![Figure 1. Endothelial cell supernumerary centrosomes disrupt migration and centrosome dynamics. (A) Enriched endothelial cells (EC) from WT mouse mammary tissue and MMTV-PyVT+/− mammary tumors stained for PECAM1 (white, endothelial cells), pericentrin (centrosomes), and DRAQ7 (DNA). (right) Higher magnification of boxed areas on left without PECAM1 channel. Bars: (main images) 50 µm; (insets) 5 µm. (B) Established WT mammary endothelial cell (nontumor [NEC]) and tumor endothelial cell (TEC) stained for PECAM1 (white, endothelial cells), γ-tubulin (centrosomes), and DRAQ7 (DNA). (right) Left images without PECAM1 channel. Bars: (main image) 50 µm; (insets) 10 µm. (C, left bars) Percentage of PECAM1-positive primary endothelial cells with greater than two centrosomes from tissues (MMTV-PyVT+/− skeletal muscle endothelial cells, n = 1 mouse; MMTV-PyVT−/− mammary tissue endothelial cells, n = 10 mice; MMTV-PyVT+/− mammary TECs, n = 15 mice). Statistical comparisons to endothelial cells from control MMTV PyVT−/− mammary tissue. χ2 test; error bars shows means ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001. (right bars) Percentage of established endothelial cells with greater than two centrosomes (WT mammary tissue endothelial cell, four clones and n = 1,082 cells; MMTV-PyVT mammary TEC, seven clones and n = 1,234 cells). χ2 test; error bars show means ± SEM. **, P = 0.013. (D) Diagram of the effect of centrosome number on cell migration. (E) 3-h migration tracks of TECs with one to two centrosomes or greater than two centrosomes; one representative experiment from three repeats. Bar, 40 µm. see also Video 1. (F) Mean total distance traveled of NECs and TECs with either one to two or greater than two centrosomes (NEC, n = 43 cells; TEC 1–2 centrosomes, n = 25 cells; TEC >2 centrosomes, n = 11 cells). Statistics: Student’s t test; error bars show means ± SEM. *, P = 0.02; **, P < 0.01. (G) Mean persistence (linear distance from origin to maximal point of migration) of NECs and TECs with either one to two or greater than two centrosomes (NEC, n = 43 cells; TEC 1–2 centrosomes, n = 25 cells; TEC >2 centrosomes, n = 11 cells). Statistics: Student’s t test; error bars show means ± SEM. *, P = 0.05; **, P < 0.01. (H) Representative endothelial cells with centrosome movements tracked over 1 h. Each track starts with blue colors and ends with pink colors. Note the divergence of tracks over time in TECs with greater than two centrosomes. Bar, 12 µm. see also Video 2. (I) Diagram of the indicated measurements. (J) Centrosome–centrosome distances in indicated groups (NEC, n = 99; TEC 1–2, n = 143; TEC >2, n = 154 centrosome pairs). Statistics: Student’s t test; error bars show means ± SD. ***, P < 0.0001. (K) Change (absolute value) in centrosome–centrosome distance in 5-min time intervals (NEC, n = 99; TEC 1–2, n = 143; TEC >2, n = 154 centrosome pairs). Statistics: Student’s t test; means ± SD. ***, P < 0.0001. (L) Centrosome–nuclear edge distances between the indicated groups (NEC, n = 130 cells; TEC 1–2, n = 142 cells; TEC >2, n = 154 measurements). Statistics: Student’s t test; means ± SD. ***, P < 0.0001. (M) Change (absolute value) in centrosome–nuclear edge distance in 5-min time intervals (NEC, n = 130; TEC 1–2, n = 142; TEC >2, n = 154 measurements). Statistics: Student’s t test; error bars show means ± SD. *, P < 0.05; ***, P < 0.0001.](https://cdn.rupress.org/rup/content_public/journal/jcb/206/2/10.1083_jcb.201311013/5/jcb_201311013r_fig1.jpeg?Expires=1767676510&Signature=iIt7rfvKUiCutMnctaRQYxfvXBGxQjYIkikLpHYjZunbY0Uhsnhjvu-jqfUCu9ZCiD2r5SAeOsQHFRO2e0XJpEbxtGAn-UAGv5R8o~C~3B6A03b7JeFXZZVxhxDXzui7eI9HlKuVzv-Fk13Sp0LstF78IrF-fWmYzOidf4SghV7zrnFTs-d2Ds1FHwtWAZpHS3B5ebc2g9M142bRDKtOl7pPBadUoLaJm4qlkefdRQBuXXYK7g6a4Pr3n91YTcX2gnGPLGxXu6WPkLu--NGB8AUUmH0JdcmhllM-XwekTr1LVRvRCFTnDfQM1Q13dQtTJmPUcuXafo4owH64PfVaUw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Endothelial cell supernumerary centrosomes disrupt migration and centrosome dynamics. (A) Enriched endothelial cells (EC) from WT mouse mammary tissue and MMTV-PyVT+/− mammary tumors stained for PECAM1 (white, endothelial cells), pericentrin (centrosomes), and DRAQ7 (DNA). (right) Higher magnification of boxed areas on left without PECAM1 channel. Bars: (main images) 50 µm; (insets) 5 µm. (B) Established WT mammary endothelial cell (nontumor [NEC]) and tumor endothelial cell (TEC) stained for PECAM1 (white, endothelial cells), γ-tubulin (centrosomes), and DRAQ7 (DNA). (right) Left images without PECAM1 channel. Bars: (main image) 50 µm; (insets) 10 µm. (C, left bars) Percentage of PECAM1-positive primary endothelial cells with greater than two centrosomes from tissues (MMTV-PyVT+/− skeletal muscle endothelial cells, n = 1 mouse; MMTV-PyVT−/− mammary tissue endothelial cells, n = 10 mice; MMTV-PyVT+/− mammary TECs, n = 15 mice). Statistical comparisons to endothelial cells from control MMTV PyVT−/− mammary tissue. χ2 test; error bars shows means ± SEM. **, P ≤ 0.01; ***, P ≤ 0.001. (right bars) Percentage of established endothelial cells with greater than two centrosomes (WT mammary tissue endothelial cell, four clones and n = 1,082 cells; MMTV-PyVT mammary TEC, seven clones and n = 1,234 cells). χ2 test; error bars show means ± SEM. **, P = 0.013. (D) Diagram of the effect of centrosome number on cell migration. (E) 3-h migration tracks of TECs with one to two centrosomes or greater than two centrosomes; one representative experiment from three repeats. Bar, 40 µm. see also Video 1. (F) Mean total distance traveled of NECs and TECs with either one to two or greater than two centrosomes (NEC, n = 43 cells; TEC 1–2 centrosomes, n = 25 cells; TEC >2 centrosomes, n = 11 cells). Statistics: Student’s t test; error bars show means ± SEM. *, P = 0.02; **, P < 0.01. (G) Mean persistence (linear distance from origin to maximal point of migration) of NECs and TECs with either one to two or greater than two centrosomes (NEC, n = 43 cells; TEC 1–2 centrosomes, n = 25 cells; TEC >2 centrosomes, n = 11 cells). Statistics: Student’s t test; error bars show means ± SEM. *, P = 0.05; **, P < 0.01. (H) Representative endothelial cells with centrosome movements tracked over 1 h. Each track starts with blue colors and ends with pink colors. Note the divergence of tracks over time in TECs with greater than two centrosomes. Bar, 12 µm. see also Video 2. (I) Diagram of the indicated measurements. (J) Centrosome–centrosome distances in indicated groups (NEC, n = 99; TEC 1–2, n = 143; TEC >2, n = 154 centrosome pairs). Statistics: Student’s t test; error bars show means ± SD. ***, P < 0.0001. (K) Change (absolute value) in centrosome–centrosome distance in 5-min time intervals (NEC, n = 99; TEC 1–2, n = 143; TEC >2, n = 154 centrosome pairs). Statistics: Student’s t test; means ± SD. ***, P < 0.0001. (L) Centrosome–nuclear edge distances between the indicated groups (NEC, n = 130 cells; TEC 1–2, n = 142 cells; TEC >2, n = 154 measurements). Statistics: Student’s t test; means ± SD. ***, P < 0.0001. (M) Change (absolute value) in centrosome–nuclear edge distance in 5-min time intervals (NEC, n = 130; TEC 1–2, n = 142; TEC >2, n = 154 measurements). Statistics: Student’s t test; error bars show means ± SD. *, P < 0.05; ***, P < 0.0001.
