Fig 2: Rh1 protein localization in fabpEY06747 mutant eyes.(A) A schematic diagram of adult Drosophila ommatidia, with rhabdomeres labeled as black circles, and endosomal vesicles as white circles. (B-E) Anti-Rh1 labeling (green) of adult Drosophila ommatidia. (B, C) Control fabp wild type eyes. (D, E) fabpEY02678 -/- eyes. Fly samples (B, D) were reared under constant light before being processed for fixation and immuno-labeling. By contrast, samples (C, E) were reared under constant darkness before processing. The scale bar in G applies for images D–G. (F, G) Assessment of Rh1 localization in fabpEY06747 -/- flies reared under light through ninaE-GFP expression (green). This line has GFP fused in frame with the Rh1 protein sequence, driven by the ninaE promoter. Rh1-GFP is found in rhabdomeres and in cytoplasmic puncta. (F) Double labeling with anti-Rab5 antibody that marks early endosomes (red). (G) Double labeling with anti-Rab7 antibody that marks late endosome/lysosomes (red). Single channel images of the red channel are in (F’, G’). White arrows point to representative regions of overlap. The scale bar in K’ represents images in J and K.

Fig 3: Clinical significance of YTHDC2-related exosome-stimulating system in LUAD and its acinar subtype.A and B, immunochemistry stain for LUAD tissue microarray slides using antibodies against YTHDC2, OAS1, IFIT2, and RAB5A. IHC score was displayed in (B). C and D, YTHDC2 protein level and OAS1, IFIT2, and RAB5A mRNA levels were measured in acinar subtype LUAD (C). The results were further presented according to the tumor stage (D). E, Survival analysis in acinar subtype LUAD. Patients were divided into two groups according to the level of YTHDC2 expression. F, YTHDC2 protein level were measured in papillary, solid, and micropapillary subtype LUAD. ∗∗p < 0.01 indicates statistical significance. NS, non-significance. Data in B, C, and F were analyzed by a student’s t test. Data in D were analyzed by a one-way ANOVA test. Data in E were analyzed by a log-rank test.

Fig 4: Knockout of TMEM239 affects colocalization of the viral p72 protein with Rab5A.(A and B) Knockout of TMEM239 affects the colocalization of the viral p72 protein with Rab5A. WSL cells (A) and TMEM239-/- cells (B) were incubated with ad-HRB1 isolates (MOI = 20) for 2 h at 4°C and then washed with ice-cold PBS to remove unbound virus particles. Then, ice-cold medium was added, and the cells were incubated at 37°C for 30, 45, and 60 minutes before being fixed in methanol. Immunofluorescence analysis was performed against p72 (red) and Rab5A (Green). Nuclear DNA was visualized by DAPI staining (blue). (C) Quantification of p72-Rab5A Co-localization. p72-Rab5A co-localization was quantified by using Pearson’s correlation coefficient method. Cells co-stained with p72 and Rab5A antibodies were randomly selected for co-localization analysis by using ImageJ software. ***p < 0.001.

Fig 5: TMEM239 interacts with Rab5A and the virus protein pE248R.(A) TMEM239 and Rab5A co-immunoprecipitation (Co-IP) assay. HEK-293T cells were transfected with pTMEM239-Flag and pRab5A-Myc either together or separately. At 36 hpt, cell lysates were collected for co-immunoprecipitation with beads conjugated with Myc antibody. Tubulin was used as an internal loading control. (B) Colocalization analysis of TMEM239 and Rab5A. Colocalization of TMEM239 and Rab5A was assessed in HEK-293T cells co-transfected with pTMEM239-Flag and pRab5A-Myc by use of confocal microscopy. Immunofluorescence analysis was performed against Flag (red) and Myc (Green). Nuclear DNA was visualized by DAPI staining (blue). (C and D) Dynasore inhibits ASFV internalization into WSL cells. WSL cells pre-treated for 15 minutes with the inhibitor for clathrin-dependent endocytosis dynasore were incubated with ad-HRB1 isolate (MOI = 5) for 2 h at 4°C, followed by extensive washing with ice-cold PBS. Subsequently, the cells were shifted to 37°C for 30 min to facilitate the internalization of viral particles, and a trypsin treatment was applied to eliminate the remaining surface-bound virus particles. Western blot analysis (p54) and quantification of viral genome copies (p72) were used to quantify virus particles internalized into cells. ***p < 0.001. (E) TMEM239 and viral proteins co-immunoprecipitation (Co-IP) assay. HEK-293T cells were transfected with pTMEM239-Myc and HA-tagged viral protein expression plasmids either together or separately. At 36 hpt, cell lysates were collected for co-immunoprecipitation with beads conjugated with Myc antibody. Tubulin was used as an internal loading control.