Fig. 1. Caffeine blocks PCSK9 expression and secretion in hepatocytes.

A HuH7 cells were treated with established inducers of PCSK9 expression, thapsigargin (TG; 100 nM) or U18 (10 µM), in the presence or absence of caffeine (CF; 200 µM) for 24 h¹³. PCSK9 expression was assessed via immunoblot analysis. B–D PCSK9 expression was also assessed in primary mouse hepatocytes (PMH) and primary human hepatocytes (PHH), as well as in HepG2 cells treated with CF and TG via real-time PCR (n = 5 biologically independent samples per group; data presented are mean ± s.d.). E–G PCSK9 ELISAs were assayed on the medium harvested from CF-treated HuH7, HepG2, PMHs, and PHHs (n = 5 biologically independent samples per group). H Coomassie blue staining of electrophoretically resolved medium harvested from CF-treated cells served to examine the effect of CF on total secreted protein levels. I Secreted PCSK9 levels from HepG2 cells treated with an increasing dose of CF (n = 4 biologically independent samples per group; data presented are mean ± s.d). J–K PCSK9 expression and secretion were assessed in HepG2 cells treated in the presence and absence of CF and a blocker of transcription, ActD (10 µM) (n = 4 biologically independent samples per group; data presented are mean ± s.d). L Finally, ELISAs were also used to measure secreted PCSK9 levels in CF-treated HepG2 cells (1 mM) transfected with a CMV-driven PCSK9 vector (n = 5 biologically independent samples per group; data presented are mean ± s.d). Statistical comparisons between two groups were conducted using unpaired two-tailed Student’s t-tests, while comparisons between 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.