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. 2022 Apr 20;8:215. doi: 10.1038/s41420-022-01009-1

Fig. 3. Negligible TP53-dependent induction of NOXA by PIs.

Fig. 3

A Cells were incubated with 5 nM PI (BTZ, CFZ, IXZ; + 10 µM Q-VD-OPh) for 8 h and expression of TP53 and NOXA were analyzed by Western blot, using GAPDH as loading control. B, C Cells were transfected with siCTRL or siTP53 and subsequently incubated with indicated inhibitors for 8 h. B Expression of PMAIP1 was analyzed by qRT-PCR and C Western blot was performed to analyze expression of TP53, MCL-1, NOXA, and β-ACTIN (loading control). D Western blot analysis of SW982/WT incubated with PIs alone or in combination with ABT-199 (+ 10 µM Q-VD-OPh) for 8 h were carried out to analyze expression of NOXA (β-ACTIN as loading control) in the absence or presence of 10 µM cycloheximide. E qRT-PCR and Western blot analysis of PMAIP1/NOXA expression in SW982/WT cells incubated with ABT-199, BTZ or ABT-199&BTZ for 8 h in the absence or presence of 1 µM actinomycin D were performed (GAPDH as loading control). F SW982/WT cells were incubated with BTZ, CFZ or IXZ in the absence or presence of ABT-199 for 8 h. Expression of NOXA (GAPDH as loading control) was analyzed by Western blot. G SW982/WT were transfected with siCTRL or siTP53 and incubated with the indicated PIs alone or in combination with ABT-199. Subsequently, PMAIP1 expression was analyzed by qRT-PCR. H SW982/WT cells were transfected with siCTRL or siTP53 and incubated with ABT-199 alone or in combination with BTZ, CFZ, or IXZ for 8 h. Cell extracts were analyzed for the expression of TP53, MCL-1 and NOXA by Western blot (β-ACTIN as loading control). Graphs show mean and individual data points. Asterisks indicate identical Western blot due to cutting/reprobing. Act-D actinomycin D, BTZ bortezomib, CFZ carfilzomib, CHX cycloheximide, IXZ ixazomib, CTRL control.