ERS leads to an ASM-dependent, rapid release of Cer. (A) Agarose gel of Xbp1 cDNA amplicons from AML12 treated with Ctrl/CM aliquots obtained at indicated time slots. (B) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper panel) and quantification of protein levels (lower panel) from AML12 treated as in A. The diamond indicates the nonspecific band. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01. (C and D) ASM activities (C) and western blot of ASM (D) in medium derived from 3T3-L1 pretreated with or without 100 nM Baf A1, 50 μM DPA, or 20 μM BAPTA-AM. The relative ASM activity was calculated by setting the activity of Ctrl_vehicle as 1. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 3. ***P < 0.001, ****P < 0.0001. (E–G) Western blot of p-IRE1α, XBP1s, p-PERK, and BiP (upper panel) and quantification of protein levels (lower panel) from AML12 treated with regular or (E) Baf A1-, (F) DPA-, and (G) BAPTA-AM–pretreated 3T3-L1–derived Ctrl/CM. The diamond indicates the nonspecific band. Lanes were numbered from left to right, with the ratio in lane 2 (Tg treatment) set as 100. The statistical analyses were calculated by one-way ANOVA. Data were shown as the mean ± SEM, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available for this figure: SourceData F4.