Fig 2: TMEM206 is present in early endosomes in HEK cells.(a) Immunofluorescence staining of endogenous TMEM206 in HEK WT cells. This staining is abolished in TMEM206 KO HEK cells. (b) Endogenous TMEM206 co-localizes with EEA1 and rab5, early endosomes markers, and partially with rab7, a marker of late endosomes. Scale bars: 10 µm, 1 µm for enlargements.

Fig 3: B. pseudomallei resides in a Rab32-labeled compartment with late endosomal features.(A) RAW264.7 cells were infected with B. pseudomallei, at an MOI of 10 for 2 h. Cells were stained with anti-EEA1, anti-Rab5, and anti-Rab7 antibodies (green), or anti-B. pseudomallei antibody (red) and colocalization was determined by confocal microscopy. Scale bar is 5 μm. (B) The percentage of EEA1, Rab5, or Rab7 colocalizing with B. pseudomallei phagosomes was enumerated by fluorescence microscopy. At least 100 bacterial phagosomes were counted for each time point. Results are represented as mean ± S.D. of three independent experiments. (C) RAW264.7 cells expressing EGFP-Rab32 were infected with B. pseudomallei for 2 h, afterwards cells were subjected to immunofluorescence for LAMP1 or LAMP2 (red) and stained with an anti-B. pseudomallei antibody (blue). (D) RAW264.7 cells expressing EGFP-Rab32 were infected with B. pseudomallei, fixed, and immunostained for LAMP1 (red) and B. pseudomallei (blue). B. pseudomallei phagosomes that colocalized with Rab32 and LAMP1 were quantified. Results are the means ± SD of three independent experiments. (E) Quantitative analysis of B. pseudomallei phagosomes that colocalized with Rab32 and LAMP2 as indicated in D. (F) RAW264.7 cells expressing EGFP-Rab32 were incubated with 50 nM Lysotracker (red) for 1 h before infection with B. pseudomallei for 2 h. Cells were stained with anti-B. pseudomallei antibody (blue) and colocalization was determined by confocal microscopy. (G) Quantitative analysis of B. pseudomallei phagosomes that colocalized with Rab32 and Lysotracker as indicated in D. Symbols represent single bacteria or distinct B. pseudomallei groups. Scale bar is 5 μm.

Fig 4: SARS-CoV-2 enters into ECs through endocytosis. (A) Representative electron microscopy images (× 80,000) for senescent HUVECs infected with SARS-CoV-2 at 50 MOI (2 hpi). Arrows indicate SARS-CoV-2 virions. Bars: 200 nm. (B) Representative electron microscopy images (× 80,000) for early passage and senescent ECs infected with SARS-CoV-2 at 1 MOI (6 hpi). Arrows indicate SARS-CoV-2 entry via caveolae-like structures. Bars: 200 nm. (C) Representative electron microscopy images (× 80,000) for early passage and senescent ECs infected with SARS-CoV-2 at 1 MOI (24 and 48 hpi). Arrows indicate SARS-CoV-2 virions. Bars: 200 nm. (D) Representative images of immunocytochemistry for SARS-CoV-2 spike protein (red fluorescence) and Rab5A (green fluorescence) in replicative senescent HUVECs infected with SARS-CoV-2 (50 MOI) at 6 hpi. SARS-CoV-2 spike proteins were largely co-localized with Rab5A. Bars: 100 μm. Spike and Rab5A double-positive areas were quantitatively analyzed (n = 6 each). Statistical analysis was performed using Mann Whitney U-test. Data are presented as mean ± SE **P < 0.01.

Fig 5: SCYL1 Ser754 phosphorylation regulates endosome distribution and EV secretion.a, b Expression of SCYL1(S754E) complements SCYL1 KO effect on endosome distribution. Fluorescent micrograph of SCYL1 KO MCF-7 cells expressing HA-SCYL1(WT), HA-SCYL1(S754A) or HA-SCYL1(S754E) variants upon 24 h (for RAB5 and RAB7) and 36 h doxycycline induction (for LAMP2), respectively. Representative pictures of n = 3 replicates are shown. Scale bar = 10 µm. c Quantification of a, b. The distance from nuclear envelope to RAB5, RAB7 and LAMP2 foci was computed and averaged per individual cell. All results were assembled in a violin plot (white dots within violins denote medians; top and bottom of violins represent the 25th and 75th percentiles; thick vertical lines within violins show the IQR, whereas thin vertical lines represent the rest of the distribution extending the 1.5xIQR). Single-cell values are indicated with white small dots. An unpaired two-tailed Student’s t-test was used for comparison; RAB5: WT/VC: p = 0.0021 (**); S754E/VC:p = 0.00022(***); WT/S754A: p = 0.0058(**); S754E/S754A: p = 0.00059(***). RAB7: WT/VC:p = 0.00077(***);S754E/VC:p = 0.00057(***); WT/S754A: p = 0.00085(***); S754E/S754A: p = 0.00082 (***). LAMP2: WT/VC: p = 0.00074(***); S754E/VC:p = 0.00099(***); WT/S754A: p = 0.00039(***); S754E/S754A: p = 0.0007(***). =20 cells were measured amongst technical triplicates of two biological replicates (n = 6). Error bars = SEM. Source data are provided as Source Data file. d HA-SCYL1 WT and S754E, but not S754A, rescue SCYL1 KO-induced EV secretion. Western blot of ultracentrifuged CM of SCYL1 KO MCF-7 cells expressing HA-SCYL1(WT), HA-SCYL1(S754A) or HA-SCYL1(S754E) mutants after 24 h doxycycline induction. Cells containing a vector-only transgene (VC) were used as negative control. To normalize protein contents, equal amounts of cells were used. One representative experiment out of n = 3 biological replicates is shown. e Quantification of d, average of n = 3 biological replicates. Error bars = SEM. An unpaired two-tailed Student’s t-test was used for comparison: WT/VC: CD9 p = 0.00031 (***), CD81 p = 0.0060 (**); S754A/VC: CD9 p = 0.0012(**), CD81 p = 0.038(*); S754E/VC: CD9 p = 0.0010(**), CD81 p = 0.036 (*); WT/S754A: CD9 p = 0.036(*), CD81 p = 0.035(*); WT/S754E: CD9 p = 0.91(ns), CD81 p = 0.66 (ns). All source data are provided as Source Data file.