Figure 1(B) 48h post-transfection of HeLa-CIITA cells with siRNAs targeting NDP52, T6BP, OPTN, and p62, AR expression was analyzed using Western Blot. Tubulin was used as control. The results are representative of at least 3 independent experiments and correspond to AR expression levels of the experiment in Figure 1B, left panel.(C) Monitoring of Gag-specific T cell activation. HeLa-CIITA cells were treated as indicated above and transfected with a plasmid encoding Gag-LC3. Left panel, a representative experiment is shown (+/- SD of three technical replicates). Right panel, three biological replicates are combined and presented as mean percentage (+/- SD). The y-axis represents the relative percentage of IFNγ spots reported to the secretion of IFNγ by the CD4+ T cell clones incubated with the siCTRL-treated HeLa-CIITA and set to 100%. The mean T cell activation levels of the three experiments using siCTRL or siT6BP were (100, 195, 119) and (35, 53, 38) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).(D) Left panel, as in Figure 1B right panel, but using DNA encoding Gag-LC3, Gag-LC3G120A, or Gag. With Gag-LC3, Gag-LC3 G120A or Gag antigens, the mean T cell activation levels using siCTRL or siT6BP were (199, 435, 384), (91, 141, 119) and (343, 415, 385) or (98, 273, 171), (27, 50, 19) and (97, 148, 131) IFNγ+ spots, respectively. 5000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Right panel, influence of T6BP silencing on peptide presentation by Hela-CIITA cells. The cognate peptide was added exogenously (gag2, 0,1µg/mL) on siRNA-treated cells (2h, 37°C), washed and T cell activation monitored using IFNγ-ELISPOT. The mean T cell activation levels using siCTRL or siT6BP were (100, 45, 42) and (71, 45, 33) IFNγ+ spots, respectively. 1000 T cell clones were seeded per well in technical triplicates. Three biological replicates are combined and presented as mean percentage (+/- SD). Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols(E) As in (D) but using cDNA encoding HCMV pp65 antigen (left panel) or pp65 peptide (0,5µg/mL; right panel) and a pp65-specific CD4+ T cell line. Results of three independent experiments are represented. The mean T cell activation levels using siCTRL or siT6BP and pp65 DNA were (66, 106, 99) and (39, 50, 33) IFNγ+ spots, respectively. For the peptide: siCTRL or siT6BP: (185, 317, 415) and (163, 292, 381) IFNγ+ spots, respectively. 1000 or 2000 T cell clones were seeded per well in technical triplicates. Data information: For all ELISPOT experiments, the background secretions of IFNγ by CD4+ T cells co-cultured with mock-treated HeLa-CIITA cells were used as negative controls and subtracted. CTRL: control. Wilcoxon's test; the symbols correspond to ** p<0.01 and # p>0.05 comparing each experimental conditions solely with its internal control (siCTRL).