Figure 1.

Knockdown of AFG3L2 and YME1L by shRNA leads to increased accumulation of complex I, IV, and V subunits and reduced growth rate of the cells. (A) Knockdown (KD) of YME1L, AFG3L2, or their combination by stable shRNA expression leads to marked reduction of corresponding protein levels. Whole-cell lysates from shRNA knockdown cell lines were immunoblotted with antibodies to YME1L, AG3L2, or mtHSP70. The control cell line was prepared by transfecting HEK293 cells with the non-silencing (scrambled) shRNAmir vector. (B) Altered accumulation of respiratory chain subunits in the KD cells. Whole cell lysates were immunoblotted with antibodies to Ndufb6, Cox1, Cox5a, Cox4, mtHSP70, and F1-alpha. (C) The quantification of western blot signal from B by densitometric analysis. The values are shown as mean ± SD. * p < 0.05; ** p < 0.01. Western blot images representative of three independent experiments are shown. (D) Stable KD cells were seeded in six-well plates at 5 × 10⁴ cells per well and cultured in DMEM (Dulbecco’s Modified Eagle Medium) containing 1 ug/mL puromycin. Viable cells were counted every 24 h for a total of 7 d and the values (mean ± SD) were plotted. * p < 0.05.