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. 2024 Jul 5;131(5):918–930. doi: 10.1038/s41416-024-02774-9

Fig. 3. Induction of proteotoxic stress, UPR, and apoptosis in MDA-MB-231 cell line treated with carfilzomib and lopinavir.

Fig. 3

a BiP-GFP FRAP analysis in MDA-MB-231 cells treated with carfilzomib, lopinavir, or their combination. FRAP images were acquired 1 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. The data represent the mean ± SD of T half (in ms) recovery of BiP-GFP fluorescence in the individual cells in three independent experiments. Statistical significance was determined with one-way ANOVA with Tukey post-test. * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001. b Induction of spliced XBP1 (sXBP1) presented as a ratio of spliced versus unspliced XBP1 RNA variants normalized to GAPDH and a time-point 30 min prior to the treatment. c Representative western blot image of the cleavage of ATF6 protein, represented by a cleaved form of ATF6 and obtained 2 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. GAPDH served as an internal loading control. d Induction of ATF3 expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. e Induction of total XBP1 (tXBP1) expression, normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. f Induction of BIP expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. g Induction of CHOP expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. h Induction of NOXA expression normalized to GAPDH and a time-point 0 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. i Representative western blot image of the induction of PARP on a protein level, represented by a cleaved form of PARP p85 and obtained 24 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. GAPDH served as an internal loading control. j Induction of early and late apoptosis represented by Anexin V + and PI− + PI+ positivity, evaluated by flow cytometry 48 h after the 1 h pulse treatment with carfilzomib or continuous treatment with lopinavir. The data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with an unpaired two-sided t-test. * represents p < 0.05, *** represents p < 0.001. In all qPCR experiments, the data represent the mean ± SD from 3 independent experiments. Statistical significance was determined with one-way ANOVA with Tukey post-test. * represents p < 0.05, *** represents p < 0.001. ATF3 Activating Transcription Factor 6, ATF6 Activating Transcription Factor 6, BiP-GFP Binding immunoglobulin Protein green-fluorescent protein, BTZ bortezomib, CFZ carfilzomib, CHOP DNA Damage-Inducible Transcript 3, GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase, LPV lopinavir, mero-GFP Mammalian Endoplasmic Reticulum-localized redox-sensitive Green-Fluorescent Protein, NOXA Phorbol-12-Myristate-13-Acetate-Induced Protein 1, PARP Poly(ADP-Ribose) Polymerase 1, XBP1 X-Box Binding Protein 1.