JCB. Supplemental material THE JOURNAL OF CELL BIOLOGY. Prospéri et al.,

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Supplemental material JCB Prospéri et al., http://www.jcb.org/cgi/content/full/jcb.201501018/dc1 THE JOURNAL OF CELL BIOLOGY Figure S1.

1 Supplemental material JCB Prospéri et al., THE JOURNAL OF CELL BIOLOGY Figure S1. Myo1b Tail interacts with YFP-EphB2 coated beads and genistein inhibits Myo1b phosphorylation. (A) YFP-EphB2 has been pulled down with GFP-Trap beads from Hek293T cells expressing this recombinant protein. The same amount of YFP-EphB2 beads were then incubated with isolated GST or recombinant GST-Myo1b-Tail and analyzed by SDS-PAGE and immunoblot using anti-gst antibodies. 1/10 of the samples have been analyzed by SDS- PAGE and anti-ephb2 antibodies. For comparison, 50 ng of GST and GST-Myo1b-Tail were detected by immunoblotting using anti-gst antibodies (Input). (B) EGFP-Myo1b has been pulled down with GFP-Trap beads from Hek293T cell lysate (Input) expressing EGFP-Myo1b and Flag-EphB2 after treatment with 1% DMSO containing or not 100 µm genistein (EMD Millipore) for 1 h at 37 C. 70% of the pull-down was loaded to detect the coip of Flag-EphB2, while 4% was loaded to detect EGFP-Myo1b and EGFP-Myo1b tyrosine phosphorylation. Note that tyrosine phosphorylation of Flag-EphB2 that coip with Myo1b is hardly detectable in these conditions. (C) The amounts of Flag-EphB2 that coip with EGFP-Myo1b and phosphorylated Myo1b in the absence or in presence of genistein treatment were quantified as described in Material and methods, normalized to the amount of EGFP-Myo1b pulled down, and expressed as a percentage of the amount detected in the presence of DMSO. Data are shown as the mean of three experiments. Error bars represent ± SEM. JCB 2015 Myosin 1b, an effector of EphB signaling Prospéri et al. S16 S17

2 Figure S2. Characterization of the cells expressing Cherry-ephrinB1 or YFP-EphB2. (A) Expression of endogenous EphB2 in Hek293T and HCT116 cells and expression of YFP-EphB2 in the isolated cellular pools were analyzed by SDS-PAGE and immunoblotted with anti-ephb2 antibodies. Tubulin was used as a loading control. Note that the level of Myo1b expression is not affected by expression of YFP-EphB2. (B) Expression of endogenous ephrinb1 in Hek293T and HCT116 cells and expression of Cherry-ephrinB1 in the isolated cellular pools were analyzed by SDS-PAGE and immunoblotted with anti-ephrinb1 antibodies. Tubulin was used as a loading control. Note that the level of Myo1b expression is not affected by expression of Cherry-ephrinB1. (C and D) YFP-EphB2 was immunoprecipitated from YFP-EphB2-HCT116 (C) and YFP-EphB2- Hek293T (D) cells treated or not for 10 min with ephrinb1-fc and analyzed by immunoblotting with anti-ephb2 or anti-phosphotyrosine antibodies. Note the increase of phosphorylation of EphB2 when the cells were stimulated with ephrinb1. (E and F) YFP-EphB2 and Cherry-ephrinB1 available at the cell surface of HCT116 (E) or Hek293T (F) cellular pools and wild-type cells were detected by incubating cells for 30 min at 4 C with clustered ephrinb1-fc or EphB2-Fc, respectively, and detected with fluorescent goat anti-igg as described in Materials and methods. Note that both YFP-EphB2 and Cherry-ephrinB1 are available at the cell surface of the cellular pools but endogenous EphB2 receptors and ephrinb1 were not detected at the surface of the wild-type cells. E and F are shown at the same magnification. Bar, 20 µm. S17

3 Figure S3. Analysis of the number of cells per islet formed. A corresponds to Fig. 3 A and shows the overlay of the phase-contrast images (gray) and fluorescent images (green) of YFP-EphB2 expressing cells (green) transfected with control or Myo1b sirnas and cocultivated with Cherry-ephrinB1-HCT116 or Cherry-HCT116 cells. (B) To identify the YFP-EphB2 islets, YFP images were first manually thresholded under ImageJ (Schneider et al., 2012) using the YFP images overlaid with the Cherry images corresponding to Cherry-ephrinB1 or Cherry-expressing cells as a visual reference. (C) Cherry cells growing occasionally on top of YFP cells (see Fig. 4 and Video 2); Cherry images were manually thresholded and subtracted from YFP masks for all the different co-cultures. (D) To eliminate sparse pixels a Gaussian filter radius of 15 pixels (9.45 µm) was applied to the resulting corrected YFP mask. Islets were then manually thresholded from this blurred image using the composite image (B) as a reference. (E) To quantify the number of nuclei present in the islets, DAPI images were used and a Cell Profiler (Lamprecht et al., 2007) pipeline was created. First DAPI intensity was enhanced by a logarithm transform. (F) Then the centers of nuclei were identified as the local minima of the Laplacien of Gaussian-filtered DAPI images for which the Gaussian radius was set to the mean size of nuclei and reconstructed by watershed. Nuclei were counted in the islets only if half of each nuclei and their center were inside the islet mask. Islets touching the image border were discarded. Bars, 150 µm. JCB 2015 Myosin 1b, an effector of EphB signaling Prospéri et al. S18 S19

4 Figure S4. Efficiency of Myo1b sirnas. (A) YFP-EphB2-HCT116 cells transfected with 10 nm of control or Myo1b sirnas were analyzed by SDS-PAGE and immunoblotting with anti-myo1b antibodies 48 and 72 h after transfection. Tubulin was used as a loading control. Note the important reduction of Myo1b in cells transfected with Myo1b sirna. (B) To quantify EphB2 at the surface of YFP-EphB2-HCT116 cells transfected with 10 nm of control or Myo1b sirnas, cells were incubated with clustered ephrinb1-fc as described in Materials and methods and the ratio of fluorescence detected at the cell surface for bound ephrinb1 over the fluorescence detected for YFP-EphB2 that represents the total amount of receptors was calculated for both experimental conditions and expressed in arbitrary unit. Data are shown as the mean of three experiments. Error bars represent ± SEM (n = 45 for cells transfected with control sirna and n = 46 for cells transfected with Myo1b sirna). Paired Student s t test was used to analyze the probabilities of these data: P = Note that the difference is not significant. (C) YFP-EphB2-Hek293T cells transfected with 10 nm of control or Myo1b sirnas were analyzed by SDS-PAGE and immunoblotting with anti-myo1b antibodies 48 and 96 h after transfection. Tubulin was used as a loading control. Note the important reduction of Myo1b in cells transfected with Myo1b sirna. (D) To quantify EphB2 at the surface of YFP-EphB2-HEK293T cells transfected with 10 nm of control or Myo1b sirnas, cells were incubated with clustered ephrinb1-fc as described Materials and methods and the ratio of fluorescence detected at the cell surface for bound ephrinb1 over the fluorescence detected for YFP-EphB2 that represents the total amount of receptors was calculated for both experimental conditions and expressed in arbitrary units. Data are shown as the mean of three experiments. Error bars represent ± SEM. n = 48 for cells transfected with control sirna and cells transfected with Myo1b sirna. Paired Student s t test was used to analyze the probabilities of these data: P = Note that the difference is not significant. (E) YFP-EphB2- HCT116 cells transfected with Myo1b or control sirnas were stimulated 10 min or not with clustered ephrinb1-fc and analyzed by SDS-PAGE and immunoblotted with anti-pmlc(ser19). Tubulin was used as loading control. (F) The amount of pmlc(ser19) in YFP-EphB2- HCT116 cells transfected with Myo1b or control sirnas and treated 10 min with clustered ephrinb1-fc has been quantified as described in Materials and methods and normalized to the amount of tubulin detected in the sample. Data are shown as the mean of three experiments. Error bars represent ± SEM. (G) To determine the efficiency of fascin sirna, YFP-EphB2-HCT116 cells transfected with 30 nm of control or fascin sirnas were analyzed by SDS-PAGE and immunoblotting with anti-fascin antibodies 48 h after transfection. Tubulin was used as a loading control. Note the reduction of fascin in cells transfected with fascin sirna. (H) To determine the efficiency of fascin sirna, YFP-EphB2-Hek293T cells transfected with 10 nm of control or fascin sirnas were analyzed by SDS-PAGE and immunoblotting with anti-fascin antibodies 48 h after transfection. Tubulin was used as a loading control. Note the important reduction of fascin in cells transfected with fascin sirna. (I) Distribution of NMM2 was analyzed by immunofluorescence labeling and 3D fluorescent microscopy in YFP-EphB2-HCT116 cells transfected with 30 nm of control or fascin sirnas stimulated or not with ephrinb1-fc for 10 min. Bar, 8.5 µm. (J) HUVECs transfected with 10 nm of control or Myo1b sirnas were analyzed by SDS-PAGE and immunoblotting with anti-myo1b antibodies 48 h after transfection. Tubulin was used as a loading control. Note the important reduction of Myo1b in cells transfected with Myo1b sirna. S19

Figure S5. HUVECs expressing endogenous EphB receptors form filopodia when they contact Cherry-ephrinB1 expressing HUVECs before cell repulsion.

confocal microscopy (Video 3, A and B). Bars, 15 µm. The colored squares represent the enlarged regions shown in B.

LifeAct before cell repulsion. The white lines outline the edge of the cells expressing LifeAct in contact with the ephrinb1-expressing cell at the beginning of the videos.

Representative videos of a YFP-EphB2-HCT116 cell that contacts a Cherry-ephrinB1- HCT116 cell (A) and another one that contacts a Cherry-HCT116 cell (B).

5 Figure S5. HUVECs expressing endogenous EphB receptors form filopodia when they contact Cherry-ephrinB1 expressing HUVECs before cell repulsion. (A) First frame of the overlay of the Cherry and GFP fluorescence of HUVECs transfected with GFP-LifeAct or Cherry-ephrinB1, cocultivated, treated or not with DMSO, and analyzed by spinning disk confocal microscopy (Video 3, A and B). Bars, 15 µm. The colored squares represent the enlarged regions shown in B. (B) The sequences of the overlay of Cherry and GFP images from time 0 to 14 min from Video 3 show the formation of protrusions at the interface of cells expressing ephrinb1 and those expressing LifeAct before cell repulsion. The white lines outline the edge of the cells expressing LifeAct in contact with the ephrinb1-expressing cell at the beginning of the videos. Note that filopodia are growing out from the cell edge. Video 1. Related to Fig. 4. Representative videos of a YFP-EphB2-HCT116 cell that contacts a Cherry-ephrinB1- HCT116 cell (A) and another one that contacts a Cherry-HCT116 cell (B). Cell behavior was monitored at 37 C and under 10% CO 2 by timelapse imaging using spinning disk confocal microscopy. The overlaid focal planes of cells expressing Cherry-ephrinB1 (red) and YFP-EphB2 (green) are shown. Images were acquired at 1 frame/5 min during 3 h 40 min. Bars,15 µm. Video 2. Related to Fig. 4. Representative videos of YFP-EphB2-HCT116 cells that contact Cherry-ephrinB1-HCT116 cells after 30-min incubation with 50 µm blebbistatin in DMSO, 1 µm PCIP in DMSO, or only DMSO. Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy. The overlaid focal planes of cells expressing Cherry-ephrinB1 (red) and YFP-EphB2 (green) are shown. Images were acquired at 1 frame/min during 3 h. Bars, 15 µm. Video 3. Related to Figs. 5 and S5. Representative videos of HUVECs transfected with the plasmid encoding GFP-LifeAct that contacts HUVECs transfected with the plasmid encoding Cherry-ephrinB1 without treatment (A) or after 30-min incubation with 2 µm PCIP (C) in DMSO or with DMSO (B). Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy. The overlaid focal planes of cells expressing Cherry-ephrinB1 (red) and GFP-LifeAct (green) are shown. Images were acquired at 1 frame/min during 3 h. Bars, 15 µm. JCB 2015 Myosin 1b, an effector of EphB signaling Prospéri et al. S20 S21

Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy.

Representative videos of YFP-EphB2-HCT116 cells transfected with control sirna, Myo1b sirna, and fascin sirna that contacts Cherry-ephrinB1-HCT116 cells.

(B) cells analyzed by fluorescence. Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using video microscopy. Images were acquired at 1 frame/5 min. Bar, 10 µm. Video 7.

Representative video of YFP-EphB2-Hek293T cells transfected with control sirna and cultivated in front of Cherry-ephrinB1-Hek293T analyzed by phase contrast (A) or fluorescence to visualize YFP-EphB2

6 Video 4. Related to Fig. 5. Representative videos of HUVECs transfected with the plasmid encoding GFP-LifeAct and control or Myo1b sirna and in contact with HUVECs transfected with the plasmid encoding Cherry-ephrinB1. Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy. The overlaid focal planes of cells expressing Cherry-ephrinB1 (red) and GFP-LifeAct (green) are shown. Images were acquired at 1 frame/ min during 3 h. Bar, 15 µm. Video 5. Related to Fig. 6. Representative videos of YFP-EphB2-HCT116 cells transfected with control sirna, Myo1b sirna, and fascin sirna that contacts Cherry-ephrinB1-HCT116 cells. Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy. The overlaid focal planes of cells expressing Cherry-ephrinB1 (red) and cells expressing YFP-EphB2 (green) are shown. Images were acquired at 1 frame/min during 3 h. Bar,15 µm. Video 6. Related to Fig. 7 A. Representative videos of YFP-EphB2-Hek293T cells transfected with control sirna and cultivated in front of other YFP-EphB2-Hek293T cells transfected with control sirna (A) or Cherry-ephrinB1-Hek293T (B) cells analyzed by fluorescence. Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using video microscopy. Images were acquired at 1 frame/5 min. Bar, 10 µm. Video 7. Related to Fig. 7 D. Representative video of YFP-EphB2-Hek293T cells transfected with control sirna and cultivated in front of Cherry-ephrinB1-Hek293T analyzed by phase contrast (A) or fluorescence to visualize YFP-EphB2 (B). Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using video microscopy. Images were acquired at 1 frame/10 s. Bars, 10 µm. Video 8. Related to Fig. 7 D. Representative video of YFP-EphB2-Hek293T cells transfected with Myo1b sirna and cultivated in front of Cherry-ephrinB1-Hek293T analyzed by phase contrast (A) or fluorescence to visualize YFP-EphB2 (B). Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using video microscopy. Images were acquired at 1 frame/10 s. Bars, 10 µm. Video 9. Related to Fig. 8 A. Representative video of YFP-EphB2-Hek293T cells transfected with control sirna and stimulated with clustered ephrinb1-fc (red arrow). Cell behavior was monitored at 37 C and under 10% CO 2 by time-lapse imaging using spinning disk confocal microscopy. Images were acquired at 1 frame/10 s. Bar, 5 µm. S21

Video 10. Related to Fig. 8 B. Representative video of YFP-EphB2-Hek293T cells transfected with a plasmid encoding MRLC- RFP and stimulated with clustered ephrinb1-fc (red arrow).

7 Video 10. Related to Fig. 8 B. Representative video of YFP-EphB2-Hek293T cells transfected with a plasmid encoding MRLC- RFP and stimulated with clustered ephrinb1-fc (red arrow). Cell behavior was monitored at 37 C and under 10% CO 2 by timelapse imaging using spinning disk confocal microscopy. Images were acquired at 1 frame/10 s. Bar, 5 µm. References Lamprecht, M.R., D.M. Sabatini, and A.E. Carpenter CellProfiler: free, versatile software for automated biological image analysis. Biotechniques. 42: Schneider, C.A., W.S. Rasband, and K.W. Eliceiri NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 9: nmeth.2089 S22 JCB 2015 Myosin 1b, an effector of EphB signaling Prospéri et al. S22

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION a 14 12 Densitometry (AU) 1 8 6 4 2 t b 16 NMHC-IIA GAPDH NMHC-IIB Densitometry (AU) 14 12 1 8 6 4 2 1 nm 1 nm 1 nm 1 nm sirna 1 nm 1 nm Figure S1 S4 Quantification of protein levels. (a) The microtubule