Figure 6. p53 inhibition by siRNAs and phosphoSer¹⁵p53 antibody prevented the reversion of mtDNA depletion.

(A) All siRNA transfections were performed with 12.5 nM siRNAs directed against p53 mRNA (p53 siRNAs) or non-targeting siRNAs (NT) and DharmaFECT4® transfection reagent used as a control (c). To check siRNA transfection efficiency, Western Blots were performed at 48 h using p53 or GAPDH (loading control) antibody. (B) The knockdown of p53 expression by siRNAs relative to GAPDH was quantified using the Bio1D software (mean values ± SEM of three experiments *p<0.05) (C) DNA was isolated from untransfected cells or transfected with NT siRNAs or with p53 siRNAs. Cells were also permeabilized with 0.1% Triton X100 and pretreated for 1 h with 1 µg/ml phosphoSer¹⁵p53 antibody (phosphoSer¹⁵Ab). Cells were then treated for 0 to 6 h with 30 ng/ml TNF-α. The quantification of mtDNA content was performed by simultaneous real-time qPCR amplification of fragments encoding mitochondrial 12S rRNA and nuclear 18S rRNA serving as a reference gene. Results are expressed in mtDNA over nDNA relative ratio (mean values ± SEM of three independent experiments with five replicates, *p<0.05).