(A) Livers from wild-type mice collected 8 hr after injection with the indicated concentrations of TM (in mg/kg) were probed by qRT-PCR for expression of Hspa5 (encoding BiP) and Ddit3 (encoding CHOP). n = 2–3 animals/group. Significance was determined by one-way ANOVA. Here and elsewhere, ***, p<0.001; **, p<0.01; *, p<0.05.; NS = not significant (B) Livers were harvested after treatment for 8 hr with vehicle or 0.025 mg/kg TM and protein lysates were probed by immunoblot to detect BiP or CHOP. Calnexin was used as a loading control. For all blots and gels in this paper, each lane represents a separate animal. Hairlines are used for visual clarity only. (C) Chronic stress treatment schematic. Mice were weighed and then injected daily with 0.025 mg/kg TM (red arrows); liver samples were collected at the indicated times (blue arrows) for downstream analysis. The naming convention is as follows: ‘D’ indicates the number of daily injections received while ‘h’ indicates the time of tissue collection after the last injection. Here and elsewhere, not treated animals (NT) received injections of vehicle at the same times that D5-24h mice received TM. (D) Formalin-fixed liver sections from animals treated as in (C) were collected, fixed, and stained by H and E at the indicated times. For each condition, representative images from two separate mice are shown. Note the extensive cytoplasmic vacuolization in the D1-24h animals. (E, F) Protein lysates from animals treated as in (C) were isolated and probed for expression of the ER-resident glycoprotein TRAPα (E) and BiP, CHOP, ADRP, and α-actin (loading control) (F).