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. 2022 Feb 9;13:770. doi: 10.1038/s41467-022-28240-9

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 6 — A The expression of PCSK9-regulated receptors, LDLR and CD36, was examined in caffeine (CF)-treated cultured hepatocytes (200 µM). B The uptake and intracellular accumulation of fluorescently labeled DiI-LDL was examined in cells treated with CF in the presence or absence of the PCSK9-inducer, U18, using a fluorescent spectrophotometer (n = 4 biologically independent samples per group; data presented are mean ± s.d.). C The effect of CF treatment (200 µM) on DiI-labeled LDL uptake was also examined in PCSK9 shRNA knockdown cells (n = 4 biologically independent samples per group; data presented are mean ± s.d.). D Immunofluorescent staining of cell-surface LDLR was carried out in live CF pre-treated HepG2 cells (200 µM). Cellular DiI-LDL accumulation was also visualized in CF-treated HepG2 cells using a fluorescent microscope. E Expression of the LDLR in CF-treated HuH7 and HepG2 cells was measured via real-time PCR (n = 4 biologically independent samples per group; data presented are mean ± s.d.). F–G The uptake of DiI-LDL was quantified in HepG2 cells transfected with siRNA targeted against CD36 and a pharmacologic inhibitor of CD36 (SSO) (10 µM) (n = 8 biologically independent samples per group; data presented are mean ± s.d.). H Knockdown was confirmed via immunoblotting. Pcsk9^+/+ and Pcsk9^−/− mice were treated with either PBS-vehicle or CF, as well as fluorescently labeled DiI-LDL (1 µg/kg). I–K Hepatic cell-surface LDLR expression was assessed via immunohistochemistry (DAPI: blue; LDLR: green; DiI-LDL: red). J Hepatic and serum DiI-LDL fluorescence intensity was quantified using a fluorescent spectrophotometer and visualized using a fluorescent microscope (n = 6 biologically independent samples per group; data presented are mean ± s.d.). L, M Native LDLc was also examined in 18-week-old male C57BL/6J mice treated with CF (30 mg/kg) every 24 h for 14 days via ELISA of the surrogate marker ApoB; serum PCSK9 levels were also assessed via ELISA (n = 10 biologically independent samples per group; data presented are mean ± s.d.). Scale bars; D 10 µm; I 50 µm; K 100 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.

Fig. 6 — A The expression of PCSK9-regulated receptors, LDLR and CD36, was examined in caffeine (CF)-treated cultured hepatocytes (200 µM). B The uptake and intracellular accumulation of fluorescently labeled DiI-LDL was examined in cells treated with CF in the presence or absence of the PCSK9-inducer, U18, using a fluorescent spectrophotometer (n = 4 biologically independent samples per group; data presented are mean ± s.d.). C The effect of CF treatment (200 µM) on DiI-labeled LDL uptake was also examined in PCSK9 shRNA knockdown cells (n = 4 biologically independent samples per group; data presented are mean ± s.d.). D Immunofluorescent staining of cell-surface LDLR was carried out in live CF pre-treated HepG2 cells (200 µM). Cellular DiI-LDL accumulation was also visualized in CF-treated HepG2 cells using a fluorescent microscope. E Expression of the LDLR in CF-treated HuH7 and HepG2 cells was measured via real-time PCR (n = 4 biologically independent samples per group; data presented are mean ± s.d.). F–G The uptake of DiI-LDL was quantified in HepG2 cells transfected with siRNA targeted against CD36 and a pharmacologic inhibitor of CD36 (SSO) (10 µM) (n = 8 biologically independent samples per group; data presented are mean ± s.d.). H Knockdown was confirmed via immunoblotting. Pcsk9^+/+ and Pcsk9^−/− mice were treated with either PBS-vehicle or CF, as well as fluorescently labeled DiI-LDL (1 µg/kg). I–K Hepatic cell-surface LDLR expression was assessed via immunohistochemistry (DAPI: blue; LDLR: green; DiI-LDL: red). J Hepatic and serum DiI-LDL fluorescence intensity was quantified using a fluorescent spectrophotometer and visualized using a fluorescent microscope (n = 6 biologically independent samples per group; data presented are mean ± s.d.). L, M Native LDLc was also examined in 18-week-old male C57BL/6J mice treated with CF (30 mg/kg) every 24 h for 14 days via ELISA of the surrogate marker ApoB; serum PCSK9 levels were also assessed via ELISA (n = 10 biologically independent samples per group; data presented are mean ± s.d.). Scale bars; D 10 µm; I 50 µm; K 100 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.