Overexpression of AFG3L2 suppresses actinonin effects on translation and OPA1 processing. (A) Growth curve of U2OS cells in actinonin after treatment with the indicated siRNA for 5 d. The data represent mean ± SD; n = 3. (B) A representative, extended 3-h 35S metabolic labeling in the presence or absence of actinonin after a 5-d siRNA knockdown of AFG3L2 in HEK293 cells. The asterisks represent aberrant translation products. (C) Quantification of the signal intensity of MT-ATP6 relative to MT-CO1 in B from independent biological experiments. Data represent mean ± SD; n = 4. (D, left) Quantification of the signal intensity of MT-ATP6 relative to MT-CO1 from 35S metabolic labeling from four independent biological experiments with AFG3L2 transient overexpression in HEK293 cells treated with different doses of actinonin for 3 h. Data represent mean ± SD; n = 4. (right) An immunoblot of cells. The asterisk is a nonspecific band detected with the AFG3L2 antibody. (E) An immunoblot of HEK293 cells treated with the indicated siRNA and actinonin doses. (F, left) A representative immunoblot of HEK293 cells treated with the indicated siRNA for 5 d with and without chloramphenicol (CA) or cycloheximide (CHX) for the indicated times. (right) Three panels of data generated from the same samples, only separated on different SDS-PAGE and transferred to independent membranes. a.u., arbitrary units.
