(A) Analysis by immunofluorescence of AGR2 expression in normal human bronchial epithelial cells (HBEC) and three lung cancer cell lines (A549, H23 and H1838) grown in 2D culture. Scale bars, 50 μm. (B) Quantification of AGR2 protein expression in cell lines according to immunofluorescence. The stacked bars show the percent contribution of high and low AGR2-positive cells relative to the total number of cells per field. (C) Expression of AGR2 protein detected by Western blot in a panel of human lung epithelial cell lines. Values correspond to three independent experiments. Data are mean ± SEM. (D) Confocal cross-sections of organoids stained with AGR2 antibody (red) and DAPI (blue) for nucleus, in normal HBECs and three lung cancer cell lines (A549, H23 and H1838). Scale bars, 50 μm. (E) AGR2 expression determined by immunohistochemistry in sections of formalin-fixed paraffin-embedded normal human lung samples and in the different lung adenocarcinoma subtypes. II = squamous cell carcinoma, IV = adenocarcinoma, VI = large cell carcinoma as compared to normal tissues (I,III,V) (x200). (F) Pulmonary lung carcinoma showing brown, nuclear immunostaining for TTF-1 expression and cytoplasmic immunostaining for AGR2 expression (dual color Multiplex TTF-1 + AGR2 immunostain; x200). (G) Kaplan-Meier survival curves of lung cancer patients. The cumulative survival was related to different levels of AGR2 expression: Group 1, low to moderately positive stains (n=17); and Group 2, strongly positive AGR2 stains (n=16), as defined in Materials and methods and Supplementary file 1A.