Proliferation of HM3KO cells.

<p>Proliferation of HM3KO cells treated with a solvent (0.1% of DMSO) (open circle in A, lane 1 in B), GDNF (100 ng/ml) (closed circle in A, lane 2 in B), SU5416 (5µM) (open square in A, lane 3 in B) and GDNF plus SU5416 (closed square in A, lane 4 in B) for 24 (A) and 48 hours (A, B) was examined by cell counting with trypan blue staining (A) and MTT assay (B). Difference between proliferation levels of DMSO-treated control cells and other cells was statistically analyzed by the Kruskal-Wallis test. *, Significantly different (P<0.05) from the control.</p

Levels of c-RET protein expression in human malignant melanoma cell lines.

<p>The levels of c-RET protein expression were examined in c-RET-transfected NIH3T3 cells as a positive control (c-RET transfectant; lane 1), primary-cultured normal human epithelial melanocytes (NHEM; lane 2), G361 (lane 3), HM3KO (lane 4) and MNT-1 (lane 5) by immunoblotting analysis with anti-RET antibody after immunoprecipitation with anti-RET antibody.</p

Augmentation of c-RET tyrosine kinase activity in HM3KO cells by c-RET ligand (GDNF).

<p>Expression of c-RET protein (A, B) and phosphorylated tyrosine 905 in c-RET (C, D) in HM3KO cells in the absence (A, C) or presence (B, D) of GDNF were examined by immunocytochemistry with anti-c-RET and anti-phosphorylated tyrosine 905 in c-RET antibodies using hematoxylin counterstaining (A–D).</p

Stage-dependent c-Ret, Gfra1 and Gdnf transcripts expression levels in tumors from RET-mice.

<p>(A, C, E) Levels of c-Ret (A), Gfra1 (C) and Gdnf (E) transcripts expression in tumors of various sizes from RET-mice. Histopathologically benign and malignant tumors are shown by open and closed squares, respectively. (B, D, F) Levels of c-Ret (B), Gfra1 (D) and Gdnf (F) transcripts expression (mean ± SD) in benign melanocytic tumors (open bar) and malignant melanoma (closed bar) from RET-mice. c-Ret (A, B), Gfra1 (C, D) and Gdnf (E, F) transcript levels measured by real-time PCR were adjusted by hypoxanthine guanine phosphoribosyl transferase (Hprt) transcript levels. Differences in expression levels of c-Ret (B), Gfra1 (D) and Gdnf (F) between benign melanocytic tumors and malignant melanoma from RET-mice were statistically analyzed by the Mann-Whitney <i>U</i> test. *, Significantly different (P<0.05) from the control.</p

Levels of c-RET, GFRa1 and GDNF transcripts expression in primary-cultured normal human epithelial melanocytes (NHEM) and human malignant melanoma cell lines.

<p>(A, B, C) Levels of c-RET (A), GFRa1 (B) and GDNF (C) transcripts expression in NHEM (lane 1) and 4 kinds of malignant melanoma cell lines (lanes 2–5; SK-Mel28, G361, MNT-1 and HM3KO). The transcript levels measured by real-time PCR were adjusted by TATA-box-binding protein (TBP) transcript levels. Differences in expression levels of c-Ret (A), Gfra1 (B) and Gdnf (C) between NHEM (lane 1; open bar) and malignant melanoma cell lines (lanes 2–5; closed bars) were statistically analyzed by the Kruskal-Wallis test. **, Significantly different (P<0.01) from the control.</p

Illustration of the mean-shift model.

<div><p><i>A</i>. Initialization of the generic kernel based on the user-defined position (x’₀,y’₀).</p> <p>The kernel (here an octagon, ndir = 8) is divided into sectors (S1 to S8), each one containing two nested triangle-shaped regions (R_b and R_w), one sensitive to dark and the other to bright pixels (“b” means “black” and “w” means “white”). Here, R_b6 and R_w6 are shown, with a total of 16 regions (R_b1 to R_b8 and R_w1 to R_w8). Only the contours of sectors 2-5 are shown in order to lighten and better visualize the figure. The cell is not presented for clarity.</p> <p><i>B</i>. <i>Adjustment of the position of the center at t₀</i>. </p> <p>Sixteen mass centers (g_b1 to g_b8 and g_w1 to g_w8) are first calculated from the intensities of the pixels from each region, (g_b1 and g_w1 are shown). Sector mass centers (C) are calculated from g_wn and g_bn, (C₈ is shown as an example). The center (x₀,y₀) is defined as the centroid of the mass centers C₁ to C₈.</p> <p><i>C</i>. <i>Adaptation of the kernels to cell morphology at t₀</i>. </p> <p>The distances (d_i) between each mass center (g_wn) and kernel center (x₀,y₀) are calculated (d₈ is shown as an example). The new outer radii (r_wn) are calculated based on d_n, the average d_n distances, the expansion factor and the anisotropy factor. r_bn is assigned according to the ratio (r_b / r_w), which is initially defined by the user.</p> <p><i>D</i>. <i>Representation of the kernels at t₁</i>. </p> <p>Information is obtained applying the processes explained in B and C. The size of the sectors will increase or decrease (indicated by the arrows) as a function of cell shape modifications. </p></div

Time-dependent characteristics of cell trajectories extracted by manual and automatic tracking.

<div><p>Cells were imaged every four minutes for 12 hours and experiments were repeated three times. The same forty independent cells were tracked manually (M) and automatically (A). The following variables were extracted from the manually and automatically retrieved sets of coordinates:.</p> <p><i>A</i>. <i>Average migration speed of WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0669 and p = 0.3266 for the average speed and standard deviation, respectively. </p> <p><i>B</i>. <i>Average acceleration of migration by WM852 human melanoma cells</i>. Manual and automatic methods were statistically significant (standard unpaired t-test comparing average acceleration, p = 10^-4). For the standard deviation of average acceleration, p = 0.2729. </p> <p><i>C</i>. <i>Percentage of pause by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.1783). </p> <p><i>D</i>. <i>Angles of displacement between two adjacent time frames (angle α) calculated for WM852 human melanoma cells</i>. Manual and automatic methods were statistically significant (standard unpaired t-test, p = 10^-4).</p> <p>E. Definition of variables .</p> <p>The polygon (gray hexagon) represents a cell migrating at three different time frames with three sets of coordinates. The angles α and β are defined relative to the horizontal line as a reference at two consecutive times. </p></div

Time benefit of automatic tracking <i>vs</i>. manual tracking.

<p>Cells were followed either manually (full circles) or using iTrack4U (white circles). It took about three minutes to track each single cell, corresponding to 181 frames, by manual tracking. Twenty cells can therefore be manually tracked in 1 hour and 200 cells during 10 hours of active work. Automatic tracking is performed in two major steps: (i) the establishment of the parameters for both pre-processing and tracking requires about two hours and (ii) the automatic tracking requires about four seconds to fully track a single cell. Using iTrack4U is beneficial for following over about 50 cells. </p

Geometric characteristics of cell trajectories associated with distances and extracted by manual and automatic tracking.

<div><p>Cells were imaged every four minutes for 12 hours and experiments were repeated three times. The same 40 independent cells were tracked manually (M) and automatically (A). The following variables were extracted from the manually and automatically retrieved sets of coordinates:.</p> <p><i>A</i>. <i>Total distance of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0774).</p> <p><i>B</i>. <i>Euclidian distance (start-end distance) of WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.9672).</p> <p><i>C</i>. <i>Persistence of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.5012).</p> <p><i>D</i>. <i>Definition of migration variables used in this figure</i>. Total distance = d_ttl, Euclidian distance = d_S-E, persistence = d_ttl / d_S-E, minimum travelled distance = d_min, maximum travelled distance = d_max.</p> <p><i>E</i>. <i>Average distance of migration by WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant (standard unpaired t-test, p = 0.0774 and p = 0.3913 for the average distance and standard deviation, respectively). </p> <p><i>F</i>. <i>Extreme values (minimum and maximum distances) of migration for WM852 human melanoma cells</i>. Manual and automatic methods were not statistically significant for the maximum distance (standard unpaired t-test, p = 0.2611). A significant difference for the minimum distance has no real meaning, as explained in the text (standard unpaired t-test, p = 0.001).</p></div

Indomethacin treatment and survival of ctnnb1Δex3 mice.

<p>Indomethacin treatment results in the closure of WT and ctnnb1Δex3 ( = <i>Tyr::Cre/°; ctnnb1Δex3</i>/+) DA and allows the survival of ctnnb1Δex3 mice. Mock (A, B) and indomethacin (indo, 10 mg/kg body weight) (C, D) intraperitoneal injections into pregnant <i>Tyr::Cre/Tyr::Cre</i>; +/+; <i>Dct::LacZ/Dct::LacZ</i> females carrying <i>Tyr::Cre/</i>°; +/+; <i>Dct::LacZ/</i>° (A, C) and <i>Tyr::Cre/</i>°; <i>ctnnb1Δex3</i>/+; <i>Dct::LacZ/</i>° (B, D) E18.5 embryos. Four hours later, embryos were isolated, fixed, X-gal stained, transversally sectioned through the DA and counterstained with eosin. We treated three pregnant females and sectioned ten embryonic hearts (five WT and five mutants). The ductus arteriosus was closed in all cases. Note that the numbers of Dct+ cells derived from ctnnb1Δex3-Dct embryos obtained from pregnant mothers injected with indomethacin or mock-injected were similar. (E) Kaplan-Meier curves of WT and ctnnb1Δex3 newborn pups treated or mock-treated with indomethacin (6 mg/kg body weight indomethacin within 12 hours of birth). Ultrasound analysis was performed on treated versus non-treated animals during the second and third months, which associated survival of treated ctnnb1Δex3 to the size of the left atrium (not shown). Indomethacin-treated ctnnb1Δex3 mice survived significantly longer than mock-treated mice (p<0.009). Note similar results were obtained when ctnnb1Δex3 mi mice were treated with indomethacin or mock. Scale bars, (A, B)  = 100 µm, (C, D)  = 50 µm.</p