Figure 6: Effects of Lp(a) on HAVIC Gel Contraction, β-Catenin Nuclear Translocation, and Lp(a) Uptake

(A) Effect of Lp(a) treatment on gel contraction. Lp(a)-treated vs. nontreated control: **p = 0.01 at 6 h, *p = 0.04 at 24 h; n = 6. (B) β-catenin nuclear translocation assessed with confocal microscopy. HAVICs were treated with or without 50 μg/ml Lp(a) for 48 h in OSM on CultureSlide (Falcon), stained with rabbit anti-CTNNB1 antibody and Alexa Fluor 488 donkey anti-rabbit IgG (Thermo Fisher Scientific), and stained with NucBlue (nuclear stain; ThermoFisher Scientific, Grand Island, New York). Tile images were taken with a Zeiss LSM780 confocal microscope (20× magnification). Nuclear masking and average integrated intensity were analyzed with Cellprofiler and are presented as mean ± SEM. Total number of nuclei analyzed: 30 min: Lp(a) n = 69, Control n = 76; 1 h: Lp(a) n = 59, Control n = 64; 4 h: Lp(a) n = 89, Control n = 70; 24 h: Lp(a) n = 74, Control n = 76. Lp(a)-treated vs. nontreated control: **p = 0.001 at 1 h and 6 h, *p = 0.04 at 24 h. (C) β-catenin nuclear translocation assessed with high content screening confocal imaging. HAVICs were treated with or without Lp(a) 50 μg/ml for 1 h in triplicates of 24-well glass-bottom optical culture plates and stained for CTNNB1 as described above. Tile images were taken using ImageXpress Micro XLS at 10× magnification. Average integrated nuclear CTNNB1 intensity was analyzed using MetaXpress as described above. *p = 0.003, Lp(a)-treated vs. nontreated control. N = total number of nuclei analyzed. (D) Time course of radiolabeled 3H-chlosterol Lp(a) uptake in HAVICs; n = 3. HAVICs cultured in triplicates of 24-well plates were loaded with 3H-chlosterol Lp(a) at ∼15,000 cpm/ml in serum-free DMEM. Cell lipid extraction was used for liquid scintillation count. Cellular uptake of labeled Lp(a) at different time intervals was calculated as percentage against initial loading. Abbreviations as in Figures 1 and 2.