JCS026625 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Immunoblotting of cell lysates prepared from the simultaneous knockdown cells. HeLa cells were transfected with the siRNAs as described in Fig. 2D. After microscopic analysis, total cell lysates were prepared and subjected to immunoblotting with antibodies to LETM1, Drp1, OPA1, and Tom40 as a loading control.

• Supplemental Figure S2 -

  Fig. S2. TMRM staining of the LETM1-knockdown cells. HeLa cells transfected with siRNA for GFP (control) or LETM1 were incubated for 15 minutes with TMRM at the indicated concentrations, and washed with medium. Live images were acquired by confocal microscopy.

• Supplemental Figure S3 -

  Fig. S3. Pulse chase experiment of LETM1 in the BCS1L-knockdown cells. HeLa cells transfected with the GFP- or BCS1L-siRNA were radiolabeled for 30 minutes and chased with methionine and cysteine in the indicated time. The cell lysates were subjected to immunoprecipitation with anti-LETM1 antibody and analysis by Bio Image Analyzer Fujifilm BAS-2500.

• Supplemental Figure S4 -

  Fig. S4. Solubilization of the subunits of the respiratory chains. The mitochondrial fractions (1 mg/ml) from cells transfected with siRNA for LETM1 (A) or BCS1L (B) were solubilized at 4°C for 60 min with 1% digitonin, centrifuged at 100,000 g for 15 minutes, and separated into supernatant (sup) and pellet (ppt) fractions. The fractions were subjected to analysis with immunoblotting with antibodies to the indicated proteins.

• Supplemental Figure S5 -

  Fig. S5. Correlation between changes in mitochondrial morphology and disassembly of the suprecomplexes of the respiratory chains. HeLa cells were transfected with the GFP- or LETM1-specific siRNAs once (#1), twice (#2) or three times (#3) in a 48-hour interval. Right panel, mitochondria prepared from the transfected cells were solubilized and analyzed by Blue-Native gel electrophoresis and immunoblotting with antibody to subunit IV of Complex IV. The ratio (LETM1-siRNA/GFP-siRNA) of the supercomplexes is marked with a red line in the graph. Mitochondrial morphologies of the transfected cells were analyzed as described in Fig. 2C. Percentages of cell with swollen mitochondria are shown as a blue line in the graph.

• Supplemental Figure S6 -

  Fig. S6. Effects of nigericin on mitochondrial morphology. HeLa cells that stably expressed Su9-DsRed were transfected with siRNA for GFP (panels a, b) or LETM1 (panels c, d). After siRNA transfection, cells were treated for 30 min with DMSO (panels a, c) or 1 µM nigericin (panels b, d). Live images were obtained by confocal microscopy. Scale bar, 10 µm.