (right). *P < 0.05; **P < 0.01. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F5.