Fig 2: Characterization of claudin-2 knockout clones expressing exogenous claudin-2.(A) Immunoblots of claudin-2 and E-cadherin in control MDCK II cells and claudin-2 knockout clones expressing exogenous claudin-2. Claudin-2 cDNA was transfected into claudin-2 knockout clone 1 (KO 1), and clones expressing FLAG tagged claudin-2 (clones F1–5) and tagless claudin-2 (clone Tl) were established. The signal intensity of claudin-2 bands was quantified, and relative signal intensity was calculated as the ratio of the signal intensity in each clone to that in control cells. N = 4 for each experiment. (B) Immunofluorescence analysis of claudin-2 and FLAG in co-culture of control MDCK II cells and F1–5 clones. Claudin-2 staining was barely detectable at TJs in F1–3 clones, whereas claudin-2 staining in the F4 and 5 clones was clearly detected at TJs but signals were weaker compared with control cells. Scale bar = 10 µm.

Fig 3: Associations between stromal/CAF-associated claudin-2 expression and PFS in the NORDIC-VII cohort. Kaplan–Meier graphs showing associations in the NORDIC-VII cohort between PFS and claudin-2 expression in cancer cells (a) or in stromal cell/CAFs (b)

Fig 4: Effects of hydrostatic pressure on transepithelial transport.(A) Effects of hydrostatic pressure on transepithelial electrical resistance (TER) in MDCK I cells. The TER was changed to decrease at two days after application of the hydrostatic pressure under the ‘Basal’ condition. (B) The ratio of P Na to P Cl (P Na/P Cl) under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The P Na/P Cl was measured at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. The value of P Na/P Cl under the ‘Basal’ condition was significantly higher than that under the ‘Apical’ condition. (C) P Na and P Cl under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The value of P Na under the ‘Basal’ condition was approximately four-fold higher than that under the ‘Apical’ condition. (D) Immunofluorescence microscopy for claudin-2 and ZO-1 in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. Claudin-2 staining was clearly detected at cell-cell contacts in a limited region under the ‘Basal’ condition. Scale bar = 10 µm. (E) Immunoblots for claudin-2 and E-cadherin in MDCK I cells at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. MDCK II cells cultured on filters for six days were used as a positive control. A faint band of claudin-2 was detected under the ‘Basal’ condition in MDCK I cells. (F) Flux of fluorescein and 4 kDa FITC-dextran under the ‘Apical’ and ‘Basal’ conditions in MDCK I cells. The flux was measured at four days after the culture under the ‘Apical’ and ‘Basal’ conditions. The flux of fluorescein under the ‘Basal’ condition was significantly lower than that under the ‘Apical’ condition. * p < 0.05, ** p < 0.01 compared with the ‘Apical’ condition.

Fig 5: Tight junction expression occurs in colonic mucosa from control mice with claudin-2 antibody; FFPE tissues. (A) Representative images of colonic mucosa from wild-type mice (n = 3) stained with E-cadherin (E-cad) antibody to identify lateral cell membranes, claudin-2 (Cldn2) antibody to identify tight junctions, and Hoechst 33342 (HOE) to identify nuclei. As has been determined before in mice,32 in our hands the 12H2 antibody to claudin-2 localized to cells in the crypt base in colon in paraffin sections. (B and C) In the crypt base, claudin-2 co-localized with the tight junction protein ZO-1 (white signal in B, arrows in C) and was also found along the basolateral membrane (arrowheads). Claudin-2 was excluded from the base of cells in colon from FFPE tissues (asterisk). Scale bars: (A) 100 µm; (B) 20 µm; (C) 10 µm.