Fig. 5: FR054 induces UPR activation and intracellular ROS increase.

a mRNA expression of HSPA5, ATF4, DDIT3, and XBP1 in MDA-MB-231 cells following 24 and 48 h of FR054 treatment. b Analysis of XBP1 mRNA splicing in MDA-MB-231 cells following 24 and 48 h treatment with FR054 or 6 h with Thapsigargin (Th). u-XBP1 indicates unspliced form and s-XBP1 indicate spliced form. Protein expression (c) and densitometric quantification of CHOP (d) and eIF2α phosphorylation (e) in MDA-MB-231 cells following 24 and 48 h treatment with FR054. Intracellular hydrogen peroxide (f) and mitochondrial superoxide (g) measured by FACS analysis after DCHF₂DA and Mitosox staining, respectively, in MDA-MB-231 cells upon treatment with 1 mM FR054 for 24 and 48 h. h Hydrogen peroxide levels measured with DCHF₂DA in MDA-MB-231 upon treatment with 1 mM FR054 for 48 h or co-treated with different doses of NAC. i Viable cell count of MDA-MB-231 cells upon treatment with 1 mM FR054 and different doses of NAC. j Caspase-3 activation and CHOP expression of the samples described in i. All data represent the average ± s.d.; *p < 0.05, **p < 0.01, ***p < 0.001 (Student’s t-test; comparison with FR054-not-treated sample); N = 3