Fig 2: Disruption of Ser724phosphorylation attenuates the RNase activation of IRE1a in hepatocytes.A, primary hepatocytes from mice of the indicated genotypes were treated with DMSO control (Con), 10 µg/ml Tm for 4 h, or 1 µM Tg for 2 h. Immunoblot analysis of IRE1a protein, with upper Phos-tag gel analysis of IRE1a phosphorylation. Actin was used as the loading control. B–E, hepatocytes of the indicated genotypes were treated with 100 ng/ml or 10 µg/ml Tm for the indicated time intervals. B and D, immunoblot analysis of IRE1a phosphorylation along with XBP1s and CHOP protein levels. Upper Phos-tag gels are shown for the band-shift analysis of IRE1a protein phosphorylation with long or short exposure time. Tubulin was used as the loading control. C and E, quantitative RT–PCR analysis of Xbp1 mRNA splicing and the RIDD target Blos1 mRNA. F and G, hepatocytes were treated with 10 µg/ml Tm. F, cell viability analysis by CCK8 assay. G, immunoblot analysis of nuclear CHOP protein after 24 h of 10 µg/ml Tm treatment. Lamin A/C was used as the nuclear protein control. All data are presented as the mean ± SD (n = 2 or 3 independent experiments). *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed unpaired Student’s t test or two-way ANOVA. CCK8, Cell Counting Kit-8; CHOP, CCAAT-enhancer binding protein homologous protein; DMSO, dimethyl sulfoxide; IRE1a, inositol-requiring enzyme 1a; RIDD, regulated IRE1-dependent decay; RNase, ribonuclease; Tg, thapsigargin; Tm, tunicamycin; XBP1s, spliced X-box binding protein 1.

Fig 3: S724A mutation of IRE1a results in blunted XBP1s production with decreased PDI but increased CHOP expression in ER-stressed livers.A–D, immunoblot analysis of the phosphorylation and expression levels of the indicated proteins in livers of Ern1S724A/S724A mice and their Ern1WT/WT littermates following treatment with Tm or vehicle for 6 h (A and B) or 24 h (C and D). Shown are representative immunoblots for three individual mice per group (A and C), and quantification of XBP1s, PDI, and CHOP protein levels after normalization to tubulin (B and D). E and F, primary hepatocytes from male Ern1S724A/S724A mice and their Ern1WT/WT littermates were infected for 30 h with adenoviruses expressing GFP control or XBP1s protein. Cells were then treated with Tm (100 ng/ml) for 24 h. E, immunoblot analysis of XBP1s protein, IRE1a phosphorylation, as well as PDI and CHOP proteins. Shown also is the quantification of PDI and CHOP protein levels after normalization to tubulin as the loading control. F, quantitative RT–PCR analysis of the mRNA abundance of the indicated genes. Results in (E) and (F) represent three independent experiments. All data are presented as the mean ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 by two-tailed unpaired Student’s t test or two-way ANOVA. CHOP, CCAAT-enhancer binding protein homologous protein; ER, endoplasmic reticulum; IRE1a, inositol-requiring enzyme 1a; PDI, protein disulfide isomerase; Tm, tunicamycin; XBP1s, spliced X-box binding protein 1.

Fig 4: Effects of S724A mutation upon cell survival under ER stress in MEF cells. MEF cells of the indicated genotypes were treated with 10 µg/ml Tm for the indicated time intervals. A, immunoblot analysis of PERK phosphorylation and CHOP protein levels. Tubulin was used as the loading control. B, immunofluorescence staining of CHOP protein along with DAPI staining in MEF cells following Tm treatment. The scale bar represents 50 µm. C, cell viability of Tm-treated MEF cells was determined by CCK8 assay. Shown is the percentage of viability after normalization to their untreated controls. D, immunoblot analysis of cleavage of caspase-3 protein. Tubulin was used as the loading control. Data are presented as the mean ± SD (n = 3 independent experiments). ***p < 0.001 by two-tailed unpaired Student’s t test. CCK8, Cell Counting Kit-8; CHOP, CCAAT-enhancer binding protein homologous protein; DAPI, 4',6-diamidino-2-phenylindole; ER, endoplasmic reticulum; MEF, mouse embryonic fibroblast; PERK, PKR-like endoplasmic reticulum kinase; Tm, tunicamycin.

Fig 5: The synergistic anti-tumor effect of morusin and TMZ in GBM. (A,B) The viability of U87 and U251 cells treated with TMZ (0, 10, 20, 50, 100 µM) combined with morusin (20 µM) for 24 h. (C,D) Synergy was analyzed by CompuSyn software. CI = 1, >1 and <1 indicate additive, antagonistic and synergistic effects, respectively. (E,F) Levels of p-eIF2a and CHOP in GBM cells after morusin and TMZ exposure for 24 h. Tubulin was used as a loading control. n = 3. (G) A schematic of xenograft tumor experiment for the investigation of anti-tumor effects of morusin and TMZ in vivo. (H) The Kaplan–Meier survival curves of tumor bearing mice after treatment. (I) H&E-stained images of mouse brain sections. Scale bar = 1 mm. (J) Representative images of IHC analysis of CHOP and Ki-67 in the intracranial tumors after morusin and TMZ treatment. Immunoreactivity was detected by DAB chromogen (brown). The figures are representative data from at least three independent experiments. Scale bar = 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.0001 compared to indicated comparator.