Mcsquant10

For determination of IFT20 expression in RCC4+pVHL-, RCC4-, RCC4 siCtl-, RCC4 siVDAC1-, RCC4 siLGMN- cells, cellular suspensions (1 × 10⁶ cells) were resuspended in 4% PFA for 15 min and washed 3 times in cold PBS/BSA 0.1%. Cell suspensions were then incubated with a polyclonal rabbit anti-IFT20 (1 µg/1 × 10⁶ cells; Proteintech) for 1 h at 4°C in PBS/BSA 0.1% buffer. After washing, cells were incubated with Alexa 488-conjugated secondary goat anti-rabbit antibody (µg/1 × 10⁶ cells;) for 30 min and washed prior to analysis. Samples were collected with Miltenyi MCSQuant10 (Bergisch Gladbach, Germany) and analyzed with FlowJo Software. Secondary antibody in the control experiment was identical to that described supra.

For determination of the cell cycle in RAS MEF (Wt, Vdac1^−/−, Cleavable#1, Cleavable#2, UnCleavable#1, UnCleavable#2), cellular suspensions (5 × 10⁵ cells) were resuspended in HBSS + FBS 10% and rinsed 3 times in cold PBS. Then, the cells were fixed with Ethanol 70% at 4 °C. Cell suspensions were then incubated with Propidium iodide solution 1 mg/mL (Sigma-Aldrich) and RNAase (Life Technologies, Carlsbad, CA, USA) for 30 min at 4 °C. Samples were collected with Miltenyi MCSQuant10 (Bergisch Gladbach, Germany) and analyzed with FlowJo Software (Version 10.6).