HeLa cells stably expressing XBP1-MS2 wild-type (WT) reporters were treated with 0.2 ug/ml doxycycline (Dox) for 15 hours before addition of 100 nM thapsigargin (TG) or 5 mg/ml tunicamycin (TM) for indicated times. (A) Western blot against PERK, ATF4, inositol-requiring enzyme 1 alpha (IRE1α), XBP1s, and GAPDH (loading control). Ectopic expression of XBP1-MS2 did not affect PERK activation or ATF4 synthesis but increased XBP1s levels under endoplasmic reticulum (ER) stress. Note that tunicamycin elicits a milder UPR. Bottom image: agarose gel documenting XBP1-MS2 reporter splicing by semi-quantitative real time (RT)-PCR analysis using mouse-specific oligonucleotides flanking the XBP1 intron. Fastest migrating band = spliced mRNA; middle band = unspliced mRNA; upper band = hybrid dsDNA product, where one strand is ‘spliced’ and the other ‘unspliced’. (B) Quantitative RT-PCR analysis determining the levels of unspliced and spliced XBP1-MS2 mRNA products to calculate splicing ratios (spliced/unspliced mRNA) in response to Dox and ER stress induction. (C) Quantitative RT-PCR analysis of endogenous transcripts: expression of XBP1-MS2 did not significantly alter the splicing ratio of endogenous XBP1 mRNA (hXBP1) or the mRNA levels of the ATF4-regulated asparagine synthetase (AsnS). And the increase of XBP1s levels in XBP1-MS2 cells led to an increase in the levels of the XBP1s-regulated ERdJ4 mRNA. (D) HeLa cells expressing XBP1 WT reporters with (XBP1-MS2) and w/o (XBP1) stem loop array were incubated in the presence of TG for the indicated time points. Splicing was visualized by semi-quantitative RT-PCR (upper panel) and quantified via qPCR-derived splicing ratios (bottom panels), as described above. In parallel, we confirmed that ectopic, murine XBP1 expression did not interfere with endogenous XBP1 splicing. In B, C and D, graphs shows the average ± SD from three independent experiments. Statistical test Kruskal-Wallis and Dunn's multiple comparison test was applied, and no significant differences were observed. For raw data see Figure 1—figure supplement 1—source data 1.