Fig. 4

Genetic depletion of AGR2 impaired 14-3-3ε and α-actinin 4 release in the CM of CMT cells. A CMT-U27e transfected with AGR2-targeting siRNA (siAGR2 1 or 2) or negative control siRNA (siNC) was grown in 1% FBS-containing RPMI for 30 h. WCL and CM samples were collected and subjected to immunoblotting analysis. Protein bands were quantified and normalized to that of GAPDH, which was used as an internal control. AGR2 expression levels in siAGR2 transfectants were presented as a ratio to that in siNC transfectants. Data were presented as the mean + SD of three independent experiments, shown in B. C CM samples collected from (A) were TCA-precipitated, and 14-3-3ε and α-actinin 4 levels were analyzed by immunoblotting and quantified as described above. Data are presented as the mean + SD of three independent experiments, shown in D. E CM collected from (A) was applied to a transwell migration. Results are presented as the mean + SD of three independent experiments. F, G CMT-U27e, a control cell clone Ctrl-S3, and two AGR2-KO clones, KO-S4 and KO-S10, were grown in 1% FBS-containing RPMI for 24 h. WCL and CM samples were collected and analyzed by immunoblotting, as shown in F and G, respectively. 14-3-3ε and α-actinin 4 levels in individual CM samples are presented as a ratio to that in CMT-U27e, as shown in H. I CM collected from individual cell clones was applied to a transwell migration assay. Results are presented as the mean + SD of three independent experiments *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (two-tailed unpaired t-test)