24(S)-Hydroxycholesterol induces ER dysfunction-mediated unconventional cell death

a–c SH-SY5Y cells were pretreated with 5？μM {"type":"entrez-nucleotide","attrs":{"text":"F12511","term_id":"708507"}}F12511 for 15？min or with 100？μM Nec-1 (a) for 1？h and then exposed to 50？μM 24S-OHC for 6？h. Cells were also treated with 3？μM thapsigargin (Thapsi) for 3？h. a Whole-cell lysates were subjected to immunoblotting with appropriate antibodies as indicated. b The unspliced (XBP1u) and spliced (XBP1s) XBP1 mRNAs were analyzed by RT-PCR. c Whole-cell lysates were immunoblotted with antibodies specific for XBP1s or β-actin. d Cells were pretreated with 20？μM MG132 for 30？min and then exposed to 50？μM 24S-OHC or 3？μM thapsigargin for 6？h. Whole-cell lysates were immunoblotted with antibodies specific for ATF6 or β-actin. Asterisks denote nonspecific bands. e, f Cells were treated as in panel a. g, h Cells were treated as in panel d. e, g Whole-cell lysates were subjected to immunoblotting with appropriate antibodies as indicated. f, h Band intensities were quantified by densitometric scanning, relative intensity is shown. Mean？±？SD n？=？3, **P？<？0.01, n.s. not significan