Fig 1: Functional deletion of PTCD2 gene affects translation and steady-state level of COIII protein in human mitochondria. (A) PTCD2 gene disruption with CRISPR/Cas9 technology. The sequence of exon 1 is shown in a black box. Start codon is green. SgRNA sequences are shown as black horizontal arrows indicating the direction from 5' to 3'. Green scissors with vertical dashed lines show the intended positions of Cas9 cleavage. Red scissors with vertical dashed lines show the real positions of Cas9 cleavage. The DNA ends joint together after Cas9 cleavage and DNA repair are shown in red and blue boxes, and the region between them was deleted, which is proved by sequencing chromatogram. (B) Western blot analysis of wild-type HeLa and PTCD2 knock-out cell lysates with antibodies against PTCD2 and Cox4. (C) Mitochondrial translation products of HeLa (WT) and knock-out cells (KO). Cytosolic translation was blocked by cycloheximide and cells were exposed to 35S-methionine as described in Materials and Methods. Newly synthesized proteins are designated at left, representative radioautograph shown (n = 4). (D) Quantification of mitochondrial translation products in wild-type HeLa and PTCD2 knock-out cells. Mean values ± SD are shown, n = 4, **—p-value < 0.01 by Student’s test. (E) Western-blot analyses of HeLa and PTCD2 knock-out cells lysates with antibodies to COIII, COII and Cox4. (F) Phenotype restoration. Western blot analyses of HeLa (WT), knock-out (KO), knock-out with insertion of PTCD2 cDNA induced with doxycycline for 24 h (KO+ 24 h), and 48 h cells lysates with antibodies against COIII and PTCD2.