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. 2018 Sep 3;217(9):3161–3182. doi: 10.1083/jcb.201802023

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© 2018 Zobel et al.

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Figure 1. — NUMB is a negative regulator of growth factor–induced CDR formation. (A) HeLa cells seeded on Matrigel-coated coverslips were transfected with four different siRNAs (hN#3, hN#2, hN#5, and hN#6) against human NUMB or scrambled oligos (siRNA Ctr, control). Cells were serum starved (SS) for 2 h, stimulated with 100 ng/ml HGF for 4 min, and stained to detect F-actin. Bar, 40 μm. (B) Number of cells forming CDRs over the total number of cells per field. (C) The expression levels of NUMB and vinculin were analyzed by immunoblotting (IB). (D) MEF cells transfected with three different siRNAs (m#2, mN#3, and mN#4) against murine NUMB or siRNA Ctr as control were seeded on gelatin-coated coverslips. Cells were serum starved for 2 h, stimulated with 1 ng/ml PDGF for 8 min, and stained to detect F-actin. Bar, 40 µm. (E) The number of cells forming CDRs over the total number of cells per field. (B and E) The data are the mean ± SEM (n > 100 cells/condition in 20 fields/each condition out of three independent experiments). (F) The expression level of NUMB and vinculin was analyzed by IB. (G) MEF cells were transfected with siRNA Ctr or with siRNA against murine NUMB (mN#3, upper panels), either alone or in combination with GFP empty vector (upper panels, right) or GFP-hNUMB isoform 1, 2, 3, or 4 (lower panels). Cells were serum starved for 2 h and stimulated with 1 ng/ml PDGF for 8 min, fixed and processed for epifluorescence to visualize GFP or GFP-NUMB (green, where indicated), and stained to detect F-actin (red). Bar, 40 µm; 20 µm in GFP or GFP-NUMB panels. (H) The number of cells forming CDRs over the total number of cells per field was counted. Representative images are shown. The data are the mean ± SEM (n > 100 cells/condition in 20 fields/each condition out of four independent experiments). (I) The expression levels of NUMB and vinculin were analyzed by IB. (C, F, and H) Molecular weight (MW) markers are shown on the left in kilodaltons. TFX, transfected. A Student’s t test was used to calculate the P values: **, P < 0.01. Dashed lines indicate that the intervening lanes have been spliced out.