Fig. 1

Ectopic expression of AGR2 modulated extracellular milieu of CMT cells, promoting CMT cell chemotaxis. A Characterization of CMT cell lines. Expression of AGR2, E-cadherin, vimentin, HER-2, or ER-⍺ in individual cell lines was analyzed by immunoblotting with specific antibodies. CMT-U27 (B) and CF41.Mg (C) were transfected with pcDNA3.1-myc.His-AGR2 or the mock vector and grown in 2% FBS-containing RPMI and DMEM, respectively, for 24 h. Whole-cell lysates (WCL) of the transfectants were analyzed by immunoblotting to confirm the expression of Myc-tagged AGR2. Conditioned media (CM) of the transfectants were collected and placed in the bottom well for a transwell migration assay, in which the responding cells were seeded in the top insert and stained with Hoechst during a 16-h incubation. Cells in the insert were fixed for image acquisition using an epifluorescence microscope with a 10 × objective. D, F The number of migrated cells was counted and presented as the mean + SD of three independent experiments. E, G Representative images of migrated cells per field were shown. H Myc-Trap-based precipitation conducted the depletion of AGR2 in the CM of AGR2-expressing CMT-U27. Expression of AGR2 in WCL or CM and Myc-Trap-precipitated AGR2 were verified by immunoblotting. I As described above, the AGR2-depleted CM (denoted deAGR2) or the untreated control was placed in the bottom well for a transwell migration assay. J, K Fresh 2% FBS-containing media supplemented with or without rcAGR2 (800 ng/mL) were placed in the bottom well for a transwell migration assay. For C, F, I–K, statistical significance was determined by a two-tailed unpaired t-test. *p < 0.05; **p < 0.01; ****p < 0.0001