FIG 2.

LDLR expression and functionality in PDAC cell lines. (A) Total protein lysates were isolated from untreated cells and analyzed for LDLR levels by an ELISA. LDLR levels were normalized to total protein levels. The assay was done in triplicate, and data represent the means ± standard errors of the means. Cell lines were compared by using 1-way analysis of variance followed by the Dunnett posttest for comparison to HPAF-II cells. **, P < 0.01; ****, P < 0.0001. (B) Cell monolayers were incubated with various amounts of VSV-ΔM51-eqFP650 (the indicated MOIs are based on virus titration on MIA PaCa-2) for 1 h at 4°C. Protein lysates were analyzed for LDLR and VSV proteins by Western blotting. Protein product sizes (kilodaltons) are indicated on the right. GAPDH and Coomassie blue stain were used to confirm equal loading. (C) For LDLR cell surface expression, cells were kept on ice and were not permeabilized or fixed. After incubation with anti-LDLR primary antibody and APC-conjugated secondary antibody, cells were analyzed by FACS analysis using the APC-A channel. Control indicates cells that were incubated with secondary antibody only. “LDLR” cells were incubated with primary and secondary antibodies. Gated populations are positive for LDLR (the percentage of LDLR-positive cells is indicated above the gate line). The MFI for each population was calculated by using FlowJo software (TreeStar). Data are representative of results from 3 independent experiments. (D) For the LDL uptake assay, cells were incubated with fluorescently labeled LDL for 4 h and then analyzed by FACS analysis using the R-phycoerythrin (PE-A) channel. “Control,” fluorescently labeled LDL was not added; “LDL,” fluorescently labeled LDL was added. Gated populations are positive for LDL uptake (the percentage of LDL-positive cells is indicated above the gate line). The MFI for each population was calculated by using FlowJo software (TreeStar). Data are representative of results from 3 independent experiments.