Fig 2: Anti-S1-RBD IgG cross-reacts with DENV proteins. (A) Recombinant DENV proteins, including envelope (E), precursor membrane (prM), and nonstructural proteins (NS1, NS4B), were coated in an ELISA plate. The cross-reactivity of anti-S1-RBD IgG to different DENV antigens was tested by an indirect ELISA. (B) The colocalization of anti-S1-RBD IgG (detected by goat-anti-Rbt-594) and different anti-DENV mAbs (detected by goat anti-mouse Alexa 488-conjugated antibodies) in DENV-infected C6/36 cells was determined by an immunofluorescent assay and visualized by Flourescence microscope DP72 (Olympus, Japan). (C) The colocalization of anti-S1-RBD IgG and anti-DENV E mAb were visualized by an FV3000-confocal laser scanning microscope (Olympus, Japan). The experiments were repeated two or three times with similar results, data from a single representative experiment was shown.

Fig 3: Effect of IRW on tight junction protein distribution on cell membrane surfaces. Cells were grown on coverslips for 21 days to allow differentiation and incubated with IRW (100 µM) for 24 h prior to LPS (20 µg/mL) treatment for 1 h. After treatment, cells were fixed with 4% paraformaldehyde, permeabilized with Triton X 100, and stained with the primary anti-occludin antibody at 4 °C on a slow shaker overnight. The antirabbit CF 594 antibody (Sigma) was used to visualize the localization of occludin by a confocal laser scanning microscope (Olympus FV3000). Images were captured under the 10× objective lens. Ctrl means the control group.

Fig 4: Activated small TFEB associates with full-length TFEB in the nucleus. (A) T4 cells were transiently co-transfected with EGFP-tagged TFEB (EGFP-TFEB-L) and mCherry-tagged small TFEB (mCherry-TFEB-S) and treated on the next day with 500 nM Torin1 for 1 h. Images were obtained using a confocal microscope (Olympus Corp., FV3000-ORS). (B) HEK293 cells were transiently co-transfected with the HA-TFEB expression plasmid and either the plasmid expressing EGFP-tagged small TFEB (GFP-TFEB-S) or the mock plasmid expressing only the GFP protein. The cells were treated with 500 nM Torin1 for 10 min. The cell lysates were used for small TFEB immunoprecipitation using an anti-GFP antibody. The protein level of small TFEB co-immunoprecipitated with TFEB-L was examined by immunoblotting using an anti-TFEB antibody. Full blots are provided in Supplementary Fig. S6. (C) Bar graph represents the relative amount of TFEB-L co-immunoprecipitated with small TFEB. The data are shown as the mean ± S.E. of three independent experiments and were analyzed using Student’s t test. (**p < 0.01).

Fig 5: SERA6 and its partner MSA180 form transient multimolecular aggregates during egress Representative IFA images demonstrating the dynamic changes in localisation of SERA6-mTAP during egress (reproducible in 4 independent experiments). SERA6-mTAP:loxP schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green; ankyrin is a major component of the RBC cytoskeleton). Scale bar, 5 µm. Z-stack series of a representative schizont demonstrating that SERA6 aggregates observed during egress are distributed throughout the schizont. SERA6-mTAP:loxP schizonts sampled 5 min following removal of C2-arrest were fixed and co-stained with anti-HA (red) and anti-ankyrin (green), then imaged on an Olympus FLUOVIEW FV3000. Scale bar, 5 µm. See also Fig EV5.Representative IFA images demonstrating localisation of MSA180-HA3 during egress. MSA180-HA3 [B11] schizonts were sampled at the indicated times following removal of C2-arrest, fixed and co-stained with DAPI (blue), anti-HA (red) and anti-ankyrin (green). Scale bar, 5 µm.