Fig. 5. Analysis of gp130 homodimerization and gp130/LIFR heterodimerization by BiFC with confocal microscopy. (A) Hek293T cells were transfected with expression vectors encoding gp130/id-YC173 and gp130/id-YN173. Cells were incubated for 30 hours at 37°C, subsequently incubated for 18 hours at 30°C and afterwards analysed by FACS. 4 hours before temperature reduction, the cells were stimulated with IL-6 (20 ng/ml) and sIL-6R (1 µg/ml) or left unstimulated. Histograms represent unstimulated cells (dark grey), stimulated cells (black) or cells that expressed only gp130/id-YC173 (light grey). The scheme (right) depicts the complementation between the fragments of the fluorophores fused to gp130. (B) Hek293T cells were transfected and stimulated as described in A. Images of unstimulated (left) and stimulated cells (right) with high (upper row) and low magnification (lower row) were taken by using the YFP channel of the confocal microscope. (C) Proportion of the area of cells with BiFC fluorescence (A_BiFC) in the total cell covered area (A_cells) in low magnification images (n=30) of unstimulated cells (light grey), stimulated cells (dark grey) and cells that had expressed only gp130/id-YC173 (white) as a BiFC negative control (±s.d.). Significant differences in fluorescence ratio (***P<0.001) were observed between groups as indicated. (D) Hek293T cells were transfected with expression vectors encoding gp130/id-YC173 and LIFR/id-CN173. Cells were incubated for 30 hours at 37°C, subsequently incubated for 18 hours at 30°C and afterwards analysed by FACS. 4 hours before temperature reduction, the cells were stimulated with LIF (20 ng/ml) or left unstimulated. Histograms represent unstimulated cells (dark grey), stimulated cells (black) or cells that expressed only gp130/id-YC173 (light grey).The scheme (right) represents the complementation between the fragments of the fluorophores fused to LIFR and gp130. (E) Hek293T cells were transfected and stimulated as described in D. Images of unstimulated (left) and stimulated cells (right) under high (upper row) and low magnification (lower row) were taken using a channel of the confocal microscope adapted to the characteristics of the new fluorophore formed by YC173 and CN173 complementation. (F) Proportion of the cell area with BiFC fluorescence (A_BiFC) in the total cell covered area (A_cells) in low magnification images (n=30) of unstimulated cells (light grey), stimulated cells (dark grey) and cells that had expressed only gp130/id-YC173 (white) as a BiFC negative control (±s.d.). Significant differences in fluorescence ratio (*P<0.05 and ***P<0.001) were observed between groups as indicated. Bar, 10 µm (upper images in E,B); 100 µm (lower images in E,B).