Fig. 5. Caffeine reduces chaperone expression and blocks hepatic PCSK9 expression in mice.

12-week-old male C57BL/6J mice were treated with caffeine (CF; 50 mg/kg) and fasted for 8 h prior to sacrifice (n = 6). A, B Plasma PCSK9 and triglyceride levels were measured using an ELISA and colorimetric assays, respectively (n = 6 biologically independent samples per group; data presented are mean ± s.d.). C The time-dependence of CF on plasma PCSK9 levels was also determined using an ELISA (n = 5 biologically independent samples per group; data presented are mean ± s.d.). D The livers of these mice were assessed for cell-surface expression of LDLR and CD36, as well as the ER stress markers GRP78 and GRP94 via immunohistochemical staining (n = 5). E Staining was quantified using ImageJ software (n = 5 biologically independent samples per group; data presented are mean ± s.d.). F, G The expression of ER stress markers (GRP78, PERK, and IRE1α) as well as cholesterol-regulatory genes (LDLR, PCSK9, HMGR, SREBP1 and SREBP2) were also examined using immunoblots and real-time PCR (n = 5 biologically independent samples per group; data presented are mean ± s.d.). H–I 12-week-old male C57BL/6J mice were treated with a single subcutaneous injection of the anti-PCSK9 neutralizing antibody, alirocumab (30 mg/kg), for 10 days (n = 10). LDLR expression was assessed using immunohistochemistry and immunoblots. J The mRNA expression of SREBP2, PCSK9, and the LDLR was assessed via real-time PCR (n = 5 biologically independent samples per group; data presented are mean ± s.d.). Bars; 50 µm. Statistical comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests, while multiple groups were compared using one-way ANOVAs with the Tukey HSD post hoc test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Source data are provided as a Source Data file.