Fig. 1.

Reduction of LDHA expression by siRNA leads to increased oxygen consumption and oxidative stress-induced cell death of P493 human lymphoma B cells. siRNAs targeting human LDHA (SMARTpool) were transfected via electroporation to knock down the LDHA expression transiently. (A) Immunoblotting was performed on whole-cell lysates, probed with rabbit monoclonal anti-LDHA, and reprobed with anti-α-tubulin as a loading control. (B) Oxygen consumption of P493 cells was determined by the use of a Clark-type oxygen electrode at 72 h posttransfection with siLDHA (slope = −1.7) or siControl (slope = −0.7). (C) Intracellular ROS production was detected with DCFDA fluorescence and monitored by flow cytometry at 72 h posttransfection with siLDHA or siControl in the presence or absence of NAC. (D) Cell death was determined by flow cytometry of annexin V- and 7-AAD-stained cells at 96 h posttransfection with siLDHA or siControl in the presence or absence of NAC (20 mM added 24 h after transfection). The number in each figure represents the average percentage (±SEM) of dead cells. The number of dead cells treated with siLDHA compared with the control group has a P value of 0.0002 using the Student’s t test; those treated with siLDHA and NAC compared with the siLDHA group have a P value of 0.001. (E) Cell population growth of siControl cells compared with cells treated with siLDHA grown in the presence or absence 5 mM NAC added daily starting 24 h after transfection. Relative cell numbers at day 4 for siControl vs. siLDHA and siLDHA vs. siLDHA + NAC have a P value of 0.008 and 0.004, respectively. For siControl vs. siControl + NAC, the P value is 0.63.