Figure 3.

Rab25 Is Involved in the Activation of EGFR Pathway

(A) Immunoblotting of EGFR and phosphorylation status of Akt at Ser⁴⁷³ and Erk at Thr²⁰² and Tyr²⁰⁴ in three pairs of lung radioresistant cells.

(B) Scores of EGFR IHC staining were shown as percentage for EGFR IHC analysis with 4 as the highest and 0 the lowest expression. Representative IHC imaging were shown in Figure S3B.

(C) Co-expression of Rab25 and EGFR in 101 NPC tissues corresponding to the patients grouped as CR, PR, and SD. The intensity of IHC staining is scored from 0 to 1 (for “no signal”) to 4 (for “strongest signal”). Rab25 and EGFR staining intensity from each patient was compared as two datasets in a column table. R value was calculated using GraphPad Prism software and the p value was calculated by two-tailed analysis and confidence interval is 95%.

(D) The relative phosphorylation of RTKs in H358R cells and H358R transfected with siRab25. Total proteins were extracted and screened simultaneously using R&D Systems Human Phospho-Receptor Tyrosine Kinase (RTK) Array Kit. The spot intensities were quantified using ImageJ software. The values for control cells (blue box) were set as 1, and the values of siRab25 (red box) were shown relative to the control, n = 3, mean ± SD, ∗∗p < 0.01.

(E) Expression of phosphorylated EGFR, HER2, c-Met, and the associated downstream signal molecules was analyzed by PathScan RTK signaling antibody array kit in CNE2R cell (control) and CNE2R transfected with Rab25 siRNA. The spot intensities were quantified using ImageJ software. The values for control cells (blue) were set as 1, and the values of siRNA (red) were shown relative to the control. Results represent mean ± SD for three analysis. ∗p < 0.05, ∗∗p < 0.01.

(F) Down-expression of Rab25 reduced the expression of EGFR and the activation of associated signaling pathways in radioresistant cells.