(A–F,H–J) Expression of µs in HeLa-µs cells was induced with 0.5 nM Mif. (A,G,H) The UPR was pharmacologically induced as a reference for 4 hr with 5 µg/ml Tm, or—for analysis of ATF6α in (A) only—for 1.5 hr with 300 nM Tg (since Tm leads to deglycosylation of ATF6α). (A,D) Immunoblotting revealed levels of µs, CHOP, BiP and α-tubulin. A shift to a lower mobility phosphorylated form, P-PERK, and the appearance of CHOP revealed activation of PERK. The release of the p50 cleavage product from the p90 precursor revealed activation of ATF6α. Cross-reaction of the secondary antibody against anti-ATF6α with µs is denoted. (A,G) Splicing of XBP1 was assessed as in Figure 1A. (A) Three different clones of HeLa-µs cells with decreasing µs expression levels: high (Hi), medium (Me), or low (Lo) were induced for 16 hr. Non-treated (nt) or Tm-treated Hi HeLa-µs served as references. (B,C,H) Immunofluorescence revealed µs (red) and CRT (blue), which marks the ER (C) at the indicated times before or after induction (B) or after 8 hr induction (C,H). Nuclei were stained with DAPI (blue) (B). (C,H) The area that is boxed is shown by 3.5-fold magnification; scale bars represent 10 µm. (D) Samples were derived from equal numbers (7 × 104) of HeLa-µs or I.29µ+ lymphomas induced with Mif or stimulated with 20 µg/ml lipopolysaccharide (LPS), respectively, for the indicated times. Ponceau staining of the blot serves as a loading control. (E) HeLa-µs cells were pulse labeled with 35S labeled methionine and cysteine for 10 min at the indicated times after induction. Immunoprecipitated µs was resolved by gel electrophoresis (F). Levels of radio-labeled µs in (E) were quantified by phosphor imaging. The maximal signal for µs (at 64 hr) was set at 100. (G) HeLa-MifON cells were treated with a high dose of Mif (10 nM) for the indicated times. (H) In HeLa-µs cells in which IRE1α was replaced with doxycycline (Dox)-inducible IRE1α-GFP (green), IRE1α-GFP expression was tuned with 10 nM Dox to levels that allowed satisfactory detection of IRE1α-GFP by fluorescence microscopy. (I) Cell growth assay as in Figure 1B of HeLa-MifON and HeLa-µs induced continuously with Mif or not. (J) Quantitation of (I), performed as in Figure 1C. Mean and s.e.m. are shown, n = 2. There is no statistical significance in a one-sample t-test of differences in growth between conditions.