IAV co-labeled with AF488 (green) and vDiD (red) was incubated with cells at 37C

IAV co-labeled with AF488 (green) and vDiD (red) was incubated with cells at 37C. (66K) GUID:?02163A40-8D96-4453-A0D6-47295960CAD7 Figure S3: Examples of fast vDiD dequenching events in A549 and MDCK cells. Relatively quick vDiD (red) dequenching events obtained by single particle tracking are shown for A549, A549-IFITM3, MDCK and MDCK-IFITM3 cells. The AF488 signal is shown in green and the ratio of vDiD and AF488 signals is shown in blue. Arrows mark sudden increases in the vDiD signal. a.u., arbitrary units.(PDF) ppat.1004048.s003.pdf (112K) GUID:?5070D925-924F-4484-A304-71F189B71F82 Figure S4: Correlation between the lag time before lipid mixing and the rate of vDiD dequenching (A) and the initial rates of vDiD dequenching (B). (A) The time of commencement of hemifusion (TH) and the initial rate of dequenching was determined as described in Materials and Methods. These parameters are uncorrelated (R2<0.19 for all). (B) The initial rates of vDiD dequenching were determined for A549-Vector, A549-IFITM3, MDCK and CHO cells. Error bars are SEM from >20 tracks. *, P<0.02.(PDF) ppat.1004048.s004.pdf (98K) GUID:?A50D0D6E-03DD-405F-AED7-22F4F6322D34 Figure S5: Relationship between IAV lipid mixing activity and infection. The fraction of A549 cells where at least one lipid mixing event was observed within 1 h at 37C, and the fraction of cells that became infected within 15 h at 37C were estimated as described in Methods S1. Infectivity data were collected from 5 image fields each, with >30 cells per field. Particle-to infectivity ratio was calculated from the fraction of infected cells and the average number of virions bound to cells. Live cell imaging experiments (n?=?10 for A549 and n?=?6 for MDCK cells) yielded the number of cells receiving at least hemifusion event.(PDF) ppat.1004048.s005.pdf (65K) GUID:?A1AC491C-5DE6-437A-9B42-327737916E2C Figure S6: Subcellular distribution of cholesterol and levels of total and free cellular cholesterol. (A) Total cellular filipin was estimated by calculating the filipin fluorescence intensity over the entire image field (after subtracting the background signal) and normalizing by the number of cells per field. Data are means and standard deviations for 4 and 6 fields Mavatrep for A549 and A549-IFITM3 cells (131 and 184 cells), respectively. (B, C) Total and free cellular cholesterol (in g/106 cells) were measured by a fluorimetric enzymatic assay using the Cholesterol Kit from Sigma-Aldrich. Data are means and standard deviations from 2 measurements performed Mavatrep with duplicate samples. ***, P<0.001; *, P<0.03.(PDF) ppat.1004048.s006.pdf (93K) GUID:?C1E58DCD-0055-4930-9032-5DB0B5438AEF Figure S7: Calibration of labeled IAV as a pH-sensor. AF488- and CypHer5E- labeled IAV particles were attached to poly-L-lysine coated coverslips, and the ratio of two fluorescence signals was measured in citrate-phosphate buffers of different acidity. (A) Top and bottom panels are images of labeled IAV at neutral pH and low pH, respectively. (B) The total signal for each dye was determined after thresholding and the CypHer5E/AF488 ratio at different pH are plotted. Error bars are standard deviations for 3 different imaged fields for each pH value. The line indicates a first order polynomial fit to the data, which served as a pH calibration curve.(PDF) ppat.1004048.s007.pdf (228K) GUID:?B4F5338E-1E43-4CFD-9C1E-39AD269CBF22 Figure S8: An example of single IAV lipid mixing event in CHO cells. (A) Image panels show entry of Mavatrep an AF488 (green) CLU and vDiD (red) labeled virus into a CHO cell that culminates in vDiD dequenching (arrow). (B) Fluorescence intensity profiles of AF488 and vDiD obtained by tracking the virion shown in panel A.(PDF) ppat.1004048.s008.pdf (148K) GUID:?095CA006-A3FB-4175-A75C-53F9C98B9E1F Figure S9: pH distribution in IAV carrying endosomes of CHO cells. Shown are the distributions of endosomal pH in CHO cells pretreated with 40 M of U18666A for 12 h or left untreated. Cells were incubated with AF488/Cypher5E-labeled IAV, and endosomal pH was measured as described in Materials and Methods. U18666A increased endosomal acidity (P<0.001).(PDF) ppat.1004048.s009.pdf (66K) GUID:?3991322A-45D6-44AE-B206-4388CC45635B Figure S10: Incoming IAV tends to colocalize with IFITM3-positive endosomes. A549-IFITM3 cells were allowed to internalize IAV for 90 min at 37C and immunostained for the IAV-NP using mouse antibody (Millipore, Billerica, MA) and for IFITM3. The enlarged boxed area is shown on the right. IAV and IFITM3 puncta were identified by thresholding and object identification. The extent of colocalization was Mavatrep estimated by counting IAV puncta, which exhibited a volumetric overlap of at least 50% with IFITM3 puncta, and normalizing over all IAV puncta. The number in the right corner is the mean % colocalization and standard deviation for 7 image fields.(PDF) ppat.1004048.s010.pdf (452K) GUID:?E185106C-8B9A-4C24-89D5-9CA6A91FF681 Figure S11: A line-fitting approach to determining the onset and the initial rate of vDiD dequenching in single IAV fusion experiments..