Supplementary Figure 1: ER stress induction with CPA and DTT.

(a) Amplification with appropriate primers of unspliced and spliced XBP1 transcripts in mock-treated cells and in cells exposed for 12 h to 10 μM CPA-induced ER stress. (b) Incorporation of ³⁵S-radiolabeled amino acids (2 h pulse with ³⁵S-methionine and-cysteine) in total MEF proteome after 8 h of Mock- or 10 μM CPA-treatment. TCE, total cell extract. (c) Same as (b) for the incorporation of ³⁵S-radiolabeled amino acids in the ER marker proteins Cnx, Grp94 and BiP as assessed by autoradiography of the respective Immunoprecipitates. (d) Quantification of (b) and (c), data from a single experiment. (e) Dithiothreitol (DTT) induces ER stress by modifying redox homeostasis. At 1 mM, DTT caused a milder stress in MEF compared to non-toxic concentrations of CPA as determined by lower induction of BiP transcription, data from a single experiment (f) Like CPA, the wash out of DTT at the end of the stress phase was followed by rapid return of BiP transcripts at or below their pre-stress levels as determined by qPCR. For CPA 10 μM, mean of two independent experiments. For others data from a single experiment. (g) Like CPA, on DTT wash out (but not in untreated cells (Mock) or during the stress (ER stress)), excess ER chaperones did accumulate in Lamp1-positive autolysosomes in cells exposed to the lysosomal inhibitor BafA1. Studies were continued with CPA, which proved less toxic to cells. Scale bars, 10 μm. Images from a single experiment. Details of statistics in the Statistics and Reproducibility section. Statistics source data in Supplementary Table 4.