Fig 1: Validation of CRISPR screen through genetic KO, chemical inhibition, and mitochondrial depletion. A, Compiled SP-2509 IC50 data and western blot images for monoclonal KO cells; mitochondrial ribosomal protein L45 (MRPL45 KO), Ubiquinone-Cytochrome C Reductase, Rieske Iron-Sulfur Polypeptide 1 (UQCRFS1 KO), Cytochrome C1 (CYC1 KO). Compiled data are from n = 3 independent experiments. Statistical analysis was performed by using a Student t test. Data that reached a significance P value of =0.05 are denoted with *. Densitometry values are indicated for each protein band and are quantified as previously described in the Materials and Methods. B, Compiled SP-2509 IC50 data for simultaneous treatment of indicated cell lines with SP-2509 and ETC inhibitors, rotenone (CI), antimycin A (CIII), or oligomycin (CV). Compiled data are from n = 3 independent experiments. Statistical analysis was performed by using a Student t test comparing each inhibitor treatment with the untreated cell lines, with * denoting a significance (P value) of = 0.05, and values that are not significant denoted as n.s. C, Western blot analysis of mtø cells for mitochondrial proteins using Membrane Integrity WB Antibody Cocktail (Abcam; ab110414). Samples are denoted with A673 cells infected with retroviral constructs for YFP-Parkin and iLuc (mock infection), that were treated with either DMSO or FCCP for 48 hours. Blots for Ubiquinone–cytochrome c reductase core protein 1 (UQCRC1) represents the mitochondrial intermembrane (IM), Cyclophilin D in the mitochondrial matrix, cytochrome C1 (CYC1) in the mitochondrial intermembrane space (IMS), and a-tubulin was used as a loading control. Depletion of these proteins in the YFP-Parkin–FCCP condition indicates depletion of mitochondria from the cells. D, Dose–response curve comparing A673 cells expressing YFP-Parkin either treated with DMSO or FCCP. E, Compiled SP-2509 IC50 data for mtø cells. Compiled data are from n = 3 independent experiments. Statistical analysis was performed by using a Student t test comparing each inhibitor treatment with the untreated cell lines, with * denoting a significance (P value) of = 0.05.
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