Figure 5.

Small influences of CRT and CNX on ATZ degradation and polymeric ATZ accumulation.A–D, Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-ATZ–encoding plasmids and treated with either 100 nm bafilomycin or 10 µg/ml MG132 or left untreated at 20 h post-transfection. At 24 h post-transfection, the cells were harvested, stained with 2C1, and analyzed. The polymeric ATZ (2C1) gate was determined by gating on forward and side scatter, live cells, then eYFP⁺ cells. The secondary antibody staining control was used as the cutoff for setting the polymeric ATZ gate. For each cell type and drug-treatment condition, ratios of signals from drug-treated/untreated cells were measured to calculate eYFP MFI ratios (A and B) or 2C1 MFI ratios (C and D). Data for CRT KO and WT were obtained from 14 independent replicates, eight of which were conducted in parallel with CNX KO. Data for CNX KO and WT were obtained from eight independent replicates. All data are shown as mean ± S.D. (error bars). RM one-way ANOVA analysis was performed, and p-values are reported for comparisons of KO and WT conditions for each drug treatment. E and F, Huh7.5 CRT KO, CNX KO, or corresponding WT cells as indicated were transfected with eYFP-AAT– or eYFP-ATZ–encoding plasmids and stained with the polymer-selective antibody 2C1 at 48 h post-transfection. 2C1 MFI (of eYFP⁺ populations) is shown (gated as described in A–D). Data quantified over nine independent experiments, conducted in parallel, are shown as mean ± S.D. (error bars). Data are normalized relative to the WT eYFP-AAT transfected signals. RM one-way ANOVA analysis was performed on the log-transformed data in E and F. *p < 0.05, ***p < 0.001.