Figure 5.
Effects of RMND1 Knockdown in HeLa Cells
HeLa cells were cultured in DMEM with 10% fetal bovine serum (FBS) until they were 70%–80% confluent. Transfection with a scramble shRNA-pLKO plasmid (negative control) and a RMND1-specific TRC shRNA-pLKO plasmid construct (TRCN0000135730, Sigma Aldrich) was mediated by Lipofectamine 2000 (Invitrogen). Five hours after transfection, cells were selected with Puromycin in DMEM 2% FBS, and transfected clones were individually expanded in DMEM 10% FBS. RMND1 knockdown was assessed in 30 clones by qRT-PCR and immunoblot analysis.
(A) Activity of mitochondrial respiratory-chain enzymes.
(B and C) Immunoblot analysis of the steady-state levels of mitochondrial respiratory enzymes (B) and pulse labeling with [35S] methionine of mitochondrial proteins (C). A total of 5 × 105 cells cultured in 10 cm culture plates were washed in methionine-free DMEM and subsequently incubated in the same medium supplemented with 15% dialyzed FBS, 1.2 mM sodium pyruvate, and glucose for 30 min. Cytosolic protein synthesis was inhibited by the addition of emetine (0.1 μg/μl) for 7 min at 37°C. Mitochondrial proteins were labeled with 50 μCi [35S]-methionine Redivue (Amersham Biosciences, Piscataway, NJ) in methionine-free medium and incubated for 1 hr at 37°C. After treatment, cells were incubated for 10 min in DMEM supplemented with 10% FBS and were collected by scraping. Protein aliquots (15 μg per sample) were electrophoresed in a 4%–12% Bis-Tris polyacrylamide gradient gel for 4 hr at 85V. The gel was dried for 60 min at 80°C and analyzed with a phosphorimager. The seven subunits of complex I (ND), three subunits of complex IV (COX), and two subunits of complex V (ATP) are indicated at the left. Values are expressed as percentages of the control and are represented as the mean ± standard deviation. One asterisk (∗) indicates a Student’s t test p < 0.05; ∗∗ indicates a Student’s t test p < 0.01.