Figure 1.

Knockdown of PDK4 in HCC cells induced apoptotic cell death and increased abnormal mitochondrial numbers and ROS production. (A) MTS assay to determine cell viability in control (shCTL) and PDK4 knockdown (shPDK4) Huh7 (left) and Hep3B (right) cells. (B) Colony formation assay for 14 days in shCTL and shPDK4 Huh7 and Hep3B cells. (C) WB of PARP protein. (D) Caspase-3, -8 and -9 activities were measured using a colorimetric method. (E) Flow cytometry using Annexin V and PI staining to determine the apoptosis rate. (F-a) Transmission electron microscopy (TEM) of mitochondria morphology in shCTL and shPDK4 Huh7 cells at day 10 after shRNA transfection. Blue arrow: condensed chromatin; yellow arrow: abnormal mitochondria; green arrow: mitochondria vacuolization; red arrowhead: normal (shCTL) or swollen and distorted (shPDK4) mitochondria cristae. A minimum of 10 cells per condition were examined. (F-b) Seahorse analysis in shCTL and shPDK4 Huh7 cells. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were monitored following the sequential addition of oligomycin (an inhibitor of ATP synthase), FCCP (uncoupling electron transport from ATP generation in the inner Mitochondria membrane) and rotenone/antimycin A (R/AA, inhibiting mitochondria complex I and III respectively) using the 24-well Seahorse XFe24 Extracellular Analyzer. Individual wells were normalized using the amount of DNA (ng). (F-c) ROS levels in shCTL and shPDK4 Huh7 cells. (F-d) PDH activities in shCTL and shPDK4 Huh7 cells. Cells were cultured in DMEM medium containing either normal (N, 4.5 g/L) or low (L, 1.0 g/L) glucose for 48 hr. D, F-c: Data are shown as mean ± SEM of three independent experiments with triplicate assays. *P < .01 vs shCTL. F-d: Data are shown as mean ± SEM. *P < .01 vs N-shCTL; ^#P < .01 vs L-shCTL.