Abstract
Mitochondrial respiratory chain inhibitors (MRCIs) are indispensable for studying cellular bioenergetics and its effects on various cellular processes. However, their off-target (those not mediated by respiratory chain inhibition) effects remain incompletely understood, even though their comprehension is crucial for the accurate interpretation of experimental outcomes. Here, we use four isogenic cell line pairs, which either have mitochondrial DNA (mtDNA) or lack it (ρ(+) or ρ(0) cells, respectively), to assess the possible off-target effects of widely used MRCIs antimycin A, oligomycin A, rotenone, and carbonyl cyanide m-chlorophenylhydrazone (CCCP). We examined clonogenic growth of ρ(0) cells and ρ(+) cells under conditions that either require the functional respiratory chain or do not. Unexpectedly, ρ(0) cells were sensitive to rotenone and antimycin A, even though these cells lack functional complex I and complex III, respectively, suggesting a nonspecific effect of these drugs. Furthermore, ρ(0) cells were more sensitive to CCCP than their ρ(+) counterparts. Intriguingly, the loss of the clonogenic potential in ρ(+) 143B cells could not be precisely correlated to the decrement of the mitochondrial inner membrane potential. These findings underscore the significance of off-target effects of MRCIs, which must be carefully considered when designing, conducting, and interpreting experiments involving these inhibitors.