Figure 5: Overexpression of ROBO1 and ROBO2 inhibited tumorigenesis, whereas some ROBO mutants impaired ROBO function.

Overexpression of wild-type ROBO1 and the ROBO1 R77H mutant (a,c), as well as wild-type ROBO2 and the ROBO2 G107E mutant (b,d), induced an increase in apoptosis in K562 (a,b) and HEL (c,d) cells. Exogenous SLIT2 (200 ng ml⁻¹) enhanced the apoptotic effect. However, these effects were not observed in K562 and HEL cells overexpressing ROBO1 (T1160A and R886H) and ROBO2 (P1058R and P522Q) mutants. Overexpression of wild-type ROBO1 and the ROBO1 R77H mutant (e,g), as well as wild-type ROBO2 and the ROBO2 G107E mutant (f,h), inhibited cell growth in K562 (e,f) and HEL (g,h) cells (top). Exogenous SLIT2 (200 ng ml⁻¹) enhanced the inhibitory effect on cell growth (bottom). However, the inhibitory effects were not obvious in K562 and HEL cells overexpressing ROBO1 (T1160A and R886H) and ROBO2 (P1058R and P522Q) mutants. In addition, overexpression of wild-type ROBO1 and the ROBO1 R77H mutant (i,k), as well as wild-type ROBO2 and the ROBO2 G107E mutant (j,l), inhibited colony formation in K562 (i,j) and HEL (k,l) cells. Exogenous SLIT2 (200 ng ml⁻¹) enhanced the inhibitory effect on colony formation. K562 and HEL cells overexpressing ROBO1 (T1160A and R886H) and ROBO2 (P1058R and P522Q) mutants displayed no significant differences in colony formation compared with the control cells. pCMV-ROBO1 M1, T1160A mutant; pCMV-ROBO1 M2, R886H mutant; pCMV-ROBO1 M3, R77H mutant; pCMV-ROBO2 M1, P1058R mutant; pCMV-ROBO2 M2, P522Q mutant; pCMV-ROBO2 M3, G107E mutant. Error bars throughout represent the s.e.m. *P<0.05, **P<0.01 and ***P<0.001 relative to the pCMV vector control group). Throughout the figure, unpaired Student’s t tests were used to calculate all P values. The data shown are representative of values from three independent experiments.