Figure 3. DDX28 is Required for mtDNA Gene Expression.
(A) Metabolic labeling of cytoplasmic or mitochondrial translation products with [35S]-methionine (in the absence or presence of emetine, a cytoplasmic protein synthesis inhibitor). Mitochondrial translation products are indicated on the right.
(B) Kinetics of in vivo mitochondrial translation performed as in panel A. The upper portion of the membrane was Ponceau-stained for loading control.
(C–E) Steady-state levels of mitochondrial rRNA and mRNA levels in control and siDDX28 cells estimated by (D) Northern blotting or (C, E) qPCR. Error bars represent the mean ± SD of three independent repetitions. * denotes p < 0.05.
(F–G) Steady-state levels of the indicated proteins by immunoblotting (F) and of mitochondrial rRNAs by northern blotting (G) in siNT and siDDX28 cells, further silenced or not for PNPase.
(H) Immunoblot analyses of the steady-state levels of mitoribosome LSU and proteins and ribosomal assembly factors (RAF) in whole cell lysates from control HEK293T and siDDX28 cells.
(I) Steady-state levels of mRNAs for mitochondrial ribosomal proteins in siNT and siDDX28 cells estimated by qPCR. Error bars represent the mean ± SD of three independent repetitions. * denotes p < 0.05.
(J) Immunoblot analyses of the steady-state levels of mitoribosome LSU and SSU proteins and ribosomal assembly factors (RAF) in mitochondria (mitos) from HEK293T, 143B and 143B-206 rho0 (ρ0) cells. See also supplementary Fig S2.