Figure 4.

ER PCSK9 retention does not induce apoptosis. HuH7 and HK-2 cells were seeded in DMEM and transfected with either PCSK9^WT or PCSK9^Q152H for 48 h. Cells were also treated with TG (100 nm) or DMSO vehicle control for 24 h. A, HuH7 cells were fixed in 4% paraformaldehyde and stained for apoptosis-induced DNA damage using a 594-labeled TUNEL stain (red). Following TUNEL staining, cells were also stained for GFP to identify cells transfected with the bicistronic pIRES2-EGFP V5-PCSK9 plasmids (green). B, transfected HK-2 cells were also examined for the apoptosis marker PUMA via immunoblot analysis. C and D, cell viability assays were carried out on transfected HK-2 and HuH7 cells using trypan blue stain and lactate dehydrogenase assays, respectively. E, a lactate dehydrogenase assay was also carried out on cells transfected with VP^G17V, a mutant known to induce ER stress as a result of ER retention (21). F, freshly isolated mouse primary hepatocytes were transfected with PCSK9 variants PCSK9^Q152H, PCSK9^Q152D, and PCSK9^S386A and examined for the apoptosis markers caspase-3 and PUMA via real-time PCR. G, apoptosis marker expression of caspase-3 and PUMA was also examined in VP-transfected mouse primary hepatocyte using real-time PCR. Differences between treatments were assessed with unpaired Student's t tests. All values are represented as means, and error bars represent S.D. C, *, p < 0.05 versus DMSO-treated WT. D, *, p < 0.05 versus DMSO-treated WT. F and G, *, p < 0.05 versus VP^WT. C, D, and E, nonsignificant (NS).