Fig. 5

Knockdown of 14-3-3ε or α-actinin 4 diminished the chemotaxis conferred by AGR2-modulated CM. A, B CMT-U27e transfected with 14-3-3ε-targeting siRNA, siYWHAE (A), α-actinin 4-targeting siRNA, siACTN4 (B), or negative control siRNA (siNC) were grown in 1% FBS-containing RPMI for 30 h. WCL samples were harvested and analyzed by immunoblotting. 14-3-3ε and α-actinin 4 levels were normalized to β-tubulin levels in individual samples, and the resulting values in siYWHAE- or siACTN4-transfectants were presented as a ratio to that in siNC. C, D 14-3-3ε and α-actinin 4 levels in the CM samples collected respectively from (A) and (B) were analyzed by immunoblotting and processed as above. E CM samples collected from (B, D) were applied to a transwell migration assay. F, G, I, J CMT-U27e cells were first transfected with the indicated siRNA (75 nM) and subsequently transfected with pcDNA3.1-myc.His-AGR2 or the mock control 6 h later. After 8 h incubation, the culture media were replaced with 1% FBS-containing RPMI, and cells were grown for another 20 h until WCL and CM were collected. Levels of indicated proteins in individual WCL (F, I) and CM (G, J) were analyzed and quantified as described above. H, K CM samples collected from (I, J) were applied to a transwell migration assay. All quantitation data shown are the mean + SD of three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed unpaired t-test)