Fig 2: The 24 h induction of XBP1s is accompanied by reduced PUMA protein levels and lower Caspase 3/7 activity in Tm-treated HeLa cells. A Representative changes in XBP1 and PUMA protein levels in HeLa cells treated with Tm (2.5 µg/ml) for 8 and 16 h as evaluated by Western blot. B The changes in XBP1 and PUMA protein levels in XBP1s-induced and -noninduced HeLa cells treated with Tm (2.5 µg/ml) 16 h were evaluated by Western blot (C) normalized to total protein levels and related to the noninduced, no-stress control, and *P < 0.05 was considered significant. XBP1s-induced (24 h) and -noninduced HeLa cells were treated with Tm (2.5 µg/ml) for 16 h in the presence or absence of 4µ8C (20 µM), a specific IRE1 activity inhibitor. D The caspase 3/7 activity was monitored by luminescence and expressed in relative light units (RLU). Cells for each time point were seeded in triplicate, and the experiments were repeated three times. Error bars represent standard deviations and *P < 0.05 was considered significant. The corresponding changes in XBP1 protein levels were evaluated by Western blot. E HeLa cells were treated with Tm (2.5 µg/ml) for 16 h in the presence or absence of a specific siRNA against BBC3 (PUMA). The caspase 3/7 activity was monitored by luminescence and expressed in RLU. Cells for each time point were seeded in triplicate, and the experiments were repeated three times. Error bars represent standard deviations, *P < 0.05 was considered significant. The corresponding changes in PUMA protein levels were evaluated by Western blot

Fig 3: ISRIB decreases G93A SOD1-dependent neuronal death.a Scheme depicting the modes of action of GSK2606414 (GSK), ISRIB, Sephin1, and 4µ8C in PERK and IRE1-dependent signaling. XBP1u (unspliced XBP1), XBP1s (spliced XBP1), and RTCB (RNA ligase enzyme that joins the exons resulting from XBP1 intron removal by IRE1). b Western blot analysis of PERK, p-eIF2a, ATF4, XBP1s, and tubulin (loading control) from HEK293 protein extracts treated with 100 nM thapsigargin (Thap) and 500 nM ISRIB, 500 nM GSK, 50 µM 4µ8C, and DMSO as control. Representative experiment from at least n = 3. c Cumulative hazard estimates of primary neurons transfected with SOD1 pathological version G93ASOD1Ch and treated with 500 nM, 100 nM, and 10 µM ISRIB (Ch as control). CPH analysis; pooled data from three independent experiments (left panel) and five independent experiments (right panel). d Cumulative hazard estimates of primary neurons transfected with G93ASOD1Ch and treated with 500 nM, and 2 and 8 µM GSK (Ch as control). CPH analysis; pooled data from three independent experiments (left panel) and two independent experiments (right panel). e Cumulative hazard estimates of primary neurons transfected with aSyn pathological version (E46KSynCh) and treated with 10 µM ISRIB. CPH analysis; pooled data from three independent experiments. f Cumulative hazard estimates of primary neurons transfected with G93ASOD1Ch and treated with 5 µM 4µ8C (Ch as control). CPH analysis; representative experiment from two independent experiments. Number of neurons (n), non-significant (n.s.), *p < 0.05 and ***p < 0.001.

Fig 4: TUSC3 alleviates ER stress in prostate cancer cell lines.(a) CHOP and ENTPD5 expression was measured after 24 hours of 5 µM tunicamycin (TM) or DMSO treatment. Decreased CHOP induction in shTUSC3 PC3 cells indicates reduced ER stress response (lane 7 vs. 8). (b) Cells were treated with 5 µM tunicamycin (TM) or DMSO for 24 hours. TUSC3 mRNA expression was assessed using qRT-PCR. (c) UPR was assessed by expression levels of BiP, IRE-1 and XBP-1s in serum starved (72 h) prostate cancer cells. XBP-1 mRNA splicing was also measured by semiquantitative RT-PCR. Intensity of the bands was quantified and ratio of spliced to unspliced isoform was calculated. Increased ratio indicates elevated levels of ER stress. Cropped blots are depicted for clarity reasons and full-length blots will be provided upon request. (d) qRT-PCR was used to evaluate the mRNA expression of BiP and spliced XBP-1 isoform. Silencing of TUSC3 leads to an increase in BiP expression in PC3 cells and conversely to an increase in spliced XBP-1 in DU145 cells, suggesting opposing effects on UPR signaling.* p = 0.03 and ** p = 0.04.

Fig 5: 6-TG and 6-TGo activate the UPR. (A) A549 cells were treated with 6-thioguanine (6-TG), 6-thioguanosine (6-TGo), 6-mercaptopurine (6-MP), 5-fluorouracil (5-FU), or ribavirin at the indicated concentrations for 6 h prior to harvesting lysates for immunoblotting for the indicated cellular proteins. Tunicamycin (TM) at 5 µg/ml served as a positive control for UPR activation. ATF6* indicates a lower-molecular-weight species that is not cleaved to its active form. (B) cDNA was generated from total RNA that was isolated from treated cells. XBP1 mRNA splicing was determined by the semiquantitative RT-PCR splicing assay. XBP1u1 and XBP1u2 indicate the cleaved products from digesting the unspliced XBP1 cDNA with PstI.