Fig. 6

PIGBOS regulates the amplitude of UPR, and apoptosis. a PIGBOS-KD and control HEK293 cells were treated with indicated concentrations of tunicamycin (TM) followed by RT-PCR analysis of XBP-1 splicing (unspliced XBP1 (XBP1u) and spliced XBP1 (XBP1s)). GAPDH was used as a loading control. A stronger UPR correlate with higher XBP1s/XBP1u ratio in PIGBOS-KD cells, which could be rescued by expression of a siRNA-resistant PIGBOS-FLAG. b XBP-1 mRNA splicing was measured in PIGBOS-KO and control HEK293 cells treated with indicated doses of brefeldin A (BFA) for 3 h. c ATF6-dependent luciferase reporter measures the activation of another branch of the UPR pathway. PIGBOS-KD led to increased luciferase activity indicative of a greater UPR, and the expression of the siRNA-resistant PIGBOS-FLAG reversed this effect. d RT-qPCR quantitation of a panel of UPR target genes in PIGBOS-KD and control HEK293 cells after an 8-hour treatment with vehicle or 1 μg/ml of TM. e Caspase-3 activity in mock and PIGBOS-KD U2OS cells treated with thapsigargin (TG) for 27 h. f Cleaved PARP and caspase-3 levels were measured by Western blot in PIGBOS-KD and control U2OS cells treated with TG for 27 h. g PIGBOS-KD and control U2OS cells were treated with indicated doses of TG for 48 h followed by cell viability measurements using MTT. h HEK293 PIGBOS-KO and WT cells were transfected with PIGBOS variants constructs as indicated. Forty-eight hours later, cells were incubated with 1 µg/ml of tunicamycin for 3 h. XBP1 splicing activity was measured by RT-PCR. Error bars, s.e.m. The p-values were calculated using two tailed unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, n = 3 experiments. Source data for Fig. 6c–e and 6g are provided as a Source Data file