Effects of TRAPPC8 knockdown on gene transduction of PsVs and infection with authentic HPV31 virions.

<p>(A) HeLa cells were transfected with individual TRAPPC8 siRNAs (KIAA1012-01, -02, -03, or -04). At 2 days after siRNA transfection the cells were inoculated with 51PsVMaL2 (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry (lower left panel). Top panels: Western blotting of cells transfected with the indicated TRAPPC8 siRNAs using anti-TRAPPC8 antibody (Anti-N1/603) and anti-α-tubulin antibodies as a loading control. Lower right panel: viability of HeLa cells transfected with the indicated TRAPPC8 siRNAs was analyzed using a WST-1 cell proliferation assay at 96 h post-transfection. (B) HeLa cells were transfected with the indicated TRAPPC8 siRNAs (KIAA1012-03, or -04). At two days after siRNA transfection the cells were inoculated with 51PsVMaL2, 16PsV or 31PsV (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry. (C) HaCaT cells were transfected with the indicated TRAPPC8 siRNAs (KIAA1012-03 or -04). At two days after siRNA transfection the cells were inoculated with HPV31b virions prepared from CIN612-9E raft tissues. Following incubation for 3 days, E1ˆE4 viral transcript was quantified by RT-qPCR. The level of the HPV31E1ˆE4 transcript was normalized to that of β-actin mRNA. E1ˆE4 transcript relative to beta-actin mRNA in control siRNA transfected cells was set as 100%. Upper panels: Western blot of HaCaT cells at 48 h post-transfection with the indicated TRAPPC8 siRNAs using anti-N1/603 and anti-α-tubulin antibody. (D) Effects of TRAPPC12 knockdown on gene transduction with 51PsVMaL2. HeLa cells were transfected with control, individual TRAPPC12 siRNAs (TTC15-01, -02, -03, or -04) or TRAPPC8 siRNA (KIAA1012-04). At 2 days after siRNA transfection the cells were inoculated with 51PsVMaL2 (MOI of ∼50 particles/cell). Following incubation for 2 days, the number of cells expressing GFP was measured by flow cytometry (left lower panel). Lysates prepared from HeLa cells at 48 h post-transfection were analyzed by Western blot using anti-TRAPPC12 antibody (left upper panel) or anti-TRAPPC8 antibody (anti-N1/603) (right panel). Alpha-tubulin was detected as a loading control. All experiments (A to D) were performed in triplicate. Error bars indicate standard deviations.