Hygromycin b

HEK293 cells were transiently transfected using calcium phosphate.
To generate the stable cell lines the eukaryotic expression vectors and an empty vector (pcDNA5), used as a control in uptake experiments, were transfected into the FlpIn-HEK293 cell line (Life Technologies, Paisley, UK) using calcium-phosphate and selected for positive clones with 100 μg/ml hygromycin B (Life Technologies, Paisley, UK).
Cell clones with the highest transport activity were chosen for further study. They were routinely cultured in Dulbecco's modified Eagle's medium (DMEM) (Lonza Verviers SPRL, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (v/v), 2 mM glutamine, and a mixture of antibiotics (100 U penicillin, 0.1 mg/ml streptomycin, and 0.25 mg/ml fungizone) (Life Technologies, Paisley, UK) in the presence of 100 μg/ml hygromycin B. They were maintained at 37°C in a humidified atmosphere containing 5% CO₂.

Cultured U2OS, HT1080, SH‐SY5Y, HeLa, or CV‐1 cells were grown in DMEM with glutamax and 4.5% (w/v) glucose (Invitrogen, Carlsbad, CA, USA) supplemented with 10,000 U/ml penicillin, 10,000 μg/ml streptomycin (Merck Millipore, Darmstadt, Germany), 100 nM Na pyruvate (Sigma‐Aldrich, St. Louis, MO, USA), and 10% (v/v) FCS (Merck Millipore, Darmstadt, Germany) at 37°C and 5% CO₂.
For Drp1 knockdown, the Drp1‐shRNA‐expressing plasmid pREP4 (Lee et al, 2004) was used. Plasmids were transiently transfected by electroporation using the nucleofector Kit V (Lonza, Basel, Switzerland). To enrich transfected cells, selection was performed 24 h after transfection using 200 μM hygromycin B (Life Technologies, Carlsbad, USA) for 2 days, followed by 5–6 days of incubation with lower amounts of hygromycin B (50 μM) in the growth medium.

Logarithmic L. major promastigotes were harvested by centrifugation at 600 × g for 10 min, washed once in sterile PBS and resuspended at 3 × 10⁷ cells/ml in 100 μl of Human T Cell Nucleofector solution (Lonza, Basel, Switzerland). Cells were transferred to Amaxa electroporation cuvettes maintained at 4 °C and already containing 10 μg of DNA. Cells were then electroporated with the program U-033 on the Nucleofector machine (Amaxa GmbH, Cologne, Germany). Following electroporation, cells were incubated overnight in their culture medium and transfectants were selected with 30 μg/ml hygromycin B (Life Technologies, France) for single transfection and with 30 μg/ml hygromycin B and 15 μg/ml blasticidin (Life Technologies, France) for double transfections.

HEK293 Flp-In T-REx cells (Life Technologies) were maintained in DMEM supplemented with 10% FBS at 37°C in a 5% CO2-humidified incubator. Stable transfection of HEK293 Flp-In T-REx cell lines was performed using the Flp-In recombinase system (Life Technologies) according to the manufacturer’s instructions to generate isogenic stable cell lines. Stable integrants were selected using 200 μg/ml hygromycin B (Life Technologies), whereas stable cell lines were maintained in 100 μg/ml hygromycin B.

Tet-inducible stable clones were generated in Flp-In-293 cells (Invitrogen, #R750-07) by co-transfection with either 10 µg of pTO-GFP-T or pTO-GFP-T-p27(3ʹUTR) plasmids and 1 µg of plasmid pOG44 with Lipofectamine 2000 (Invitrogen, #11,668-027). Stable clones were selected in the presence of 200 µg/ml of hygromycin B (Invitrogen, #10,697-010) and maintained in 50 µg/ml hygromycin B. GFP-T and GFP-T-p27(3ʹUTR) HEK293 cells were cultured in 12-well plates and transfected with small-interfering RNAs (siRNAs) and control siRNAs (Scr [71]) at ~60% cell confluency using Lipofectamin RNAiMAX reagent (Life Technologies, #13,778-100). In brief, 3 µl of Lipofectamine RNAiMAX was diluted in 50 µl of Opti-Mem (Gibco, # 31,985,070) and combined with 10 pmol of respective siRNA supplied in 50 µl of Opti-Mem and incubated for 15 min at room temperature (RT). The mixture was added to cells, which were further grown in media supplemented with 1 µg/ml of tet (Fisher, #BP-912-100) and treated with 20 µM of CP for 15 h. Plasmid transfections were performed at 70% cell confluency with 2 µg of pEGFPC1-6XHis-FLKSRP (Addgene, #23,001) and Lipofectamine 2000 (Invitrogen, #11,668-027).

Knockdowns of Mic10, Mic13, Mic19, Mic25, Mic26, Mic27, Mic60, OPA1, or ATP5ME were achieved by transfection with the respective siRNA pool (siTOOLs Biotech). Cells were cultivated for 2–5 days after transfection.
Knockdown of DRP1 was achieved by transfection with the shRNA expression plasmid pREP4 (Lee et al, 2004). After transfection, cells were selected with DMEM supplemented with 250 μM hygromycin B (Life Technologies, Carlsbad, USA) for 2 days. Afterward, cells were selected for 5 days with 50 μM hygromycin B (Life technologies). All knockdowns were verified by Western blotting and immunofluorescence microscopy.