FIGURE 1:

Stable knockdown of C7orf30 in human fibroblasts results in OXPHOS assembly defects and mitochondrial fragmentation. (A) SDS–PAGE immunoblot analysis of OXPHOS proteins in fibroblasts transduced with lentiviral shRNA constructs targeting C7orf30. The shRNA-A1 construct, which was not effective in knocking down C7orf30, was used as a control; shRNA-A2-C1 and -C2 are two clonal lines established from the shRNA-A2 construct. ND1, subunit of complex I; COX, subunits of complex IV; UQCRC1, subunit of complex III; SDHA, subunit of complex II; VDAC1, loading control. (B) BN–PAGE analysis of control or C7orf30-shRNA lines alone or expressing C7orf30-FLAG. Antibodies against individual subunits of OXPHOS complexes I–V were used for immunoblotting. (C) Northern blot analysis of RNA from control and C7orf30-shRNA fibroblasts. Hybridization was performed with probes specific for mitochondrial 12S and 16S rRNA (mt-rRNA), and the mitochondrial mRNAs encoding ATP6 and ATP8 of complex V, COX1 of complex IV, and ND5 of complex I. (D) Control and C7orf30-shRNA fibroblast lines stained with MitoTracker Green. Knockdown of C7orf30 (shRNA-A2) disrupts the mitochondrial network.