Semiquantitative XBP1 mRNA Splicing Assay

The semiquantitative XBP1 mRNA splicing assay was performed as described previously (51 (link)). Specifically, total RNA was isolated from treated A549 cells with the RNeasy kit (Qiagen). Five hundred nanograms of total RNA was reversed transcribed using the Maxima H RT kit (Thermo). A 473-bp PCR product spanning exon/intron boundaries was generated using the XBP1 forward primer 5′-AAACAGAGTAGCAGCTCAGACTGC-3′ and the XBP1 reverse primer 5′-TCCTTCTGGGTAGACCTCTGGGA-3′. The PCR product was digested overnight with PstI-HF to cleave the unspliced XBP1 product into XBP1u1 and XBP1u2. The digested PCR product was resolved on a 2.5% agarose gel made with 1× Tris-acetate-EDTA and stained with ethidium bromide (Sigma-Aldrich). The gel was imaged on a ChemiDoc imaging station (Bio-Rad).