Fig 2: L1CAM is upregulated in the 5-FU-resistant cells.(A) Gene Ontology analysis of 319 genes upregulated in the B1V clone compared to the Nt clone of the Panc 03.27 cell line. (B) RNA levels (relative quantity) of L1CAM in the chemosensitive (Nt, Nw) and the chemoresistant (B1Q, B1V) cell lines, as measured by RT-PCR. Error bars represent standard deviation. Statistically significant difference between the chemosensitive and the chemoresistant clones (P <0.05) is indicated by *. (C)Western blot showing levels of L1CAM in all cell lines. (D) Flow cytometric analysis using anti L1CAM-PE antibody on all cell lines. Unstained cells are shown in grey and antibody-stained cells are shown in black. (E) Immunostain of L1CAM. Increased membrane localization of L1CAM can be seen in the of the chemoresistant lines (B1Q, B1V). Images are taken with 40x magnification.

Fig 3: Endocytosis of dendritic receptors in the AIS of human neurons.(a) Dendritically polarized TfR and axonal L1CAM are polarized to their appropriate domains in induced human neurons that were fixed and stained at DIV26. (b) Induced human neurons have a single ankyrinG-labelled AIS when co-cultured with glial cells. (c, d) Confocal images of human neurons fixed and stained at DIV26 for endocytic proteins. Arrows designate the AIS. (e) AIS of induced human neurons that were fixed and stained at DIV26 for ankyrinG, AP-2, and clathrin heavy chain. (f) Confocal images of human neurons fixed and stained at DIV26 for the indicated endogenous proteins. Bottom images show the AIS (brightness is increased for better visibility). Note that extra-neuronal fluorescence represents endogenous staining in co-cultured glial cells. (g) Zoomed in region from f of an endocytic vesicle in the AIS. Brightness is increased for better visibility. (h) Histogram analysis of axonal branches in the 50 microns distal to the AIS in the indicated animals (n = 15 animals/genotype). (i) Confocal images of cultured mouse neurons pretreated with vehicle control (top, DMSO) or endocytic inhibitor (bottom, Dyngo 4a) for 5 h prior to being fixed and stained at DIV14. Arrows designate the axon. (j) Endogenous TfR fluorescence in the axon from neurons described in i. Arrows designate the cropped region. (k) Average TfR fluorescence in the axon compared to the dendrite. All data are shown as mean ± s.e.m. N-values are indicated on bar graphs and represent the number of individual cells scored for each condition. P values in k were calculated using a two-way ANOVA with Šidák multiple comparison test. Scale bars, 20 µm (a, b, c, d, f top, i), 5 µm (f bottom, j), 2 µm (e), 1 µm (g). Source data

Fig 4: L1CAM is involved in proliferation and invasiveness of the 5-FU-resistant clones.(A) Control western blot showing the levels of L1CAM following transfection with esiRNA targeting L1CAM or control esiRNA, as described in Materials and methods. Actin is used as loading control. (B) Graph displaying number of invading cells (24 h invasion assays) following transfection with esiRNA targeting L1CAM or control esiRNA. Experiments were performed twice with similar results. Representative results are shown. (C) Graph displaying numbers of invading cells (48 h invasion assays) of cells treated with 2 μg/ml anti-L1CAM antibody (clone UJ127.11) or control isotype antibody for 1 h at room temperature before they were added to the invasion assay plate. (D) Incucyte growth curves (96 hours) of all cell lines following transfection with esiRNA targeting L1CAM or control esiRNA. The experiment was performed in triplicates. (E) The chemosensitive cell line Nt and the chemoresistant cell line B1V (F) were treated with 1 ug/ml 5-FU for 72 h following transfection with esiRNA targeting L1CAM or control, and Annexin V flow cytometric assays were performed. Each assay was repeated twice with similar results. Percentage in the graphs shows Annexin V-positive cells.

Fig 5: Analysis of L1CAM ectodomain shedding after ADAM10/17 single and double knockdown in Y79 and WERI-Rb1 cell lines. Western blot analysis of ADAM mediated L1CAM ectodomain (L1–200) shedding in cell culture supernatant of Y79 and WERI-Rb1 cells 72 h after ADAM10 (shADAM10), ADAM17 (shADAM17) single and ADAM10/17 (shADAM10/17) double knockdown in comparison to control cells (ctr). Representative western blots of L1 shedding upon ADAM knockdown (a) and quantification of the L1CAM ectodomain expression reveals a significant reduction of L1-200 shedding after ADAM10/17 single and double knockdown in the RB cell lines Y79 and WERI-Rb1 (b). Indicated intensity ratios relative to ß-actin, used as a loading control, were calculated using MICRO MANAGER 1.4 software. Values are means of three independent experiments ± SEM. * p < 0.05 and ** p < 0.01 statistical differences compared to the control (ctr) group calculated by Student’s t-test.