Fig 2: Wound healing of U3DT cells.(A) Control cells fixed immediately after scratching. The wide of the scratched area was ca. 500µm. (B?D) Mock (B), non-targeting siRNA (NT)-treated (C), and GPC5-siRNA (KD)-treated (D) cells were cultured in appropriate medium containing 25 nM FGF2 for 23 h after scratching. Cells were fixed and immunofluorescence images were acquired using a Leica SP-8 microscope equipped with a 20x objective. (E) The number of cells that moved into the scratched area or the removed insert area was counted after incubation of control (without siRNA) cells (yellow), NT cells (red), and KD cells (blue) in appropriate medium containing (red and blue columns) or lacking (brown and sky blue columns) FGF2 at 37°C for 23 h. (F?H) Immunofluorescence images of cells treated with mock (F), non-targeting siRNA (G), or GPC5-targeting siRNA (H) for 72 h after removing an insert from a µ-Dish. Cells were stained with anti-GPC5 (red) and anti-FGFR1 (green) antibodies. (I) Quantification of the percentage of cells with blebs at telophase. Control (mock) cells (yellow), NT cells (red) and KD cells (blue) were incubated in appropriate medium containing FGF2 at 37°C for 23 h. (J?L) Immunofluorescence images of mock (J), non-targeting siRNA-treated (K), and GPC5-siRNA-treated (L) cells after 72 h. Cells were stained with WGA (green). White arrows indicate blebs in a telophase cell (J, K). The numbers in parentheses in (E, I) are the number of images observed. Scale bar: (F?H), 100 µm; (J?L), 2 µm.

Fig 3: Alterations in GPC5 expression during long-term culture.Gene expression of GPCs (GPC1 to GPC6) at four culture stages were analyzed using data in the DDBJ database of the National Institute of Genetics (http://www.ddbj.nig.ac.jp/), accession number: DRA000533 [3]. Each value is shown relative to the corresponding value in U3-A cells (culture stage 1). GPC1, red; GPC2, green; GPC3, no expression; GPC4, purple; GPC5, blue; GPC6, light blue. As shown in the previous report [3], PDLs of U3-A, U3-B, U3-C and U3DT cells are 60?90, 91?150, 151?230 and 231?295, respectively.

Fig 4: EVs are incorporated to UE6E7T-3 cells.(A, D) Images of control cells. (B, E) Images of EV-treated cells. (D, E) Merged images of UE6E7T-3 cells (D) and EV-treated cells (E) were stained for GPC5 (red) and FGFR1 (green). (C) Quantitation of GPC5 (pixel sum per cell) in control UE6E7T-3 cells (blue) or cells cultured with EVs for 1 day (red). n = 67 (blue) and 78 (red). (F, G) FACS pattern of GPC5 in UE6E7T-3 cells (F) and in cells cultured with EVs for 1 day (G). ? indicates GPC5-positive cells. Scale bar, 5 µm.

Fig 5: Localization of GPC5 and Rab11 in U3DT cells treated with RAB11A-siRNA.Immunofluorescence images of cells were obtained using a Leica SP-8 confocal microscope, and the pixel sum was estimated with the SP8 software (RAS X). (A,B) Images of Rab11 in untreated (A) and RAB11A-siRNA-treated U3DT cells (B). (C) Quantitative analysis of U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (D,E) Images of GPC5 in U3DT cells not treated (D) or treated (E) with 100 nM RAB11A-siRNA. (F) Quantitative expression of GPC5 in U3DT cells not treated (blue) or treated (red) with 100 nM RAB11A-siRNA. (G,H) Images of GPC5 (red) and Rab11 (green) in trypsinized-U3DT cells not treated (G) or treated (H) with RAB11A-siRNA. (G?J) Immunofluorescence images (G,H) and the pixel sum (I,J) of trypsinized cells were obtained using a Leica SP-8 immunofluorescence microscope the same as in C and F. (I) The mean intensity (pixel sum) of Rab11 relative to that of DAPI is shown for each untreated (blue) and RAB11A-siRNA-treated U3DT cell (red). (J) The mean fluorescence intensity (pixel sum) of GPC5 relative to that of DAPI staining per cell is shown for each untreated (blue) or RAB11A-siRNA-treated U3DT cell (red). (K?M) FACS analysis. (K) Contour display of merged three images. U3DT cells were stained with (blue) or without (red) anti-GPC5 and anti-Rab11 antibodies. RAB11A-siRNA-treated cells were stained with anti-GPC5 and anti-Rab11 antibodies (green). (L) Histogram of Alexa Fluor 488 fluorescence intensity (Rab11) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). (M) Histogram of Alexa Fluor 594 fluorescence intensity (GPC5) of RAB11A-siRNA-treated cells (green) or not treated cells (blue). The same preparation was used for immunofluorescence (G, H) and for FACS analyzes (L, M). Red arrows indicate the peak position of immunostaining control cells (L, M). (N, O) Immunofluorescence images of Rab11 (red) and WGA (green) in not treated cells (N) and RAB11A-siRNA-treated cells (O). (P) The mean distribution of telophase cells with three or more blebs (>ca. 2 µm diameter) in telophase are shown for not treated cells (blue) and RAB11A-siRNA-treated cells (red). The numbers in parentheses in Figure (C, F, I, J and P) are the number of images. White arrows indicate blebs in a telophase cell (N). Scale bar, 5 µm.