[PubMed] [CrossRef] [Google Scholar] 28. (siNT). (A) Silencing of CHC and ATP6V12 was Berbamine hydrochloride assessed 72?h posttransfection by immunoblotting. (B) Cell surface expression of transferring receptor (CD71) was assessed by flow cytometry 72?h posttransfection using an anti-CD71 conjugated with phycoerythrin (PE). (C) Serum-starved cells were incubated 10?min at 37C with transferrin conjugated with Alexa Fluor 555 72?h posttransfection. (D) HeLa cells were transfected with an siRNA pool to CHC (siCHC), ATP6V12 (siATPB6V1B2), or adaptator-related protein complex 2 (siAP2M1) or the negative control (siNT). Cells were infected 72?h posttransfection with 17D (MOI of 1 1) and Asibi (MOI of 30). Infection levels were assessed 24?h postinfection by flow cytometry using the 2D12 Berbamine hydrochloride anti-E MAb and Rabbit Polyclonal to CSGALNACT2 normalized to infection in siNT-transfected cells. (E) HeLa cells transfected with siCHC and siNT were infected 72?h posttransfection with 17D (MOI of 1 1) and Asibi (MOI of 30) viruses produced from molecular clones. Infected cells were stained 24?h postinfection with the 2D12 anti-E MAb followed by anti-mouse Ab conjugated with Alexa Fluor 488 (green). (F) HeLa cells were transfected with an siRNA pool to ATP6V12 (siATPB6V1B2) or EPS15 (siEPS15) or the control siNT. Cells were infected 72?h posttransfection with 17D (MOI of 1 1) and Asibi (MOI of 30). Infection levels were assessed 24?h postinfection by flow cytometry using the 2D12 anti-E MAb and normalized to infection in siNT-transfected cells. (A to E) The Berbamine hydrochloride data shown are representative of three independent experiments. Download Figure?S2, TIF file, 1.1 MB mbo001162676sf2.tif (1.1M) GUID:?4E5448BA-5C16-4730-9095-4EAD100B0BE7 Figure?S3&#x000a0: Transfection of dynamin-2 siRNA impairs transferrin receptor (CD71) internalization. HeLa cells were transfected with an siRNA pool to dynamin-2 (siDyn-2) or the nontargeting negative control (siNT). Cell surface expression of CD71 was assessed 72?h posttransfection by Berbamine hydrochloride flow cytometry. Download Figure?S3, TIF file, 0.1 MB mbo001162676sf3.tif (124K) GUID:?5316D13E-CE84-462E-84B7-F890D747D4A4 Figure?S4&#x000a0: siRNAs directed against the clathrin heavy chain and caveolin-1 do not impair 17D infection in several human cell lines. Cells of the HeLa, 293T, and Huh7.5 cell lines were transfected with an siRNA pool targeting clathrin heavy chain (siCHC) or caveolin-1 (siCav1) or the nontargeting negative control (siNT). (A) Silencing of CHC and caveolin-1 were assessed 72?h posttransfection by immunoblotting. (B) Cells were infected 72?h posttransfection with 17D (MOI of 1 1) and normalized to infection in siNT-transfected cells. Download Figure?S4, TIF file, 0.6 MB mbo001162676sf4.tif (641K) GUID:?E88B8ABF-1CC3-47AC-A92B-FCEA4F3859BE Figure?S5&#x000a0: 17D infection is independent of the IL-2R clathrin-independent pathway. Hep2 cells were transfected with siRNA targeting the clathrin heavy chain (siCHC), ATP6V12 (siATPB6V1B2), Pak1, Rac1, or cortactin (siCTTN) or the nontargeting negative control (siNT). (A) Silencing was assessed by immunoblotting 72?h posttransfection. (B) Quantification of intracellular IL-2R and transferrin (TF) endocytosis in transfected cells. Results are expressed as percentages of intracellular fluorescence intensity normalized to siNT-transfected cells. The data shown are means SD from two independent experiments. (C) Cells were infected 72?h posttransfection with YFV 17D (MOI of 1 1), and the percentage of infected cells was assessed by flow cytometry using the 2D12 anti-E MAb. The data shown are representative of two independent experiments performed in duplicate. Download Figure?S5, TIF file, 0.3 MB mbo001162676sf5.tif (301K) GUID:?71A33A92-FB1A-47FC-BB69-0B823E601807 Figure?S6&#x000a0: E380 mutant Asibi RVPs are highly sensitive to CHC depletion. HeLa cells were transfected with an siRNA pool targeting the clathrin heavy chain (siCHC) or ATP6V12 or a nontargeting siRNA (siNT) as a negative control and then infected with the indicated RVPs 72?h posttransfection. Infection was assessed 24?h later by flow cytometry using the anti-YFV E 2D12 MAb. The data shown are representative of three independent experiments performed in duplicate. Download Figure?S6, TIF file, 0.1 MB mbo001162676sf6.tif (121K) GUID:?B807F2A5-6650-49FB-9957-9117156754CB Table?S1&#x000a0: Primers used for semiquantitative and real-time PCR. Table?S1, TIF file, 0.2 MB mbo001162676st1.tif (255K) GUID:?F34996A4-35BE-4353-9D8D-638AC6F28D6A ABSTRACT The live attenuated yellow fever virus (YFV) vaccine 17D stands as a gold standard for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its.