BH3-only sensors Bad, Noxa and Puma are Key Regulators of Tacaribe virus-induced Apoptosis
Fig 5
Protein expression, phosphorylation and nuclear translocation of p53 in TCRV-infected cells.
(A) Transcript levels of p53. TCRV infection was performed in Vero76 cells at an MOI of 2 and samples were harvested each day for RNA extraction. Expression of p53 was quantified using RT-qPCR with a gene specific primer set (S1 Table) at the indicated time points between 0–4 days post infection (dpi). GAPDH levels were used for standardization and fold change in mRNA levels of TCRV-infected cells (compared to mock-infected cells) was calculated using the 2-ΔΔCt method. Values represent the means and standard deviations from three independent replicates. (B) Protein expression levels and phosphorylation of p53. Cell lysates from TCRV-infected cells between 1 to 4 dpi were analysed by Western blot for total p53 expression, as well for phosphorylation at Ser15 or Ser392 with the respective antibodies. Vinculin served as a loading control. Mock cells served as a negative control, while CPT treatment (10 μM) was used as a positive control (Ctrl ind.). An arrowhead indicates an unidentified cross-reactive band that is also detected with the anti-p53 antibody. Western blots were evaluated by measuring pixel intensities with normalization to the associated loading control. Quantifications are based on at least two independent experiments. Statistical significance was determined using two-way ANOVA (*p≤0.05, **p≤0.01, ns not significant). (C) p53 nuclear translocation. Mitochondria were stained 3 dpi with TCRV using MitotrackerRed (red), followed by antibody staining using anti-p53 (green) and anti-TCRV NP (magenta), and labeling of nuclei with DAPI (blue). Mock cells served as a negative control, while CPT treatment (10 μM) was used as a positive control (Ctrl ind.). Arrowheads highlight morphological changes consistent with cells in the late stages of apoptosis. Scale bars show a distance of 10 μm. (D) Impact of p53 inhibition on Puma and Noxa expression. Vero76 cells were treated daily with PFT-α (3, 10 and 30 μM) or DMSO only and either Mock- or TCRV-infected (MOI = 1). Cell lysates were harvested 4 dpi and analysed for Puma and Noxa expression via Western blot, while Vinculin served as a loading control. Puma and Noxa expression were quantified based on two independent experiments and normalized to the associated loading control. Statistical significance was determined using two-way ANOVA (**p≤0.01, ***p≤0.001, ns not significant).