Fig. 4.

Tspan5 activates Notch signalling in HCC cells. (A) Western blotting showing that Tspan5 regulates the expressions of ADAM10, NICD1 (Val1744) and Hes5 in tumour cells. GAPDH was used as the protein loading control. Numbers indicate relative protein ratio measured by image j software and normalized to GAPDH. (B) Representative images of IF for NICD1 showing that Tspan5 affects the expression and nuclear translocation of NICD1. 630× magnifications, scale bar: 10 μm. Quantification of the protein expression was performed by Zeiss zen microscope software. Mean ± SD, n = 9, Student's t‐test; ***P < 0.001. (C) Representative IHC images of NICD1 expression and nuclear localization in xenograft sections of metastatic foci in mouse lungs. 600× magnifications, scale bar: 50 μm. Mean ± SD, n = 4–6, Student's t‐test; **P < 0.01, ***P < 0.001. (D) Western blotting showed that DBZ treatment (60 nm for BEL7402 and 4 nm for MHCC97L) does not significantly affect the expression of ADAM10 but fully abolishes the upregulation of NICD1 (Val1744) and Hes5 by Tspan5. GAPDH was used as a protein loading control. Numbers indicate relative protein ratio measured by image j software and normalized to GAPDH.