Fig 2: Rab31 inhibits MAPK6 degradation. (A) The interaction of endogenous Rab31 and MAPK6 in SiHa cells was detected using the Co-IP assay. (B) Transfection of plasmids expressing Rab31 (Flag tag) and MAPK6 (HA tag) into SiHa cells and the interaction between exogenous Rab31 and MAPK6 was detected. (C) Detection of MAPK6 protein expression after Rab31 knockdown. (D) Western blotting of MAPK6 cellular localization following Rab31 knockdown was shown. (E) Cells with Rab31 knockdown were incubated with 5 µg/mL of CHX and harvested at the indicated times. MAPK6 stability was monitored by the line chart, and the half-life of MAPK6 was evaluated on the right. (F) Cells with Rab31 overexpression were incubated with 5 µg/mL of CHX and harvested at the indicated times. MAPK6 stability and the half-life of MAPK6 were shown. (G-H) The mRNA level of MAPK6 was detected following Rab31 knockdown or Rab31 overexpression using qRT-PCR. *P <0.05, **P <0.01, ***P <0.001.

Fig 3: Rab31 knockdown inhibits the metastasis of cervical cancer cells in vivo. (A) In vivo fluorescence imaging of nude mice showed that fluorescence was mainly located in the chest and abdomen. (B) Visible lung and liver metastatic nodules and fluorescence imaging of the lung and liver were shown. (C-D) H&E staining of paraffin-embedded sections of lung and liver metastatic nodules was shown. The pictures below are enlargements of the rectangle in the pictures above. The arrows indicate the metastatic nodules.

Fig 4: Depletion of RAB22A in HuH7 cells.(A) Western blot showing endogenous RAB22A in HuH7 and Trf4, as well as in HuH7 expressing empty vector (Ctrl) or RAB22A shRNAs (sh1 and sh2), as detected with anti-RAB22A antibody. Actin was a loading control. The data are representative of blots from three independent lysates of stable HuH7 sh1 and sh2 populations. The ratio of RAB22A to actin bands determined by NIH ImageJ was not significantly different for HuH7 versus HuH7 vector or Trf4 but RAB22A was significantly reduced in sh1 (p<0.04) and sh2 (p<0.0001). (B) The independent lysates from (A) were examined with anti-RAB31 antibody. There was no significant difference RAB31 expression between HuH7, Trf4 and HuH7 cells expressing sh1 or sh2. (C) Surface Tfn binding was analyzed after incubation of Alexa Fluor 488-labeled Tfn with cells on ice for 30 min followed by flow cytometry. Results were normalized to surface Tfn in HuH7 cells and are shown as mean ± SEM, n = 4. (D) MTT assay of cells incubated with different concentrations of PEx at 37°C for 4 days. Cells were treated with MTT solution at 37°C for 3 hours. Cell viability was reflected by the color of the formazan crystals solubilized in DMSO. The data are representative of two independent experiments. Statistical significance was determined by Student’s t-test. *p<0.05, ***p<0.001.

Fig 5: Active RAB31 drives EGFR entry into CD63-positive MVEs to form ILVs and exosomes.a Up panels, immunofluorescence of EGFR-HA (red) and Flag-RAB31 (magenta) with CD63-GFP (green) in the indicated stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under serum starvation (SS). Low panel left, the ratio of co-localization of EGFR-HA with CD63-GFP-positive LE/MVE in Vector (n = 6 fields), RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). Low panel middle, the ratio of entry of EGFR-HA into CD63-GFP-positive LE/MVE in RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). Low panel right, the ratio of entry of Flag-RAB31 into CD63-GFP-positive LE/MVE in RAB31WT (n = 8 fields) and RAB31Q65L (n = 11 fields). b–d Immunofluorescence of the localization of EGFR-HA (red) with CD63-GFP (green) (b), Flag-RAB31Q65L (red) with CD63-GFP (green) (c), and Flag-RAB31Q65L (green) with EGFR-HA (red) (d) in Flag-RAB31Q65L stable HeLa cells transiently expressing EGFR-HA and CD63-GFP under SS using three-dimensional structured illumination microscopy (3D-SIM). e Immunoelectron microscopy of the localization of EGFR-HA in Vector stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. f Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31WT in Flag-RAB31WT stable HeLa cells transiently expressing EGFR-HA under SS. NE, nuclear envelope; N, nucleus; C, cytoplasm. g Immunoelectron microscopy of the localization of EGFR-HA and Flag-RAB31Q65L in Flag-RAB31Q65L stable HeLa cells transiently expressing EGFR-HA under SS. h Western blotting analyses of the concentrated conditional media from the indicated stable HeLa cells under SS. i NanoSight nanoparticle tracking analysis of the concentrated conditional media from the indicated stable HeLa cells under SS. j Transmission electron microscopy analysis of the concentrated conditional media from Flag-RAB31Q65L stable HeLa cells under SS. All data are means ± SD. Unpaired t-test was used to analyze the difference between the two groups. ****P < 0.0001. Scale bars, 10 µm (a–d), 200 nm (e–g), 100 nm (j).