Fig 2: Angiogenic factors released by RMG-1 and HUVEC. (A) VEGF, (B) osteopontin, (C) Ang1, and (D) Ang2 production from RMG-1 and HUVEC in response to IL-6 signal blockade determined by ELISA assay. Data of HUVEC was calculated by the subtraction the data of RMG-1 mono-culture from that of RMG-1 + HUVEC. Error bars are SEs. *P < 0.05, **P < 0.005.

Fig 3: The role of IL-6 signal for the anti-angiogenic and the anti-tumor effects of Bev in 3D co-culture system. (A) Representative micrograph of 3D co-culture with or without IL-6 blockade and/or Bev observed through light microscope and fluorescent microscope at experimental day 4. HUVEC was stained by Dil, emitting red fluorescence. RMG-1/GFP was seen as green fluorescence. Overlaid images of Dil and GFP were shown at the rightmost. (B) Measured area at day 4 of each color by hybrid cell count software. Dil-stained area, regarded as the tube area, is shown on the left and GFP area, regarded as the tumor area, is shown on the right side. Error bars are SEs. All the images were observed by fluorescence microscope (BZ-X800, Keyence). Then, these obtained images were analyzed by the BZ-H4C analytic application (Keyence) for hybrid cell count and the BZ-H4CM application (Keyence) for macro cell count.

Fig 4: Attenuation of the anti-angiogenic function of Bev in culture media of IL-6 blocked OCCC cell. (A) Representative images of tube formation of HUVEC with the indicated culture media and reagents (magnification × 100). Fluorescent microscope observation was made after 18 h of incubation and Calcein AM staining. (B) Tube areas in each well with indicated condition were measured by hybrid cell count software. Data were average of triplicated well. (C) The tube reduction rate by Bev treatment with or without IL-6 blockade. IL-6 blockade weakened Bev function. Data are shown from one of two independent experiments with similar results. Error bars are SEs. All the images were observed by fluorescence microscope (BZ-X800, Keyence, Osaka, JAPAN). Then, these obtained images were analyzed by the BZ-H4C analytic application (Keyence) for hybrid cell count and the BZ-H4CM application (Keyence) for macro cell count.

Fig 5: The role of Ang1 in IL-6 mediated Bev anti-angiogenic effect enhancement. (A) Images of tubes formed by HUVEC in the conditioned media from RMG-1 treated by indicated siRNA and anti-IL-6 antibody (magnification × 40). Fluorescent microscope observation was made after 18 h of incubation and Calcein AM staining. (B) Tubes in each well with indicated condition were measured by hybrid cell count software in order to elucidate the tube area. Data were average of triplicated well. (C) Reduction rate of tube area by Bev treatment with or without IL-6 blockade and siRNA, IL-6 blockade weakened Bev function in siCTL whereas siAng1 restored the anti-angiogenic function. Data are shown from one of two independent experiments with similar results. siCTL: siRNA without gene silencing ability (control). siAng1: angiopoietin-1 silencing siRNA. Error bars are SEs. All the images were observed by fluorescence microscope (BZ-X800, Keyence, Osaka, JAPAN). Then, these obtained images were analyzed by the BZ-H4C analytic application (Keyence) for hybrid cell count and the BZ-H4CM application (Keyence) for macro cell count.