Fig. 4.

Silencing of ABCA1 results in decreased formation of large pre-β HDL concomitant with increased TG secretion. Control and ABCA1 siRNA transfected McA cells were incubated with [³H]oleate (5 µCi/ml) and [¹²⁵I]apoA-I (10 µg/ml; 10⁵ cpm/µg) in the presence of 0.4 mM oleate in serum-free medium for 24 h. A: ABCA1 expression levels were assessed by Western blot analysis (top). Aliquots of conditioned medium were lipid extracted, lipids (PL, TG, and CE) were separated by TLC, and [³H]oleate incorporated into the lipid fractions was determined (mean ± SEM, n = 4). B: Conditioned media and lipid-free [¹²⁵I]apoA-I were separated on agarose gels and radioactivity was visualized using a phosphorimager. Human HDL and LDL were used as markers of α and β migration. C: Aliquots (15µl) of conditioned medium were fractionated by NDGGE and nascent HDL particles were visualized using a phosphorimager. Nascent pre-β migrating HDL formation was quantified by phosphorimager analysis and normalized to control siRNA transfected McA cells for each pre-β HDL fraction. Results are expressed as mean ± SEM of triplicate gels. Image is representative data from three independent experiments. * (P < 0.05), ** (P < 0.01), and *** (P < 0.001) by Student's t-test.