Fig 2: Suppression of TFF1 expression in CRC cells overexpressing L1. (A) The level of TFF1 RNA was determined in L1 overexpressing LS 174T and DLD-1 CRC cells; (B) L1 RNA levels were determined by RT-PCR in the cells described in (A); (C,D) the expression of TFF1 and L1 protein was determined by Western blotting in the cell lines described in (A). Tubulin served as loading control; (E) the intensity of the TFF1 bands shown in the Western blots of the different CRC cell lines (D) was determined by densitometric analysis of the gels shown in (D). Relative integrated band densities of TFF1 were calculated from triplicate gels, and the significance was determined using Student’s unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. The uncropped blots are shown in Supplementary File S1.

Fig 3: Subtype 2 tumors are associated with a higher risk of metastasis.a Immunostaining of CRX, ARR3, and TFF1 in normal retina and retinoblastoma. Immunohistochemistry experiments were performed on 55 samples (subtype 1, n = 18; subtype 2, n = 37) from the initial series of 102 retinoblastomas. Representative images are shown: one subtype 1 tumor (RB1) and two subtype 2 tumors (RB635, RBsjd8). The subtype 2 tumors presented either a co-staining (RB635) or a mirror pattern (RBsjd8) for ARR3 and TFF1. b Boxplots showing the quick score (QS) for TFF1 in 55 tumors of the initial series (subtype 1, n = 18; subtype 2, n = 37), and in 112 tumors of the HRPF series. In the boxplots, the central mark indicates the median and the bottom and top edges of the box the 25th and 75th percentiles. The whiskers are the smaller of 1.5 times the interquartile range or the length of the 25th percentiles to the smallest data point or the 75th percentiles to the largest data point. Data points outside the whiskers are outliers. Two-sided Wilcoxon tests were used to assess the difference of the QS for Subtype 1 vs Subtype 2, p = 1.1 × 10-7, and metastatic vs non-metastatic, p = 0.007. c Immunostaining of TFF1 for primary tumors of metastatic retinoblastoma (left) and their metastatic sites (right), at low and high magnification. TFF1 expression could be assessed by immunohistochemistry for 6 of 7 available primary-metastasis tumor pairs. Representative images of four are shown.

Fig 4: The inverse relationship between the expression of TFF1 and NF-?B signaling components in L1-expressing CRC cells. (A) The levels of p65 and I?B protein were determined in the CRC cell clones described in Figure 3; (B) the level of TFF1 RNA was determined in L1-expressing CRC cell clones in which NF-?B signaling was blocked by either an shRNA to p65 (L1 + shp65) or by the expression of the I?B super-repressor (L1 + I?B-SR); (C,D) quantitative immuno-dot-blot analysis of TFF1 protein levels in the CRC cell clones described in (B). * p < 0.05, ** p < 0.01. The uncropped blots are shown in Supplementary File S1.

Fig 5: Isolation and localization of L1 and TFF1 in CRC cell clones overexpressing both L1 and TFF1. (A) Serial dilutions of cell extracts analyzed by immuno-dot-blotting using LS 174T CRC cell clones overexpressing both L1 (L1) and TFF1 (L1 + TFF1 cl1 and cl2); (B) densitometric analysis of the dot blots shown in (A); (C) localization of L1 and TFF1 in CRC cell clones overexpressing both L1 (red) and TFF1 (green) by double immunofluorescence microscopy. Nuclei were stained with DAPI (blue). * p < 0.05, ** p < 0.01. The uncropped blots are shown in Supplementary File S1.