Article Figures & Data
Figures
- FIG 1
IFN-β induces cholesterol accumulation in acidic endosomes in A549 cells. (A) A549-WT cells or (B) A549-IFITM3KO cells were left untreated or exposed to IFN-β (10 ng/ml, 16 h) and loaded with LysoTracker Red DND-99 to label acidic endosomes. Unesterified cellular cholesterol was stained using filipin (blue). Filipin pixel intensities were pseudocolored according to the LUT and are shown as a heat map, with the gradient of colors ranging from blue (lowest intensity) to white (highest intensity). Bar, 20 µm. Colocalization coefficients of LysoTracker with filipin were quantitated from z-stacks. Mean values ± SEM were calculated from 35 individual cells per condition from at least two independent experiments. **, P ≤ 0.01; ****, P ≤ 0.0001 (unpaired Student’s t test). (C) A549-WT and A549-IFITM3KO cells were exposed to IFN-β (10 ng/ml, 16 h). The fractions of early endosomes (EE) and late endosomes/lysosomes (LE) were enriched from postnuclear supernatants (PNS) through sucrose gradient separation and analyzed for their cholesterol levels. Data are expressed as mean cholesterol concentrations (in micrograms per milliliter) ± SEM from at least three independent gradients. **, P ≤ 0.01; ns, not significant (two-way ANOVA with Tukey’s multiple-comparison test).
- FIG 2
LE/L positioning is linked to LE/L cholesterol. (A) A549-WT cells transfected with GFP or YFP-NPC1 were left untreated or exposed to IFN-β (10 ng/ml, 16 h). A549-IFITM3KO cells were left untreated or exposed to U18666A (U18) (2 µg/ml, 16 h). All cells were then loaded with LysoTracker Red DND-99 to label acidic endosomes. Bar, 5 µm. (B) From each LysoTracker-positive object, the number of neighbors in a 30-px radius was determined with the help of the ImageJ plugin collection “BioVoxxel Toolbox.” Data are expressed as the frequencies of distribution determined for at least 5 cells per condition.
- FIG 3
Interferon-independent LE/L cholesterol accumulation impairs early IAV infection. (A) A549-WT cells transfected with GFP, AnxA6-GFP, or AnxA6-GFP together with YFP-NPC1 were infected with IAV (PR8M; MOI of 5, 4 h). Cells were stained with anti-NP antibodies, and nuclei were labeled with DAPI. Bar, 20 µm. Numbers of transfected cells with NP-positive nuclei were determined from 150 transfected cells per condition from at least three independent experiments. Data are expressed as mean percentages ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001; ns, not significant (one-way ANOVA with Tukey’s multiple-comparison test). (B) A431-WT and A431-AnxA6 cells were transfected with GFP or YFP-NPC1 as indicated and were infected with IAV (PR8M; MOI of 20, 4 h). Numbers of transfected cells with NP-positive nuclei were determined from 150 transfected cells per condition from three independent experiments. Data are expressed as mean percentages ± SEM. ****, P ≤ 0.0001 (two-way ANOVA with Tukey’s multiple-comparison test). (C) A431-WT cells expressing GFP or YFP-NPC1 were stimulated with IFN-β (10 ng/ml, 16 h) as indicated and then infected with IAV (MOI of 20, 4 h). Numbers of cells with NP-positive nuclei were determined from 150 transfected cells per condition from at least three independent experiments, and the mean percentages ± SEM of NP-positive nuclei were quantitated. *, P ≤ 0.05 (unpaired Student’s t test). (D) A549-WT and A549-IFITM3KO cells were exposed to U18666A (U18) (2 µg/ml, 16 h) and were infected with IAV (PR8M; MOI of 2, 4 h), stained for NP, and analyzed by FACS (10.000 cells per sample). Data are expressed as the mean percentages ± SEM of NP-positive nuclei from three independent experiments. ****, P ≤ 0.0001 (two-way ANOVA with Tukey’s multiple-comparison test).
- FIG 4
Impact of LE/L cholesterol accumulation on the sequential steps of IAV host cell entry. (A) A431-WT and A431-AnxA6 cells were infected with IAV (PR8M; MOI of 50) at 4°C for 1 h. An acidic bypass was applied to induce fusion at the plasma membrane, and infection through endosomal uptake was blocked by bafilomycin A1 (Baf) treatment. Cells were further incubated for 8 h, stained for NP, and analyzed by FACS for NP-positive cells. Data are expressed as the mean percentages ± SEM of NP-positive cells from three independent experiments. (B) A431-WT and A431-AnxA6 cells were infected with IAV (PR8M; MOI of 10). M1 protein levels were monitored by Western blotting, and blots were probed for tubulin to verify equal levels of loading. Mean M1 signal intensities ± SEM of results from three independent experiments were calculated relative to the mean M1 intensity detected in A431-WT cells 30 min p.i. (C) A431-WT and A431-AnxA6 cells were infected with IAV (×31; MOI of 20). Cells were fixed and stained 1 h p.i. with the A1 antibody to detect the acid-induced conformation of HA (HAac). The signal intensity per cell was quantified by ImageJ analysis. Mean values ± SEM were calculated from 55 individual cells per condition from at least two independent experiments. ns, not significant (two-way ANOVA followed by Tukey’s multiple-comparison test).
- FIG 5
LE/L cholesterol accumulation inhibits IAV/host membrane fusion. (A) A549-WT and A549-IFITM3KO cells were either left untreated or exposed to IFN-β (10 ng/ml, 16 h), U18666A (U18) (2 µg/ml, 16 h), or bafilomycin A1 (Baf) and were infected with dually labeled IAV (PR8M; MOI of 10). SP-DiOC-positive cells were identified by FACS analysis at 1 h p.i. (10,000 cells per condition). Data are expressed as percentages of the mean number of SP-DiOC-positive cells in the untreated A549-WT cell sample and represent mean values ± SEM. **, P ≤ 0.01; ****, P ≤ 0.0001 (two-way ANOVA followed by Tukey’s multiple-comparison test). (B) A431-WT and A431-AnxA6 cells transfected with control (siC) or IFITM3-specific (siIFITM3) siRNA were either left untreated or incubated with bafilomycin A1 (Baf) and were infected with dually labeled IAV (PR8M; MOI of 10). SP-DiOC-positive cells were identified by FACS analysis at 1 h p.i. (10,000 cells per condition). Data are expressed as percentages of the mean number of SP-DiOC-positive cells in the untreated A431-WT cell sample and represent mean values ± SEM. *, P ≤ 0.05 (two-way ANOVA followed by Tukey’s multiple-comparison test).
Supplemental Material
FIG S1
IFN-β induces endosomal/lysosomal cholesterol accumulation. (A) A549-WT cells left untreated or exposed to IFN-β (10 ng/ml, 16 h) were stained for LE/L marker protein CD63. Unesterified cellular cholesterol was visualized using filipin (blue). Filipin pixel intensities were pseudocolored according to the LUT and are shown as a heat map, with the gradient of colors ranging from blue (lowest intensity) to white (highest intensity). Bar, 20 µm. Colocalization coefficients of CD63 with filipin were quantitated from z-stacks. Mean values ± SEM were calculated from 35 individual cells per condition from at least two independent experiments. ****, P ≤ 0.0001 (unpaired Student’s t test). (B) Numbers of voxels positive for both filipin and LysoTracker or for filipin or CD63 individually were related to the total number of LysoTracker-positive (or CD63-positive) voxels. Mean values ± SEM were calculated from 35 individual cells per condition from at least two independent experiments. **, P ≤ 0.01 (unpaired Student’s t test). Download FIG S1, PDF file, 2.2 MB.
Copyright © 2018 Kühnl et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S2
A549 IFITM3 knockout by CRISPR/Cas9 editing. (A) IFITM3 protein levels in lysates of A549-WT and A549-IFITM3KO cells either left untreated or exposed to IFN-β (10 ng/ml, 16 h) were analyzed by immunoblotting. Equal levels of protein loading were verified using GAPDH antibody. STAT1 served as a positive control for IFN-induced gene expression. (B) An antibody recognizing both IFITM2 and IFITM3 was used to analyze IFITM2 protein levels in the A549-IFITM3KO cells. GAPDH served as a loading control. (C) A549-IFITM3KO cells exposed to U18666A (U18) (2 µg/ml, 16 h) were stained for cellular cholesterol using filipin and were analyzed by confocal microscopy. Filipin pixel intensities were pseudocolored according to the LUT and are shown as a heat map, with the gradient of colors ranging from blue (lowest intensity) to white (highest intensity). Bar, 20 µm. Download FIG S2, PDF file, 1.2 MB.
Copyright © 2018 Kühnl et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S3
Characterization of endosomal pH. Endosomal/lysosomal pH was measured by ratio imaging of OG488/TMR-dextran in A549-WT cells incubated overnight with the indicated concentrations of U18666A (U18) (A) as well as in A549-WT cells transfected with myc-IFITM3 or empty vector (B) and in A431-WT and A431-AnxA6 cells (C). pH values are means ± SEM of results from 30 cells from five independent experiments. ****, P ≤ 0.0001; ns, not significant (one-way ANOVA followed by Tukey’s multiple-comparison test). Download FIG S3, PDF file, 0.04 MB.
Copyright © 2018 Kühnl et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S4
Characterization of impaired IAV endosomal escape. (A) A549-WT cells transfected with myc-IFITM3 or empty vector as a control were infected with IAV particles labeled with SP-DiOC and R18 (PR8M; MOI of 10). To monitor lipid mixing of virus envelope and endosomal membranes, dequenching of SP-DiOC was measured by FACS analysis at 1 h p.i. (10,000 cells per condition). Data are expressed as percentages of the mean numbers of SP-DiOC-positive cells in the empty vector control and represent mean values ± SEM of results from three independent experiments. *, P ≤ 0.05 (Student’s t test). (B) A431-WT and A431-AnxA6 cells were either left untreated or incubated with bafilomycin A1 (Baf) and were infected with single-labeled IAV (PR8M; MOI of 10). SP-DiOC-positive cells or DiD-positive cells were identified by FACS analysis at 1 h p.i. (10,000 cells per condition). Data are expressed as percentages of the mean number of SP-DiOC-positive cells or DiD-positive cells in the untreated A431-WT cell sample and represent mean values ± SEM. (C) A549-WT cells were either left untreated or exposed to U18666A (U18) (2 µg/ml, 16 h) and/or bafilomycin A1 (Baf) and were infected with dually labeled IAV (PR8M; MOI of 10). SP-DiOC-positive cells were identified by FACS analysis at the indicated times p.i. (10,000 cells per condition). Data were expressed as geomeans of SP-DiOC signals in the respective samples, and the slopes of linear regression were calculated. Histograms of the original FACS results are depicted at the bottom of the panel. Histogram marker M1 defines the SP-DiOC-positive cell subpopulation. Download FIG S4, PDF file, 0.4 MB.
Copyright © 2018 Kühnl et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
