fig3

Figure 3. AKT signaling was involved in anoikis regulation by ROR2 in LM8. A: fluorescence intensities of live and dead cells labeled with different dyes were measured using appropriate filters after 24 h of culture under low adhesion conditions as described in the Methods. The dead cell ratio was calculated by dividing the fluorescence intensity of the dead cells by the total fluorescence intensity. n= 3, *P < 0.05, **P < 0.01, ***P < 0.001; B: pAKT and AKT levels in LM8 sublines cultured under adhesion (left) and low adhesion (right) conditions analyzed by western blotting. The pAKT/AKT at the bottom of the figure shows the ratio of pAKT to AKT levels normalized by GAPDH levels; C: pAKT and AKT levels in LM8-H treated with MK2206 for indicated time under low adhesion conditions. The western blotting experiments were repeated three times, and representative data are shown; D: dead cell ratio of LM8-H cells cultured with MK2206 or solvent only (DMSO) for 24 h under adhesion or low adhesion conditions. Dead cell ratios are shown as normalized values for cells treated with MK2206 vs. those for untreated cells. n = 3, *P < 0.05. ROR2: receptor tyrosine kinase-like orphan receptor 2; LM8-H: LM8 cell line with high metastatic ability; LM8-L: LM8 cell line with low metastatic ability; H/Ror2-KO: LM8-H knocked out of Ror2; n.s: not significant; DMSO: dimethyl sulfoxide; L/ROR2: LM8-L expressing ROR2; KO/ROR2: H/Ror2-KO expressing ROR2