Figure 2: The effects of high density lipoprotein (HDL) treatment on TTR and TTR-T4 uptake in HepG2 cells. Cells were serum starved for 4 hours before uptake experiments. 500 ug/ml HDL was added to the serum free medium during this 4-hour period for the HDL ‘pretreated’ group. Medium was removed and replaced with serum free medium containing unconjugated Alexa dye, Alexa-labelled TTR, or Alexa-labelled TTR-T4 (± 500/1000 ug/ml HDL) and cells were transferred to an Essen IncuCyte incubator where intracellular fluorescence and cell confluence (%) were measured every 2 hours. Alexa-TTR uptake is expressed as red objects/% confluence. (a) Relative uptake of dye, Alexa-TTR, and Alexa-TTR-T4 at 24 hours. Pretreatment with 500 ug/ml HDL for 4 hours resulted in greater uptake of Alexa-TTR-T4 at 24 hours (p<0.05, n=4). Cotreatment with 500 ug/ml HDL resulted in reduced uptake of Alexa-TTR and Alexa-TTR-T4 in all conditions tested. Coincubation with 1000 ug/ml HDL reduced uptake of Alexa-TTR in cells pretreated with HDL only. 1000 ug/ml HDL reduced uptake of Alexa-TTR-T4 in both control and pretreated cells (n=4, p<0.05, p<0.01). (b) Uptake of Alexa-TTR over 48 hours. Coincubation with HDL reduced Alexa-TTR uptake under all conditions (n=4). (c) Uptake of Alexa-TTR-T4 over 48 hours. Coincubation reduced Alexa-TTR-T4 uptake under all conditions (n=4).