(A) HXB2 pseudotyped viruses co-labeled with Gag-mKO/DiD (top panel) or Gag-mKO/DiO (lower panel, DiO is coloured reddish) were adhered to poly-L-lysine coated coverslips

(A) HXB2 pseudotyped viruses co-labeled with Gag-mKO/DiD (top panel) or Gag-mKO/DiO (lower panel, DiO is coloured reddish) were adhered to poly-L-lysine coated coverslips. Additional file 2 Number S2. Transferrin uptake is definitely clogged by dynasore and partially inhibited by dynole. TZM-bl cells were left untreated (A, B) or LDC4297 were pretreated with 80 M dynasore from different manufacturers (C-G), dynole 34-2 (H) or with dynole 31-2 (inactive control, I) dissolved in DMEM for 30 min at 4C or 37C. Cells LDC4297 were then incubated with 20 g/ml of transferrin-Alexa488 (Invitrogen) in the chilly, washed and further incubated for 10 min at 4C (black histogram) or at 37C (reddish collection) in the absence or in the presence of dynamin inhibitors. Residual transferrin-Alexa488 in the cell TMPRSS2 surface was eliminated by pronase treatment (2 mg/ml, 10 min on snow), cells were washed with chilly PBS supplemented with 10% FBS and resuspended in an appropriate volume of chilly PBS. Transferring uptake was measured by circulation cytometry (FACS LSRII, BD Biosciences) gating on live cells bad for the propidium iodide staining. 1742-4690-8-99-S2.PDF (228K) GUID:?7E39176B-9D6F-47B8-93E6-2942613AD99A Additional file 3 Figure S3. Dynole, but not dynasore, affects the cell viability at concentrations that inhibit transferrin endocytosis. (A) The dose-dependent effect of dynasore from different manufacturers on LDC4297 TZM-bl cell viability was as determined by the MTS assay, using CellTiter 96 Aqueous reagent (Promega) according to the manufacturer’s specifications. The producing absorbance measured at 490 nm in triplicate wells was normalized to the transmission from untreated cells. Error bars are SEM. (B) The effect of dynole 34-2 and dynole 31-2 (inactive compound) from Ascent on TZM-bl cell viability determined by the MTS assay. 1742-4690-8-99-S3.PDF (37K) GUID:?093E2905-9583-42DE-AF5A-A6CCB96F4688 Additional file 4 Figure S4. The effect of dynole within the uptake of HIV-1 pseudoviruses. Representative images of HXB2 pseudovirus uptake by TZM-bl cells are demonstrated. Cells were pretreated with 60 M dynole 34-2 (A) or with 60 M dynole 31-2 (inactive compound) (B) in HBSS++ for 30 min followed by binding of pseudoviruses co-labeled with HIV Gag-Cherry and EcpH-ICAM-1 (a chimera consisting of the Ecliptic pHluorin and the ICAM-1 transmembrane website [23,25]). Cells were washed to remove unbound viruses and either imaged immediately (0 min) or incubated for 60 min at 37C (60 min) in the presence of a dynamin inhibitor, using the Zeiss LSM780 confocal microscope. Level bar is definitely 20 m. (C) Quantification of pseudovirus uptake exemplified in panels A and B. Z-stacks of cells from at least 5 random areas were acquired. Virus access into acidic endosomes upon incubation at 37C was manifested by quenching of the EcpH fluorescence (green) while the transmission from mCherry-tagged viral cores (reddish) was not significantly altered. The total EcpH intensity from several hundreds of cell-associated double-labeled viruses was determined, and the ratio of the sum of EcpH transmission to the sum of mCherry transmission (normalized to that at time = 0) was plotted. 1742-4690-8-99-S4.PDF (190K) GUID:?D6582CF4-7388-433C-9850-3AF37558F4E3 Additional file 5 Movie 1. Dynasore does not impact HXB2 disease motility. HXB2 pseudoviruses co-labeled with Gag-mKO (green) and DiD (reddish) were bound to TZM-bl cells at 4C, and cells were transferred to 37C to initiate fusion, at which point the and imaging begun. After 5 min, 60 M dynasore (Santa Cruz) was added to cells, and acquisition continued for an additional 15 min. Images were acquired using the Personal DeltaVision system. 1742-4690-8-99-S5.QT (1.1M) GUID:?76BF3097-F8BE-424B-A1BE-20F20F87357C Additional file 6 Movie 2. Rab5 motility is not modified by dynasore treatment. TZM-bl cells were transfected with Rab5-GFP. Twenty four hours after transfection, cells were imaged at 37C. After 5 min, 60 M of dynasore (Santa Cruz) was launched and imaging was continued for more 15 min. Images were acquired using the Personal DeltaVision system and processed with the Spot Enhancing Filter 2D plugin from ImageJ. 1742-4690-8-99-S6.QT (1.1M) GUID:?D2FBC41A-CA96-486B-A206-DE1636F72D49 Abstract Background We recently provided evidence that HIV-1 enters HeLa-derived TZM-bl and lymphoid CEMss cells by fusing with endosomes, whereas its fusion with the plasma membrane does not proceed beyond the lipid mixing step. The mechanism of restriction of HIV-1 fusion in the cell surface and/or the factors that aid the virus access from endosomes remain unclear. Results We examined HIV-1 fusion having a panel of target cells lines and with LDC4297 main CD4+ T cells. Kinetic measurements of fusion combined with time-resolved imaging of solitary viruses further reinforced the notion.