FIG 6.

6-TG treatment does not block global translation or decrease surface expression of human leukocyte antigen A/B/C (HLA-A/B/C) glycoproteins. (A) A549 cells were treated with 10 μM 6-TG and 1 μM thapsigargin (Tg) for the indicated times or with 500 μM sodium arsenite (As) for 45 min prior to harvesting cell lysates and Western blotting for BiP, eIF2α, phosphorylated eIF2α, or actin. (B) Protein synthesis in cells treated as described above for panel A was analyzed using a puromycylation assay. (C) The puromycin signal (B, top) was quantified and normalized to total protein loading (B, bottom, Stain-free) from 3 independent replicates. Error bars indicate standard deviations. One-way ANOVA and Dunnett multiple-comparison tests were done to determine statistical significance (*, P value of <0.05; **, P value of <0.01; ***, P value of <0.001). (D) A549 cells were treated as indicated, and the surface expression of HLA-A/B/C was analyzed using flow cytometry. The histograms from one representative experiment are depicted on the left, where the vertical dotted line indicates the median fluorescence intensity (MFI) of HLA-A/B/C in the DMSO-treated sample. The histograms on the right display the MFI of HLA-A/B/C on the cell surface for each treatment shown, with the individual data points (colored circles) with black lines indicating the means ± standard deviations (n = 4, except for the isotype control [n = 2]). The vertical dotted line in the graph indicates the mean MFI from the DMSO-treated samples.