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. 2018 Jul 18;6:22. doi: 10.1038/s41413-018-0023-x

Fig. 5.

Fig. 5

OxPLs induce LRP6 endocytosis in MSCs through clathrin-mediated pathway. a Localization of LRP6 in MSCs at 0, 1, 3, 5, 10, and 30 min after oxLDL treatment detected by immunofluorescence staining of LRP6 (in green). (Scale bar: 20 μm). b Time-lapse imaging of co-endocytosis of LRP6-eGFP and DiI-oxLDL. 293 T cells transfected with LRP6-eGFP were incubated with DiI-oxLDL for 2 h at 4 °C, and the cells were placed into the stage chamber at 37 °C and time-lapse imaging were examined with 30-s intervals. Images of merged LRP6-eGFP (green) and DiI-oxLDL (red) are presented. Insets are high magnification images of the region indicated by white rectangles, White arrows point on the examples of colocalization of LRP6-eGFP and DiI-oxLDL. (Scale bars: 20 μm). c Redistribution of LRP6 between the lipid raft and nonlipid raft fractions in response to oxLDL. MSCs were treated with 20 μg/ml nLDL or oxLDL for 30 min. Cell lysates were fractionated by sucrose density gradient centrifugation, and aliquots were probed with anti-LRP6. Caveolin-1 and clathrin indicate the positions of the lipid raft and nonlipid raft fractions, respectively. d MSCs were incubated with 20 μg·mL-1l nLDL or oxLDL together with DMSO (vehicle), Filipin III (3 μg·mL-1 in DMSO) or MDC (100 μmol·L-1" in DMSO) for 30 min. Cell surface proteins were labeled with Sulfo-NHS-SS-Biotin. Cells were lysed, and biotinylated proteins were precipitated with streptavidin-agarose. Presence of LRP6 and Na,K-ATPase was evaluated by Western blot analysis. e Co-localization of LRP6 and clathrin in MSCs at 0, 10, and 30 min after POVPC treatment detected by immunofluorescence staining of LRP6 (in green) and clathrin (in red). DAPI was used to stain the nuclei.(Scale bar: 20μm). f, g Representative images of the flow cytometry analysis (f) and the percentage of LRP6+ cells (g) in human MSCs treated with vehicle (Control) or oxLDL in the absence or presence of MDC for 30 min. n = 6, data are represented as mean ± s.e.m. *P < 0.01, vs. Control group; #P < 0.01, vs. Vehicle + oxLDL group, as determined by Student’s t-tests