Figure 4

Ub-proteasome- and ERAD-dependent gp100^mel_{47–52/40–42} epitope production is enhanced by β5i subunit.

(A) HeLa cells were transfected with HLA-A*03:01 and gp100^mel and were subjected to epoxomicin treatment (250 nM; 2 hrs) or DMSO (control) and compared to HeLa cells transfected with HLA-A*03:01 treated with epoxomicin (250 nM; 2 hrs) and pulsed with the synthetic peptide gp100^mel_{47–52/40–42} (50 μM). Values are means and SD of 2 replicates in a representative assay of 2 independent experiments. Significant differences (between the samples with or without epoxomicin) in the means of replicates are marked by * (student t-test; E:T ratio: 1:1, p = 0.002; 2:1, p = 0.001; 4:1, p < 0.001; 8:1, p < 0.001). (B) HeLa 33/2 cells were exposed to β5i subunit or control siRNAs (30 nM for 24 hrs) and subsequently transfected with gp100^mel. Cell extracts were analyzed by western blotting (representative of 4 independent experiments). β5i subunit-depleted HeLa 33/2 cells were subjected to a 16 hr CTL assay. Values are means and SD of 2 replicates in a representative assay of 4 independent experiments. Significant differences in the means of replicates are marked by * (student t-test; p = 0.047). (C) HeLa cells were transfected with plasmids encoding HLA-A*03:01, gp100^mel full-length protein, β5i subunit or the empty vector. The HeLa cell ability to process the gp100^mel_{47–52/40–42} was assessed by CTL assay. Values are means and SD of 2 replicates in a representative assay of 4 independent experiments. Significant difference in the means of replicates is marked by * (student t-test; p = 0.001). (D) HeLa cells were exposed to 30 nM siRNA specific for the 19S Rpn10 subunit or p97/VCP (or control siRNA) prior to transfection with the HLA-A*03:01 and the full-length gp100^mel protein plasmids and pulsed or not pulsed with the synthetic peptide gp100^mel_{47–52/40–42} (30 μM). Values are means and SD of 2 replicates in a representative assay of 3 independent experiments. Significant differences in the means of replicates are marked by * (student t-test) for Rpn10 siRNA (E:T ratio: 1:1, p = 0.038; 2:1, p = 0.029; 4:1, p = 0.047; 8:1, p = 0.002) and for p97/VCP siRNA (E:T ratio: 1:1, p = 0.016; 2:1, p = 0.020; 4:1, p = 0.024; 8:1, p = 0.001). The knock-down efficiencies were monitored by determining the steady-state level of Rpn10 or p97/VCP proteins by western blotting.