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. 2017 Nov 27;59(2):207–223. doi: 10.1194/jlr.M078360

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Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.

Author’s Choice—Final version free via Creative Commons CC-BY license.

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Fig. 6. — A: PCSK9 is associated with VLDL and LDL from Ldlr^−/− and LDb mice. We used FPLC to separate plasma of Ldlr^−/−, LTp, and LDb mice into lipoproteins. VLDL, LDL, HDL. VLDL and LDL fractions were pooled and concentrated using the Ultrafree 15 centrifugal filter device. We applied 20 μg of proteins to 4–20% SDS-PAGE. PCSK9 was detected by Western blot analysis using anti-PCSK9 (Biolegend; 1:3,000 dilution). The positions of PCSK9 are indicated. There is no detectable PCSK9 in LTp mice. B: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). C: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tail unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). D: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). E: The comparison of binding and uptake of LDb-LDL and LTp-LDL to MCECs. Control DiI, DiI-LDb-LDL, or DiI-LTp-LDL was incubated with MCECs to study the binding and uptake of LDLs to MCECs. The detailed methods are described in the Materials and Methods. Binding of LDLs to MCECs was examined using a confocal microscope. The red dots represent the binding of LDLs to ECs. Both DiI-LDb-LDL and DiI-LTp-LDL bound to ECs after incubation at 4°C for 30 min. The uptake of LDLs to MCECs was carried out at 37°C for 4 h. There were strong uptakes of both LDLs to MCECs. There was no difference in the number of cells taken up by MCECs as determined by flow cytometry (LDb-LDL vs. LTp-LDL; 58 ± 3.2 vs. 56 ± 3.7). ns, not significant. F: LDb-LDL stimulated gene expression levels of pro-atherosclerosis molecules in MCECs. MCECs were plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). G: Protein expression levels of LOX-1, p62, and TRAF6 in MCECs after treatment with either LDb-LDL or LTp-LDL. Protein levels were analyzed by SDS-PAGE followed by Western blot analysis. The results are expressed as mean ± SD. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. H: Protein levels of CCL-2 and IL-6 in the cell media after treatment with PBS (circles), LDb-LDL (squares), or LTp-LDL (triangles). ELISA was used to determine the protein levels of CCL-2 (in nanograms per milliliter) and IL-6 (in picograms per milliliter) in cell media. Individual measurements are shown as well as mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction.

Fig. 6. — A: PCSK9 is associated with VLDL and LDL from Ldlr^−/− and LDb mice. We used FPLC to separate plasma of Ldlr^−/−, LTp, and LDb mice into lipoproteins. VLDL, LDL, HDL. VLDL and LDL fractions were pooled and concentrated using the Ultrafree 15 centrifugal filter device. We applied 20 μg of proteins to 4–20% SDS-PAGE. PCSK9 was detected by Western blot analysis using anti-PCSK9 (Biolegend; 1:3,000 dilution). The positions of PCSK9 are indicated. There is no detectable PCSK9 in LTp mice. B: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). C: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tail unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). D: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). E: The comparison of binding and uptake of LDb-LDL and LTp-LDL to MCECs. Control DiI, DiI-LDb-LDL, or DiI-LTp-LDL was incubated with MCECs to study the binding and uptake of LDLs to MCECs. The detailed methods are described in the Materials and Methods. Binding of LDLs to MCECs was examined using a confocal microscope. The red dots represent the binding of LDLs to ECs. Both DiI-LDb-LDL and DiI-LTp-LDL bound to ECs after incubation at 4°C for 30 min. The uptake of LDLs to MCECs was carried out at 37°C for 4 h. There were strong uptakes of both LDLs to MCECs. There was no difference in the number of cells taken up by MCECs as determined by flow cytometry (LDb-LDL vs. LTp-LDL; 58 ± 3.2 vs. 56 ± 3.7). ns, not significant. F: LDb-LDL stimulated gene expression levels of pro-atherosclerosis molecules in MCECs. MCECs were plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 μg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 μg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with β-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). G: Protein expression levels of LOX-1, p62, and TRAF6 in MCECs after treatment with either LDb-LDL or LTp-LDL. Protein levels were analyzed by SDS-PAGE followed by Western blot analysis. The results are expressed as mean ± SD. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. H: Protein levels of CCL-2 and IL-6 in the cell media after treatment with PBS (circles), LDb-LDL (squares), or LTp-LDL (triangles). ELISA was used to determine the protein levels of CCL-2 (in nanograms per milliliter) and IL-6 (in picograms per milliliter) in cell media. Individual measurements are shown as well as mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction.