Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: The Kir3.4-Gly387Arg Mutation Is Associated with Congenital Long QT Syndrome

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Mutagenesis

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Clinical Characteristics of the Pedigree Members with Kir3.4-Gly387Arg or Sudden Death

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques:

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Electrophysiological Analyses of Kir3.4-Gly387Arg

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques:

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Cell Surface Expression of Kir3.4-Gly387Arg and Kir3.1

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Expressing

Journal: American Journal of Human Genetics

Article Title: Identification of a Kir3.4 Mutation in Congenital Long QT Syndrome

doi: 10.1016/j.ajhg.2010.04.017

Figure Lengend Snippet: Human Atrial and Ventricular Expression of Kir3.1 and Kir3.4

Article Snippet: Proteins were transferred onto Hybond-P polyvinylidene difluoride transfer membranes (Amersham Biosciences, 0.45 μm), and membranes were blocked in 5% nonfat milk in TBST (0.1% Tween 20 in Tris-buffered saline solution) for 2 hr at room temperature and then incubated with primary antibody against Kir3.1 (1:1,000; Alomone Labs), Kir3.4 (1:1,000; Alomone Labs), calnexin (1:2,000; Nordic Biosite), or Na + /K+-ATPase α1 (1:500; Santa Cruz Biotechnology) overnight.

Techniques: Expressing

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of ECM on the differentiation of kidney organoids in a static culture condition. A Fluorescent images of immunostained kidney organoids cultured with various conditions of ECMs for 6 days. The cells were immunostained by NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue). B Quantitative analysis of kidney antibody-specific markers from kidney organoids (* indicates p <0.05 for experimental group vs. control group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Cell Culture

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of the shear stress on the differentiation of kidney organoids in a kidney organoid-on-a-chip. A Fluorescent images showing hPSCs-derived kidney organoid-related cell differentiation under a shear stress condition. Kidney organoids were cultured within kidney organoid-on-a-chip for 6 days. After culturing for 6 days, the cells were stained for NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue) to identify the kidney-related cells, respectively. B Quantitative analysis of the kidney-related markers from kidney organoids. (* indicates p < 0.05 for LTL expression of Matrigel-coated group vs. Matrigel/VEGF-coated group, *** indicates p < 0.001 for PECAM expression of Matrigel-coated group vs. Matrigel/VEGF-coated group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Derivative Assay, Cell Differentiation, Cell Culture, Staining, Expressing

Journal: The Journal of Neuroscience

Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

doi: 10.1523/JNEUROSCI.3567-14.2015

Figure Lengend Snippet: Proteomic analysis of the PSD fraction of GluN2B−/− cortical neurons. A, PSDs were successfully purified from high-density rat cortical cultures (14–15 DIV). Western blot for markers of the presynaptic (Synaptophysin) and postsynaptic fractions (PSD-95, SynGAP, NMDAR receptor subunits GluN1 and GluN2A, and AMPAR subunit GluA1) validated PSD isolation of PSDs. Transferrin receptor (Transf R) and β-actin were used as controls. Hom, Homogenate; S1, non-nuclear fraction; P2 crude synaptosomes. Equal amounts (13 μg) of each fraction were applied. B, PSD-localized proteins (PSD-95, GluN1, SynGAP) are increased relative to the homogenate in the PSD fraction, whereas presynaptic synaptophysin (Synapto.) or the trans membrane receptor of transferrin are decreased compared with the P2 fraction, consistent with their absence or low expression, respectively, at the PSD. Actin levels did not change in any of the fractions analyzed. Data are presented as mean ± SEM from two independent experiments. C, Setup of the 8-plex iTRAQ experiments. Isolated PSDs from GluN2B+/+, GluN2B+/−, and GluN2B−/− cortical neurons, 10 μg each, were digested with trypsin and samples (three wild-types, two heterozygous, and three knock-outs) were tagged with a set of 8-plex iTRAQ reagents. Peptides were pooled together and fractionated by 2D liquid chromatography and subject to tandem mass spectrometric analysis. After using peptides for protein identification, the individual contribution of each sample to each peptide can be measured by the intensity of the reporter ion peaks and used for relative quantification. Example spectra correspond to one of the GluN1 peptides identified in the PSD preparations. D, Chymotrypsin-like proteasome activity was measure in protein extracts from GluN2B+/+ and GluN2B−/− cortical neurons. Proteasome activity is significantly decreased (p < 0.05) in GluN2B−/− neurons compared with control neurons. The control proteasome activity, determined in GluN2B+/+ neurons, was set to 100%. Data are presented as mean ± SEM from three independent experiments. *p < 0.05, paired t test.

Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07-294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phospho-CaMKII α/β subunit (Thr286/287) (1:1000, #06–881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and anti-phospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and anti-PSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII β (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII α (6G9) (1:2000, #C265) was from Sigma-Aldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti-β-actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

Techniques: Purification, Western Blot, Isolation, Expressing, Two-dimensional Liquid Chromatography, Activity Assay

Journal: The Journal of Neuroscience

Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

doi: 10.1523/JNEUROSCI.3567-14.2015

Figure Lengend Snippet: GluN2B is required for homeostatic synaptic scaling in response to blockade of neuronal activity. A, B, Low-density cortical cultures from wild-type and +/− (A) and GluN2B−/− mice (B) were incubated for 24 h at 14 DIV with TTX (2 μm) and immunostained for surface-GluA1 (live-staining, green merge). After fixation, cells were stained for PSD-95 (red merge), VGLUT (blue merge), and MAP2. Scale bar, 5 μm. C, The integrated intensity of the surface GluA1 (sGluA1) clusters per dendritic length was analyzed. Data are presented as mean ± SEM, n = 37 to 41 cells from two independent experiments. **Comparison between GluN2B+/+ and GluN2B−/−, p < 0.01; ###comparison between control and TTX conditions, p < 0.0001, n.s., p > 0.05, one-way ANOVA followed by Bonferroni's multiple-comparisons test.

Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07-294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phospho-CaMKII α/β subunit (Thr286/287) (1:1000, #06–881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and anti-phospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and anti-PSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII β (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII α (6G9) (1:2000, #C265) was from Sigma-Aldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti-β-actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

Techniques: Activity Assay, Incubation, Staining

Journal: The Journal of Neuroscience

Article Title: GluN2B-Containing NMDA Receptors Regulate AMPA Receptor Traffic through Anchoring of the Synaptic Proteasome

doi: 10.1523/JNEUROSCI.3567-14.2015

Figure Lengend Snippet: AMPAR synaptic expression is restored to control levels in GluN2B−/− hippocampal neurons upon activation of the proteasome. A, B, Low-density hippocampal cultures from control (A; GluN2B+/+ and GluN2B+/−) and GluN2B−/− (B) mice were fixed at 14 DIV and stained for the AMPAR subunits GluA1 or GluA2 (green, merge), the excitatory postsynaptic marker PSD-95 (green, merge), and the presynaptic marker VGLUT (red, merge). MAP2 staining was used as a dendritic marker (blue, merge). Scale bar, 5 μm. Hippocampal neurons were incubated with 25 or 50 μm IU1-47 for 24 h before neuronal fixation and staining. C–E, The integrated intensity of the synaptic (apposed to VGLUT) GluA1 (C), GluA2 (D)or PSD-95 (E) clusters per dendritic length was analyzed. Data are presented as mean ± SEM, n = 29–48 cells from three independent experiments for GluA1 clustering; n = 39–60 cells from two or three independent experiments for GluA2 clustering; n = 55–76 from three or four independent experiments for PSD-95. ***p < 0.001 and **p < 0.01 for comparison between genotypes; #p < 0.05 and ###p < 0.0001 for comparison between IU1-47-treated and control neurons for each genotype, one-way ANOVA, followed by Bonferroni's multiple-comparisons test.

Article Snippet: Primary: anti-GluN1 (1:500 WB, #MAB363), anti-GluN2A (1:1000, #AB1555P), anti-GluA1 (1:1000 WB or 1:100 ICC, #AB1504), anti-GluA2 (1:1000 WB or 1:500 ICC, #MAB397), anti-phospho-GluA1 Ser845 (1:1000, #AB5849), anti-phospho-GluA2 Ser880 (1:1000, #07-294), phospho-stargazin (Ser239/240) (1:500, #ab3713), phospho-CaMKII α/β subunit (Thr286/287) (1:1000, #06–881) were from Millipore; anti-GluN1 (1:1500 ICC, #MAb 54.1) was from Invitrogen; N-terminal of GluA1 (1:50) was a kind gift from Dr. Andrew Irving; anti-PSD-95 (D27E11) (1:2000 WB or 1:100 ICC, #3450) and anti-phospho-protein kinase C (PKC) substrates (1:1000, #2261S) were from Cell Signaling Technology; anti-SynGAP (1:1000, #PA1-046) and anti-PSD-95 (1:500, #MA 1.046 C7E3-1B8) were from Affinity Bioreagents; anti-Synaptophysin (1:10000, #101 011) was from Synaptic Systems; anti-CaMKII β (K-19) (1:500, #sc-100366) was from Tebu-Bio, Santa Cruz Biotechnology); CaMKII α (6G9) (1:2000, #C265) was from Sigma-Aldrich; anti-phospho-GluA1 Ser 831 (1:5000, #2041) was from Tocris Bioscience; anti-phospho-GluA1 T840 was a kind gift from R. Huganir (JH2811); anti-Transferrin receptor antibody (1:1000, #13-6800) was from Invitrogen; anti-vesicular glutamate transporter 1 (VGLUT1; 1:5000, #AB5905) was from Millipore; anti-MAP2 (1:5000, #ab5392) was from Abcam; and anti-β-actin (1:5000, #1378996) was from Roche Molecular Biochemicals.

Techniques: Expressing, Activation Assay, Staining, Marker, Incubation

Journal: International Journal of Oncology

Article Title: Differential effects of α-catenin on the invasion and radiochemosensitivity of human colorectal cancer cells

doi: 10.3892/ijo.2018.4279

Figure Lengend Snippet: DLD-1 subpopulations display an altered morphology and invasion in 3D. (A) Phase-contrast images of the DLD-1 subpopulation grown in col-I. The DLD-1 α-cat cells (left panel) grew as round colonies, while the DLD-1 Δα-cat cells (right panel) grew as single cell groups. (B) Representative western blots of cell-cell contact proteins in DLD-1 subpopulations. (C) Fluorescence images indicate differential localization of E-cadherin (left panel) and β-catenin (right panel) in DLD-1 subpopulations. (D) Relative proliferation rates of DLD-1 subpopulation cultured in either lrECM or col-I. (E) Adhesion of DLD-1 subpopulations on col-I-coated plates. (F) Representative phase-contrast images of spheroids embedded in col-I and invasion at different time-points. (G) Invasion distance of DLD-1 subpopulations at the indicated time-points. Results represent the means ± SD (n=3; ** P<0.01; *** P<0.001; n.s., not significant).

Article Snippet: Antibodies against β-actin (A5441; Sigma-Aldrich, Taufkirchen, Germany), α-catenin (3236S), β-catenin (9587), p-β-catenin S675 (4176S), TGF-β (3709) (all from Cell Signaling Technology, Frankfurt am Main, Germany), E-cadherin (610181), fibronectin (610077) (both from BD Biosciences, Heidelberg, Germany), Zonula occludens-1 (ZO-1; 40-2300; Zymed Laboratories, San Francisco, CA, USA) and horseradish peroxidase-conjugated donkey anti-rabbit (NA934) and goat anti-mouse (NA9310) antibodies (GE Healthcare, Buckinghamshire, UK), as well as Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (A11001 and A1037; Invitrogen, Karlsruhe, Germany) were purchased as indicated. lrECM (MatrigelTM) and col-I were from BD Biosciences, and 5-fluorouracil (5-FU) was from Medac (Wedel, Germany).

Techniques: Western Blot, Fluorescence, Cell Culture

Journal: International Journal of Oncology

Article Title: Differential effects of α-catenin on the invasion and radiochemosensitivity of human colorectal cancer cells

doi: 10.3892/ijo.2018.4279

Figure Lengend Snippet: Deletion of α-catenin has no effect on the radiochemosensitivity of DLD-1 subpopulations. (A) Basal clonogenic survival, (B) radiation survival, and (C) survival following 5-FU treatment of DLD-1 subpopulations cultured in lrECM. Results represent the means ± SD (n=3; n.s., not significant). (D) Representative western blots, (E) basal clonogenic survival and (F) radiation survival of DLD-1 subpopulations after siRNA-mediated α-catenin knockdown (means ± SD; n=2–3; n.s., not significant).

Article Snippet: Antibodies against β-actin (A5441; Sigma-Aldrich, Taufkirchen, Germany), α-catenin (3236S), β-catenin (9587), p-β-catenin S675 (4176S), TGF-β (3709) (all from Cell Signaling Technology, Frankfurt am Main, Germany), E-cadherin (610181), fibronectin (610077) (both from BD Biosciences, Heidelberg, Germany), Zonula occludens-1 (ZO-1; 40-2300; Zymed Laboratories, San Francisco, CA, USA) and horseradish peroxidase-conjugated donkey anti-rabbit (NA934) and goat anti-mouse (NA9310) antibodies (GE Healthcare, Buckinghamshire, UK), as well as Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (A11001 and A1037; Invitrogen, Karlsruhe, Germany) were purchased as indicated. lrECM (MatrigelTM) and col-I were from BD Biosciences, and 5-fluorouracil (5-FU) was from Medac (Wedel, Germany).

Techniques: Cell Culture, Western Blot, Knockdown

Journal: International Journal of Oncology

Article Title: Differential effects of α-catenin on the invasion and radiochemosensitivity of human colorectal cancer cells

doi: 10.3892/ijo.2018.4279

Figure Lengend Snippet: Effect of α-catenin and upregulated genes in DLD-1 Δα-cat cells on spheroid morphology and invasion. (A) Representative western blots of siRNA-mediated α-catenin knockdown in DLD-1 α-cat cells or reconstitution of α-catenin in DLD-1 Δα-cat cells. (B) Representative phase-contrast images, (C) analysis of spheroid size and (D) invasion in col-I after 48 h after α-catenin modulation. Experiments were performed in triplicate and the results represent the means ± SD ( * P<0.05; n.s., not significant). Effect of esiRNA-mediated knockdown of upregulated genes in DLD-1 α-cat cells on (E) spheroid size and (F) invasion in col-I after 48 h. Results represent the means ± SD (n=2; n.s., not significant; n.a., not applicable due to spheroid instability).

Article Snippet: Antibodies against β-actin (A5441; Sigma-Aldrich, Taufkirchen, Germany), α-catenin (3236S), β-catenin (9587), p-β-catenin S675 (4176S), TGF-β (3709) (all from Cell Signaling Technology, Frankfurt am Main, Germany), E-cadherin (610181), fibronectin (610077) (both from BD Biosciences, Heidelberg, Germany), Zonula occludens-1 (ZO-1; 40-2300; Zymed Laboratories, San Francisco, CA, USA) and horseradish peroxidase-conjugated donkey anti-rabbit (NA934) and goat anti-mouse (NA9310) antibodies (GE Healthcare, Buckinghamshire, UK), as well as Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (A11001 and A1037; Invitrogen, Karlsruhe, Germany) were purchased as indicated. lrECM (MatrigelTM) and col-I were from BD Biosciences, and 5-fluorouracil (5-FU) was from Medac (Wedel, Germany).

Techniques: Western Blot, Knockdown, esiRNA

Journal: International Journal of Oncology

Article Title: Differential effects of α-catenin on the invasion and radiochemosensitivity of human colorectal cancer cells

doi: 10.3892/ijo.2018.4279

Figure Lengend Snippet: E-cadherin and α-catenin inversely correlate with the prognosis of patients with rectal cancer. (A) Examples of rectal cancer biopsies with high and low immunohistochemical detection of E-cadherin and α-catenin. Original magnification, ×400; scale bar, 50 µ m. (B) Cumulative incidence of overall survival, (C) locoregional failure and (D) distant failure according to a low E-cadherin and α-catenin (individual WS ≤6) expression vs. a high E-cadherin and α-catenin (WS >6) expression in pretreatment biopsies of 33 patients with rectal carcinoma treated with neoadjuvant chemoradiotherapy.

Article Snippet: Antibodies against β-actin (A5441; Sigma-Aldrich, Taufkirchen, Germany), α-catenin (3236S), β-catenin (9587), p-β-catenin S675 (4176S), TGF-β (3709) (all from Cell Signaling Technology, Frankfurt am Main, Germany), E-cadherin (610181), fibronectin (610077) (both from BD Biosciences, Heidelberg, Germany), Zonula occludens-1 (ZO-1; 40-2300; Zymed Laboratories, San Francisco, CA, USA) and horseradish peroxidase-conjugated donkey anti-rabbit (NA934) and goat anti-mouse (NA9310) antibodies (GE Healthcare, Buckinghamshire, UK), as well as Alexa Fluor 488 and Alexa Fluor 594 secondary antibodies (A11001 and A1037; Invitrogen, Karlsruhe, Germany) were purchased as indicated. lrECM (MatrigelTM) and col-I were from BD Biosciences, and 5-fluorouracil (5-FU) was from Medac (Wedel, Germany).

Techniques: Immunohistochemical staining, Expressing