Figure 6 ( A , B ) VCaP cells stably expressing control ( sh - control ) or p53 - specific shRNA were infected with lentivirus for empty vector ( EV ) or deletion mutants of EZH2 . Cell growth in 2D ( A ) and 3D ( B ) conditions were determined by MTS assay and measurement of clone size , respectively . Statistical significance was determined by two - tailed Student ' s t - test . * P < 0 . 05 , * * * P < 0 . 001 . ( C , D ) VCaP cells ( 1x107 ) infected with lentivirus were injected subcutaneously into NSG mice ( n = 8 / group ) . Tumors were measured by caliper twice a week . Tumors at the end point of measurement were isolated and photographed ( C ) , and data are shown as means ± SD ( D ) . Statistical significance was determined by two - tailed Student ' s t - test for tumors at day 56 . * * * P < 0 . 001 . ( E - G ) Luciferase - expressing MIA PaCa - 2 cells ( 2 × 106 ) infected with lentivirus were injected via tail vein into NSG mice ( n = 6 / group ) . At 12 weeks after injection , mice were subjected to bioluminescent imaging , and images were recorded ( E ) and bioluminescent signals were quantified ( F ) . Bioluminescent flux ( photons s - 1 sr - 1 cm - 2 ) was determined for lesions in lung , the ends of the box are the upper and lower quartiles and the box spans the interquartile range ; the median is marked by a vertical line inside the box ; the whiskers are the two lines outside the box that extend to the highest and lowest observations . Lungs were isolated from mice , stained with Bouin ' s solution , and photographed ( G ) . The white spots on lungs ( stained in yellow ) are metastatic tumors . * P < 0 . 05 ; NS , no significance .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) VCaP cells stably expressing control (sh-control) or p53-specific shRNA were infected with lentivirus for empty vector (EV) or deletion mutants of EZH2. Cell growth in 2D (A) and 3D (B) conditions were determined by MTS assay and measurement of clone size, respectively. Statistical significance was determined by two-tailed Student's t-test. * P < 0.05, *** P < 0.001.(C, D) VCaP cells (1x107) infected with lentivirus were injected subcutaneously into NSG mice (n = 8/group). Tumors were measured by caliper twice a week. Tumors at the end point of measurement were isolated and photographed (C), and data are shown as means ± SD (D). Statistical significance was determined by two-tailed Student's t-test for tumors at day 56. *** P < 0.001.(E-G) Luciferase-expressing MIA PaCa-2 cells (2×106) infected with lentivirus were injected via tail vein into NSG mice (n = 6/group). At 12 weeks after injection, mice were subjected to bioluminescent imaging, and images were recorded (E) and bioluminescent signals were quantified (F). Bioluminescent flux (photons s-1 sr-1 cm-2) was determined for lesions in lung, the ends of the box are the upper and lower quartiles and the box spans the interquartile range; the median is marked by a vertical line inside the box; the whiskers are the two lines outside the box that extend to the highest and lowest observations. Lungs were isolated from mice, stained with Bouin's solution, and photographed (G). The white spots on lungs (stained in yellow) are metastatic tumors. * P < 0.05; NS, no significance.


Figure 7 ( B ) Kaplan - Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort . Tumors were separated into two groups according to EZH2 expression levels ( high or low ) and three groups based on the mutation status of p53 ( WT , loss or mutated ) . VCaP and C4 - 2 cells were treated with GSK - 126 , DZNep ( 5 µM for VCaP and 10 µM for C4 - 2 ) , and EZH2 ASO , followed by western blots with indicated antibodies ( C ) Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 . VCaP and C4 - 2 cells were treated with GSK - 126 , DZNep ( 5 µM for VCaP and 10 µM for C4 - 2 ) , and EZH2 ASO , followed by MTT assay ( D ) . Data shown as means ± SD ( n = 6 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 . ( E , F ) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts . Mice ( n = 10 ) bearing the Ptenpc - / - ; Trp53pc - / - ( E ) and Ptenpc - / - ; Trp53pc - / R172H allografts ( F ) were treated with control ASOs ( 50 mg kg - 1 ) or Ezh2 ASOs at different doses ( 25 mg kg - 1 and 50 mg kg - 1 ) . Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels . Each allograft was isolated by the end of ASO treatment and photographed ( top panels ) . Data shown as means ± SD ( n = 10 ) . Statistical significance was determined by two - tailed Student ' s t - test . * * * P < 0 . 001 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(B) Kaplan-Meier plots showing the association of EZH2 overexpression with biochemical recurrence of prostate cancer in patients from the TCGA cohort. Tumors were separated into two groups according to EZH2 expression levels (high or low) and three groups based on the mutation status of p53 (WT, loss or mutated).VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by western blots with indicated antibodies (C) Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.VCaP and C4-2 cells were treated with GSK-126, DZNep (5 µM for VCaP and 10 µM for C4-2), and EZH2 ASO, followed by MTT assay (D). Data shown as means±SD (n=6). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.(E, F) Effect of Ezh2 ASOs on growth of murine prostate cancer allografts. Mice (n=10) bearing the Ptenpc-/-;Trp53pc-/- (E) and Ptenpc-/-;Trp53pc-/R172H allografts (F) were treated with control ASOs (50 mg kg-1) or Ezh2 ASOs at different doses (25 mg kg-1 and 50 mg kg-1). Tumor growth was measured twice a week for 3 weeks and the data are shown in the bottom panels. Each allograft was isolated by the end of ASO treatment and photographed (top panels). Data shown as means±SD (n=10). Statistical significance was determined by two-tailed Student's t-test. *** P < 0.001.


Figure 1 Western blot analysis of Shp2 in control and Shp2 ko cells . Immunofluorescence analysis of Ki67 on control and Shp2 ko cells at day 7 . Scale bar , 50 μm . Quantification of the numbers of Ki67 + cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . phere formation by control and Shp2 ko cells at day 7 . Scale bar , 200 μm . Quantification of the numbers of spheres formed by control and Shp2 ko cells shown in ( E ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Growth kinetics of spheres formed by control and Shp2 ko cells by counting sphere cells at day 4 , 7 , and 12 . Error bars represent SEM ( n = 3 ) . SA ‐ β ‐ gal staining on spheres formed by control and Shp2 ko cells at day 7 . Scale bar , 100 μm . Quantification of the numbers of senescent cells in the spheres shown in ( H ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Western blot analysis of H3K9me3 , p27 , p53 pS18 ( Ser18 ‐ phosphorylated p53 ) , total p53 , Shp2 , and α ‐ tubulin in control and Shp2 ko cells . SA ‐ β ‐ gal staining on spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM at day 7 . Scale bar , 100 μm . Quantification of the numbers of senescent cells shown in ( K ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Quantification of the numbers of spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Statistical significance was assessed by Student ' s unpaired t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Western blot analysis of Shp2 in control and Shp2 ko cells.  Immunofluorescence analysis of Ki67 on control and Shp2 ko cells at day 7. Scale bar, 50 μm. Quantification of the numbers of Ki67+cells shown in (C). Error bars represent SEM (n = 3). **P 0.01.  phere formation by control and Shp2 ko cells at day 7. Scale bar, 200 μm. Quantification of the numbers of spheres formed by control and Shp2 ko cells shown in (E). Error bars represent SEM (n = 3). **P 0.01.  Growth kinetics of spheres formed by control and Shp2 ko cells by counting sphere cells at day 4, 7, and 12. Error bars represent SEM (n = 3).  SA‐β‐gal staining on spheres formed by control and Shp2 ko cells at day 7. Scale bar, 100 μm. Quantification of the numbers of senescent cells in the spheres shown in (H). Error bars represent SEM (n = 3). **P 0.01.  Western blot analysis of H3K9me3, p27, p53 pS18 (Ser18‐phosphorylated p53), total p53, Shp2, and α‐tubulin in control and Shp2 ko cells.  SA‐β‐gal staining on spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM at day 7. Scale bar, 100 μm. Quantification of the numbers of senescent cells shown in (K). Error bars represent SEM (n = 3). **P 0.01. Quantification of the numbers of spheres formed by control cells and those treated by the Shp2 inhibitor GS493 at 15 μM. Error bars represent SEM (n = 3). **P 0.01. Statistical significance was assessed by Student's unpaired t‐test.


Figure 2 qRT - PCR analysis of mRNA levels of Skp2 , Aurka , Dll1 , and Hey1 in control , Shp2 ko , and Shp2 ‐ inhibited cells . Error bars represent SEM ( n = 3 ) . SA ‐ β ‐ gal staining on control , Shp2 ko , and Shp2 ko cells with overexpression of Skp2 , Aurka , or N3IC cDNA . Scale bar , 100 μm . Quantification of the numbers of senescent cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; * * * P 0 . 001 . Quantification of the numbers of spheres formed by cells shown in ( C ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01 ; and * * * P 0 . 001 . Statistical significance was assessed by Student ' s unpaired t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control, Shp2 ko, and Shp2‐inhibited cells. Error bars represent SEM (n = 3).  SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNA. Scale bar, 100 μm. Quantification of the numbers of senescent cells shown in (C). Error bars represent SEM (n = 3). **P 0.01; ***P 0.001. Quantification of the numbers of spheres formed by cells shown in (C). Error bars represent SEM (n = 3). *P 0.05; **P 0.01; and ***P 0.001. Statistical significance was assessed by Student's unpaired t‐test.


Figure 3 SA ‐ β ‐ gal staining on control , Shp2 ko , and Shp2 ko cells with shRNA knockdown of p27 or p53 . Scale bar , 50 μm . Quantification of the numbers of senescent cells shown in ( A ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Quantification of the numbers of spheres formed by cells shown in ( A ) . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 . Western blot analysis of p27 , p53 , and α ‐ tubulin in control , Shp2 ko , and Shp2 ko cells with overexpression of Skp2 , Aurka , or N3IC cDNAs . The numbers below indicate the relative ratios of band intensities between p27 and α ‐ tubulin or between p53 and α ‐ tubulin . Western blot analysis of p27 , p53 , and α ‐ tubulin in PyMT cells treated with control ( DMSO ) or inhibitors against Skp2 ( MLN4924 ) , Aurora A ( VX ‐ 680 ) , or Notch ( DAPT ) at the indicated concentrations . The numbers below indicate the relative ratios of band intensities between p27 and α ‐ tubulin or between p53 and α ‐ tubulin . Quantification of the numbers of senescent cells in PyMT cells treated with control , MLN4924 , MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; NS , not significant . Quantification of the numbers of senescent cells in PyMT cells treated with control , VX ‐ 680 , VX ‐ 680 plus p27 knockdown , or VX ‐ 680 plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; NS , not significant . Quantification of the numbers of senescent cells in PyMT cells treated with control , DAPT , DAPT plus p27 knockdown , or DAPT plus p53 knockdown . Error bars represent SEM ( n = 3 ) . * * P 0 . 01 ; NS , not significant . Statistical significance was assessed by Student ' s unpaired t ‐ test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 SA‐β‐gal staining on control, Shp2 ko, and Shp2 ko cells with shRNA knockdown of p27 or p53. Scale bar, 50 μm. Quantification of the numbers of senescent cells shown in (A). Error bars represent SEM (n = 3). **P 0.01. Quantification of the numbers of spheres formed by cells shown in (A). Error bars represent SEM (n = 3). **P 0.01.  Western blot analysis of p27, p53, and α‐tubulin in control, Shp2 ko, and Shp2 ko cells with overexpression of Skp2, Aurka, or N3IC cDNAs. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin.  Western blot analysis of p27, p53, and α‐tubulin in PyMT cells treated with control (DMSO) or inhibitors against Skp2 (MLN4924), Aurora A (VX‐680), or Notch (DAPT) at the indicated concentrations. The numbers below indicate the relative ratios of band intensities between p27 and α‐tubulin or between p53 and α‐tubulin.  Quantification of the numbers of senescent cells in PyMT cells treated with control, MLN4924, MLN4924 plus p27 knockdown or MLN4924 plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant.  Quantification of the numbers of senescent cells in PyMT cells treated with control, VX‐680, VX‐680 plus p27 knockdown, or VX‐680 plus p53 knockdown. Error bars represent SEM (n = 3). *P 0.05; NS, not significant.  Quantification of the numbers of senescent cells in PyMT cells treated with control, DAPT, DAPT plus p27 knockdown, or DAPT plus p53 knockdown. Error bars represent SEM (n = 3). **P 0.01; NS, not significant. Statistical significance was assessed by Student's unpaired t‐test.


Figure 4 SA ‐ β ‐ gal staining on MLN4924 tumorcells treated with control , Shp2 inhibitor GS493 ( 15 μM ) , Src inhibitor PP2 ( 2 . 5 μM ) , Fak inhibitor TAE226 ( 0 . 5 μM ) , or Mek1 inhibitor U0126 ( 20 μM ) . Scale bar , 100 μm . Quantification of the numbers of senescent cells in the presence or absence of the inhibitors , as shown in ( A ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01 . Quantification of the numbers of spheres in the presence or absence of the inhibitors , as in ( A ) . Error bars represent SEM ( n = 3 ) . * P 0 . 05 ; * * P 0 . 01 Western blot analysis of pY527 , pY416 and total Src , pY925 and total Fak , pT202 / pT204 and total Erk1 / 2 , and α ‐ tubulin in MLN4924 tumorcells with or without treatment with 5 μM GS493 . qRT - PCR analysis of mRNA levels of Skp2 , Aurka , Dll1 , and Hey1 in control cells and those treated with the indicated inhibitors . Error bars represent SEM ( n = 3 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 SA‐β‐gal staining on MLN4924 tumorcells treated with control, Shp2 inhibitor GS493 (15 μM), Src inhibitor PP2 (2.5 μM), Fak inhibitor TAE226 (0.5 μM), or Mek1 inhibitor U0126 (20 μM). Scale bar, 100 μm.  Quantification of the numbers of senescent cells in the presence or absence of the inhibitors, as shown in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01. Quantification of the numbers of spheres in the presence or absence of the inhibitors, as in (A). Error bars represent SEM (n = 3). *P 0.05; **P 0.01  Western blot analysis of pY527, pY416 and total Src, pY925 and total Fak, pT202/pT204 and total Erk1/2, and α‐tubulin in MLN4924 tumorcells with or without treatment with 5 μM GS493.  qRT-PCR analysis of mRNA levels of Skp2, Aurka, Dll1, and Hey1 in control cells and those treated with the indicated inhibitors. Error bars represent SEM (n = 3).


Figure 5 A Kaplan - Meier survival analysis of humanpatients ( n = 3 , 935 ) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 ( see ) . B - D Kaplan - Meier survival analyses of the humanpatients ( n = 3 , 935 ) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A : cell cycle genes , DNA replication genes , or p53 target genes . B - D Kaplan - Meier survival analyses of the humanpatients ( n = 3 , 935 ) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A : cell cycle genes , DNA replication genes , or p53 target genes . E , F Kaplan - Meier survival analyses of humanpatients ( n = 3 , 935 ) divided into two groups based on the gene expression level of Skp2 or Aurka . HR , hazard ratio .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5 A Kaplan-Meier survival analysis of humanpatients (n = 3,935) with invasive breast cancers divided into two groups based on the gene expression level of Shp2 (see ).  B-D Kaplan-Meier survival analyses of the humanpatients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes.  B-D Kaplan-Meier survival analyses of the humanpatients (n = 3,935) divided into two groups based on the expression level of the genes of each overrepresented class determined in Fig A: cell cycle genes, DNA replication genes, or p53 target genes.  E, F Kaplan-Meier survival analyses of humanpatients (n = 3,935) divided into two groups based on the gene expression level of Skp2 or Aurka. HR, hazard ratio.


Figure 6 Mammary gland whole ‐ mounts from control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice at indicated ages . The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads ; premalignant lesions are marked by red arrowheads . Scale bar , 4 mm . Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice , as shown in ( B ) . Error bars represent SEM ( n = 6 ) . * P 0 . 05 ( Student ' s unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 Mammary gland whole‐mounts from control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice at indicated ages. The characteristic lymph node of the mammary glandfat pad is marked by white arrowheads; premalignant lesions are marked by red arrowheads. Scale bar, 4 mm. Quantification of premalignant lesions in mammary glands of control MLN4924 and MLN4924;MMTV‐Cre;Shp2fl/flmice, as shown in (B). Error bars represent SEM (n = 6). *P 0.05 (Student's unpaired t‐test).


Figure 7 Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice at hyperplasia , adenoma , and carcinoma stages . Scale bar , 100 μm . n = 8 . Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors ( top ) and metastases ( bottom ) from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . Scale bar , 100 μm . n = 8 . qRT - PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924 ; Shp2fl / fl ( n = 8 ) , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ( n = 6 ) , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ( n = 6 ) mice . Error bars represent upper and lower quartiles . Western blot analysis of Shp2protein levels in tumor tissues from MLN4924 ; Shp2fl / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 Immunostaining of EYFP and CK8 on mammary gland sections from MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice at hyperplasia, adenoma, and carcinoma stages. Scale bar, 100 μm. n = 8.  Immunohistochemistry analysis of Cre recombinase on paraffin sections of primary tumors (top) and metastases (bottom) from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice. Scale bar, 100 μm. n = 8.  qRT-PCR analyses of Shp2mRNA levels in tumor tissues from MLN4924;Shp2fl/fl (n = 8), MLN4924;MMTV‐Cre;Shp2+/fl (n = 6), and MLN4924;MMTV‐Cre;Shp2fl/fl (n = 6) mice. Error bars represent upper and lower quartiles.  Western blot analysis of Shp2protein levels in tumor tissues from MLN4924;Shp2fl/fl, MLN4924;MMTV‐Cre;Shp2+/fl, and MLN4924;MMTV‐Cre;Shp2fl/flmice.


Figure 8 Immunohistochemistry analysis of EYFP ( top ) and SA ‐ β ‐ gal staining ( bottom ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Note that SA ‐ β ‐ gal activity was present in the EYFP ‐ positive ( Shp2 ‐ negative ) areas ( right , arrows ) and was absent in the EYFP ‐ negative ( Shp2 ‐ positive ) areas ( right , arrowhead ) of mammary glandtumors of MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . Consecutive sections of EYFP and SA ‐ β ‐ gal staining for each group are shown . Scale bar : 100 μm . n = 5 . Immunofluorescence analysis of Ki67 , EYFP and CK8 ( pan ‐ epithelial marker ) ( top panel ) , p27 and EYFP ( middle panel ) , and p53 and EYFP ( bottom panel ) on cryosections of mammary glandtumors from control MLN4924 ; Shp2fl / fl ; EYFP − / fl , MLN4924 ; MMTV ‐ Cre ; Shp2 + / fl ; EYFP − / fl , and MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / flmice . DAPI was used for nuclear counterstain . Ki67 was absent in EYFP ‐ positive cells ( top right , arrows ) and was present in EYFP ‐ negative cells ( top right , arrowheads ) ; p27 was present in EYFP ‐ positive cells ( middle right , arrows ) and was absent in EYFP ‐ negative cells ( middle right , arrowhead ) ; and p53 was present in EYFP ‐ positive cells ( bottom right , arrows ) and was absent in EYFP ‐ negative cells ( bottom right , arrowhead ) . Scale bar : top panel , 100 μm ; middle and bottom panel , 50 μm . n = 5 . qRT - PCR analysis of mRNA levels of Shp2 , Skp2 , Aurka , Dll1 , and Hey1 in EYFP + and EYFP − cells isolated by FACS from MLN4924 ; MMTV ‐ Cre ; Shp2fl / fl ; EYFP − / fltumors . Error bars represent SEM ( n = 5 ) . Kaplan - Meier analysis of tumor formation in control ( blue , n = 8 ) and GS493 ‐ pretreated ( red , n = 8 ) MLN4924 ; MMTV ‐ Cre ; Shp2fl / flmice . The bar indicates the duration of treatment . Tumorgrowth curves for control ( blue , n = 5 ) and GS493 ‐ treated ( red , n = 5 ) MLN4924 mice . Note that after day 24 , all mice in the control group were sacrificed due to the tumor size . Error bars represent SEM ( n = 5 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 Immunohistochemistry analysis of EYFP (top) and SA‐β‐gal staining (bottom) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Note that SA‐β‐gal activity was present in the EYFP‐positive (Shp2‐negative) areas (right, arrows) and was absent in the EYFP‐negative (Shp2‐positive) areas (right, arrowhead) of mammary glandtumors of MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. Consecutive sections of EYFP and SA‐β‐gal staining for each group are shown. Scale bar: 100 μm. n = 5.  Immunofluorescence analysis of Ki67, EYFP and CK8 (pan‐epithelial marker) (top panel), p27 and EYFP (middle panel), and p53 and EYFP (bottom panel) on cryosections of mammary glandtumors from control MLN4924;Shp2fl/fl;EYFP−/fl, MLN4924;MMTV‐Cre;Shp2+/fl;EYFP−/fl, and MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/flmice. DAPI was used for nuclear counterstain. Ki67 was absent in EYFP‐positive cells (top right, arrows) and was present in EYFP‐negative cells (top right, arrowheads); p27 was present in EYFP‐positive cells (middle right, arrows) and was absent in EYFP‐negative cells (middle right, arrowhead); and p53 was present in EYFP‐positive cells (bottom right, arrows) and was absent in EYFP‐negative cells (bottom right, arrowhead). Scale bar: top panel, 100 μm; middle and bottom panel, 50 μm. n = 5.  qRT-PCR analysis of mRNA levels of Shp2, Skp2, Aurka, Dll1, and Hey1 in EYFP+ and EYFP−cells isolated by FACS from MLN4924;MMTV‐Cre;Shp2fl/fl;EYFP−/fltumors. Error bars represent SEM (n = 5).  Kaplan-Meier analysis of tumor formation in control (blue, n = 8) and GS493‐pretreated (red, n = 8) MLN4924;MMTV‐Cre;Shp2fl/flmice. The bar indicates the duration of treatment.  Tumorgrowth curves for control (blue, n = 5) and GS493‐treated (red, n = 5) MLN4924 mice. Note that after day 24, all mice in the control group were sacrificed due to the tumor size. Error bars represent SEM (n = 5).


Figure 1mRNA expression of individual patients normalized to 18S - rRNA and relative to a pooled organ donor sample . Lines between dots connect samples from the same patient . CP : chronic pancreatitis ; PDAC : pancreatic ductal adenocarcinoma ; significant difference ( P < 0 . 05 ) between tumor and normal tissue P = 9 . 39 × 10 − 5 determined by paired two - tailed Student ' s t - test , n = 17 patients . Association of microarray - based TBL1 expression and survival in 105 patients with PDAC : High TBL1 expression is significantly associated with shorter post - resection survival . P - value determined by log - rank test as described in Materials and Methods . Immunohistochemistrystaining for TBL1 in human paraffin - embedded tissue sections . 1 , 2 : healthy tissue with acini and duct showing no or only very weak staining . 3 , 4 : PanIN - 1 lesions with occasional nuclear and cytoplasmicstaining . 5 , 6 : PanIN - 2 lesion with nuclear and occasional cytoplasmicstaining . 7 - 10 : PanIN - 3 lesion with nuclear and increased occurrence and intensity of cytoplasmaticstaining . 11 , 12 : invasive pancreatic ductal adenocarcinoma cells with nuclear and cytoplasmicstaining . Scale bars : 100 μm . Images are representative of 12 patient samples . Immunohistochemistrystaining for TBL1 in paraffin - embedded tissue sections of wild - type or p48 + / Cre ; Kras + / LSL - G12Dmice . 1 , 2 : healthy tissue with acini , islet of Langerhans , and duct ( zoom - in in panel 2 ) showing occasional weak nuclearstaining . 3 , 4 : PanIN - 1 lesions with nuclearstaining . 5 , 6 : PanIN - 2 lesions with nuclearstaining . 7 , 8 : PanIN - 3 lesion with nuclearstaining . 9 - 12 : invasive pancreatic ductal adenocarcinoma cells with nuclear and occasional faint cytoplasmicstaining . Scale bars : 100 μm . Images are representative of 32 wild - type and 29 p48 + / Cre ; Kras + / LSL - G12Dmice .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1mRNA expression of individual patients normalized to 18S-rRNA and relative to a pooled organ donor sample. Lines between dots connect samples from the same patient. CP: chronic pancreatitis; PDAC: pancreatic ductal adenocarcinoma; significant difference (P < 0.05) between tumor and normal tissue P = 9.39 × 10−5 determined by paired two-tailed Student's t-test, n = 17 patients.Association of microarray-based TBL1 expression and survival in 105 patients with PDAC: High TBL1 expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.Immunohistochemistrystaining for TBL1 in human paraffin-embedded tissue sections. 1, 2: healthy tissue with acini and duct showing no or only very weak staining. 3, 4: PanIN-1 lesions with occasional nuclear and cytoplasmicstaining. 5, 6: PanIN-2 lesion with nuclear and occasional cytoplasmicstaining. 7-10: PanIN-3 lesion with nuclear and increased occurrence and intensity of cytoplasmaticstaining. 11, 12: invasive pancreatic ductal adenocarcinoma cells with nuclear and cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 12 patient samples.Immunohistochemistrystaining for TBL1 in paraffin-embedded tissue sections of wild-type or p48+/Cre; Kras+/LSL-G12Dmice. 1, 2: healthy tissue with acini, islet of Langerhans, and duct (zoom-in in panel 2) showing occasional weak nuclearstaining. 3, 4: PanIN-1 lesions with nuclearstaining. 5, 6: PanIN-2 lesions with nuclearstaining. 7, 8: PanIN-3 lesion with nuclearstaining. 9-12: invasive pancreatic ductal adenocarcinoma cells with nuclear and occasional faint cytoplasmicstaining. Scale bars: 100 μm. Images are representative of 32 wild-type and 29 p48+/Cre; Kras+/LSL-G12Dmice.


Figure 2Left panel : EdU incorporation in siRNA - transfected Capan - 1 cells ( 72 h post - transfection ) , n = 6 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : siTBL1 # 1 8 . 24 × 10 − 4 ( * ) , 5 . 43 × 10 − 4 ( # ) , 7 . 93 × 10 − 6 ( $ ) ; siTBL1 # 2 1 . 66 × 10 − 11 ( * ) , 1 . 27 × 10 − 11 ( # ) , 8 . 79 × 10 − 13 ( $ ) , one - way ANOVA with Bonferroni post - test . Right panel : EdU incorporation of Panc02 cells with stable expression of shRNA ; n = 6 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( shNC ) # P = 0 . 0398 ; Welch ' s t - test . Cellgrowth of siRNA - transfected Capan - 1 or Panc8680 cells ; n = 3 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 $ ; P - values : Capan - 1 cells 48 h time - point : 0 . 0408 ( * ) for siTBL1 # 1 / 0 . 0464 for siTBL1 # 2 , Capan - 1 cells 72 h time - point : 8 . 60 × 10 − 5 ( * ) , 1 . 82 × 10 − 3 ( # ) , 3 . 24 × 10 − 5 ( $ ) for siTBL1 # 1 / 2 . 39 × 10 − 10 ( * ) , 3 . 34 × 10 − 9 ( # ) , 1 . 08 × 10 − 10 ( $ ) for siTBL1 # 2 ; Panc8680 72 h time - point : 1 . 55 × 10 − 4 ( * ) , 2 . 73 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - test . Uptake of radio - labeled 3H - 2 - deoxyglucose in siRNA - transfected Capan - 1 cells ( 48 h post - transfection ) ; n = 6 cell culture wells per group ; statistically different ( P < 0 . 05 ) from control ( siNC # 2 ) $ P = 0 . 03 ; Welch ' s t - test . Extracellular flux measurement of siRNA - transfected Capan - 1 cells with glycolysis stress test assay ( 48 h post - transfection ) ; 2 - DG : 2 - deoxyglucose ; ECAR : extracellular acidification rate . n = 6 - 8 cell culture wells per group ; significantly different ( P < 0 . 05 ) from control ( no siRNA ) * , siNC # 1 # , siNC # 2 ) $ . Upper symbols : statistics for siTBL # 1 , P - values : 2 . 18 × 10 − 3 ( * ) time - point 8 , 9 . 83 × 10 − 3 ( * ) time - point 9 , 0 . 0102 ( * ) time - point 10 , 0 . 044 ( * ) time - point 11 ; 2 . 42 × 10 − 6 ( # ) time - point 8 , 1 . 47 × 10 − 4 ( # ) time - point 9 , 3 . 17 × 10 − 4 ( # ) time - point 10 , 0 . 0277 ( # ) time - point 11 ; 9 . 44 × 10 − 7 ( $ ) time - point 8 , 1 . 23 × 10 − 6 ( $ ) time - point 9 , 1 . 33 × 10 − 6 ( $ ) time - point 10 , 7 . 14 × 10 − 5 ( $ ) time - point 11 . Lower symbols : statistics for siTBL # 2 , P - values : 3 . 94 × 10 − 3 ( * ) time - point 5 , 0 . 0143 ( * ) time - point 6 , 0 . 0342 ( * ) time - point 7 , 1 . 45 × 10 − 3 ( * ) time - point 8 , 1 . 27 × 10 − 3 ( * ) time - point 9 , 2 . 38 × 10 − 3 ( * ) time - point 10 ; 0 . 01 ( # ) time - point 5 , 0 . 0362 ( # ) time - point 6 , 1 . 22 × 10 − 6 ( # ) time - point 8 , 1 . 28 × 10 − 5 ( # ) time - point 9 , 5 . 84 × 10 − 5 ( # ) time - point 10 ; 0 . 0118 ( $ ) time - point 5 , 0 . 0208 ( $ ) time - point 6 , 0 . 0291 ( $ ) time - point 7 , 4 . 52 × 10 − 7 ( $ ) time - point 8 , 6 . 04 × 10 − 8 ( $ ) time - point 9 , 1 . 43 × 10 − 7 ( $ ) time - point 10 , 4 . 83 × 10 − 3 ( $ ) time - point 11 . Two - way ANOVA with Bonferroni post - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Left panel: EdU incorporation in siRNA-transfected Capan-1 cells (72 h post-transfection), n = 6 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: siTBL1#1 8.24 × 10−4 (*), 5.43 × 10−4 (#), 7.93 × 10−6 ($); siTBL1 #2 1.66 × 10−11 (*), 1.27 × 10−11 (#), 8.79 × 10−13 ($), one-way ANOVA with Bonferroni post-test. Right panel: EdU incorporation of Panc02 cells with stable expression of shRNA; n = 6 cell culture wells per group; significantly different (P < 0.05) from control (shNC) #P = 0.0398; Welch's t-test.Cellgrowth of siRNA-transfected Capan-1 or Panc8680 cells; n = 3 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2 $; P-values: Capan-1 cells 48 h time-point: 0.0408 (*) for siTBL1 #1 / 0.0464 for siTBL1 #2, Capan-1 cells 72 h time-point: 8.60 × 10−5 (*), 1.82 × 10−3 (#), 3.24 × 10−5 ($) for siTBL1 #1 / 2.39 × 10−10 (*), 3.34 × 10−9 (#), 1.08 × 10−10 ($) for siTBL1 #2; Panc8680 72 h time-point: 1.55 × 10−4 (*), 2.73 × 10−5 ($); two-way ANOVA with Bonferroni post-test.Uptake of radio-labeled 3H-2-deoxyglucose in siRNA-transfected Capan-1 cells (48 h post-transfection); n = 6 cell culture wells per group; statistically different (P < 0.05) from control (siNC #2) $P = 0.03; Welch's t-test.Extracellular flux measurement of siRNA-transfected Capan-1 cells with glycolysis stress test assay (48 h post-transfection); 2-DG: 2-deoxyglucose; ECAR: extracellular acidification rate. n = 6-8 cell culture wells per group; significantly different (P < 0.05) from control (no siRNA) *, siNC #1 #, siNC #2) $. Upper symbols: statistics for siTBL#1, P-values: 2.18 × 10−3 (*) time-point 8, 9.83 × 10−3 (*) time-point 9, 0.0102 (*) time-point 10, 0.044 (*) time-point 11; 2.42 × 10−6 (#) time-point 8, 1.47 × 10−4 (#) time-point 9, 3.17 × 10−4 (#) time-point 10, 0.0277 (#) time-point 11; 9.44 × 10−7 ($) time-point 8, 1.23 × 10−6 ($) time-point 9, 1.33 × 10−6 ($) time-point 10, 7.14 × 10−5 ($) time-point 11. Lower symbols: statistics for siTBL#2, P-values: 3.94 × 10−3 (*) time-point 5, 0.0143 (*) time-point 6, 0.0342 (*) time-point 7, 1.45 × 10−3 (*) time-point 8, 1.27 × 10−3 (*) time-point 9, 2.38 × 10−3 (*) time-point 10; 0.01 (#) time-point 5, 0.0362 (#) time-point 6, 1.22 × 10−6 (#) time-point 8, 1.28 × 10−5 (#) time-point 9, 5.84 × 10−5 (#) time-point 10; 0.0118 ($) time-point 5, 0.0208 ($) time-point 6, 0.0291 ($) time-point 7, 4.52 × 10−7 ($) time-point 8, 6.04 × 10−8 ($) time-point 9, 1.43 × 10−7 ($) time-point 10, 4.83 × 10−3 ($) time-point 11. Two-way ANOVA with Bonferroni post-test.


Figure 3Gene expression microarray from Capan - 1 cells transfected with siRNA . Heatmap shows genes annotated to KEGG pathway hsa04110 " Cell Cycle " and / or gene ontology cluster GO : 0007049 " Cell Cycle " . Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice . Chromatin immunoprecipitation from Capan - 1 cells with or without siRNA - mediated knockdown of TBL1 . IgG and primers for PIK3CA coding region served as a negative control ; histone H3 served as a positive control . Proliferation time course assay in control and TBL1 shRNA - transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K . Cells were stained with crystal violet , and absorbance was measured at 595 nm . n = 9 cell culture wells per group ; statistically significant at P < 0 . 05 for indicated comparisons : shNC pBabe - puro vs . shNC pBabe - PI3K ( * ) , shTBL1 pBabe - puro vs . sbTBL1 pBabe - PI3K ( # ) , shNC pBabe - puro vs . shTBL1 pBabe - puro ( $ ) , shNC pBabe - PI3K vs . shTBL1 pBabe - PI3K ( † ) ; P - values : day 2 : 2 . 01 × 10 − 3 ( # ) , 4 . 11 × 10 − 9 ( $ ) , 5 . 77 × 10 − 3 ( † ) ; day 3 : 4 . 50 × 10 − 5 ( * ) , 1 . 01 × 10 − 9 ( # ) , 1 . 71 × 10 − 30 ( $ ) , 1 . 22 × 10 − 6 ( † ) ; day 4 : 5 . 61 × 10 − 22 ( * ) , 1 . 10 × 10 − 45 ( # ) , 6 . 65 × 10 − 62 ( $ ) ; two - way ANOVA with Bonferroni post - test . Matrigel invasion assay in same cells as in ( D ) . Invading cells per microscopy field were counted . n = 6 microscopy fields per group ; statistically significant at P < 0 . 05 ; P - values : 6 . 64 × 10 − 4 ( * ) , 1 . 35 × 10 − 7 ( # ) , 6 . 94 × 10 − 5 ( $ ) ; two - way ANOVA with Bonferroni post - test . mRNA expression of PIK3CA in human patient samples ; significant difference ( P < 0 . 05 ) between tumor and normal tissue * P = 2 . 13 × 10 − 4 determined by paired two - tailed Student ' s t - test , n = 18 patients . Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue ( normalized to 18S rRNA and relative to a pooled organ donor sample ) ; n = 17 patients ; P - value determined by testing for slope of regression line ≠ 0 . Survival analysis in 105 patients with PDAC : High PIK3CA expression is significantly associated with shorter post - resection survival . P - value determined by log - rank test as described in Materials and Methods .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Gene expression microarray from Capan-1 cells transfected with siRNA. Heatmap shows genes annotated to KEGG pathway hsa04110 "Cell Cycle" and/or gene ontology cluster GO:0007049 "Cell Cycle".Protein expression of Panc02 cells with stable shRNA expression prior to implantation into mice.Chromatin immunoprecipitation from Capan-1 cells with or without siRNA-mediated knockdown of TBL1. IgG and primers for PIK3CA coding region served as a negative control; histone H3 served as a positive control.Proliferation time course assay in control and TBL1 shRNA-transfected Panc02 cells overexpressing constitutively active PI3K p100α mutant E545K. Cells were stained with crystal violet, and absorbance was measured at 595 nm. n = 9 cell culture wells per group; statistically significant at P < 0.05 for indicated comparisons: shNC pBabe-puro vs. shNC pBabe-PI3K (*), shTBL1 pBabe-puro vs. sbTBL1 pBabe-PI3K (#), shNC pBabe-puro vs. shTBL1 pBabe-puro ($), shNC pBabe-PI3K vs. shTBL1 pBabe-PI3K (†); P-values: day 2: 2.01 × 10−3 (#), 4.11 × 10−9 ($), 5.77 × 10−3 (†); day 3: 4.50 × 10−5 (*), 1.01 × 10−9 (#), 1.71 × 10−30 ($), 1.22 × 10−6 (†); day 4: 5.61 × 10−22 (*), 1.10 × 10−45 (#), 6.65 × 10−62 ($); two-way ANOVA with Bonferroni post-test.Matrigel invasion assay in same cells as in (D). Invading cells per microscopy field were counted. n = 6 microscopy fields per group; statistically significant at P < 0.05; P-values: 6.64 × 10−4 (*), 1.35 × 10−7 (#), 6.94 × 10−5 ($); two-way ANOVA with Bonferroni post-test.mRNA expression of PIK3CA in human patient samples; significant difference (P < 0.05) between tumor and normal tissue *P = 2.13 × 10−4 determined by paired two-tailed Student's t-test, n = 18 patients.Correlation of mRNA expression of TBL1 and PIK3CA in human patient tumor tissue (normalized to 18S rRNA and relative to a pooled organ donor sample); n = 17 patients; P-value determined by testing for slope of regression line ≠ 0.Survival analysis in 105 patients with PDAC: High PIK3CA expression is significantly associated with shorter post-resection survival. P-value determined by log-rank test as described in Materials and Methods.


Figure 4A - D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6 / N mice . Six days later , tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA . ( A - C ) : Data plotted as mean ± SEM ; n = 4 animals in control ( shNC ) group and n = 3 animals in knockdown ( shTBL1 ) group ; significantly different ( P < 0 . 05 ) between control ( shNC ) and knockdown ( shTBL1 ) * . ( A ) Tumor volume determined by transcutaneous measurement with digital calipers ; P - values : 9 . 21 × 10 − 3 at day 15 , 6 . 02 × 10 − 4 at day 18 , 2 . 49 × 10 − 4 at day 20 ; two - way ANOVA with Bonferroni post - test . ( B ) Luminescence of tumors after intraperitoneal injection of firefly d - luciferin ; P = 0 . 0133 at day 20 ; two - way ANOVA with Bonferroni post - test . ( C ) Tumor mass at necropsy ; P = 0 . 0299 , one - sided Welch ' s t - test . ( D ) Luminescence images of one representative mouse in each group . E - G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection ; n = 4 animals in shNC + GEM group , n = 5 animals in all other groups ; significantly different ( P < 0 . 05 ) between groups : shNC vs . shNC + GEM ( * ) , shNC vs . shTBL1 ( # ) , shNC vs . shTBL1 + GEM ( $ ) , shNC + GEM vs . shTBL1 ( % ) , shNC + GEM vs . shTBL + GEM ( † ) ; shTBL1 vs . shTBL1 + GEM ( ‡ ) ; two - way ANOVA with Bonferroni post - test . ( E ) Tumor volume determined by transcutaneous measurement with digital calipers . P - values : day 17 : 8 . 48 × 10 − 3 ( # ) , 1 . 07 × 10 − 3 ( $ ) , day 20 : 3 . 07 × 10 − 3 ( * ) , 9 . 37 × 10 − 6 ( # ) , 1 . 56 × 10 − 8 ( $ ) , 0 . 0239 ( † ) . ( F ) Tumor volume relative to the volume at day 7 post - implantation ; P - values : day 17 : 3 . 94 × 10 − 3 ( $ ) , day 20 : 1 . 09 × 10 − 3 ( * ) , 1 . 39 × 10 − 7 ( $ ) , 0 . 01195 ( % ) , 3 . 12 × 10 − 6 ( ‡ ) . ( G ) Area under curve for relative tumor volume ; P = 0 . 0195 ( ‡ ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-D Panc02 cells with stable expression of luciferase were implanted subcutaneously into C57Bl6/N mice. Six days later, tumors were injected three times per week with 108 ifu of adenovirus encoding shRNA. (A-C): Data plotted as mean ± SEM; n = 4 animals in control (shNC) group and n = 3 animals in knockdown (shTBL1) group; significantly different (P < 0.05) between control (shNC) and knockdown (shTBL1) *. (A) Tumor volume determined by transcutaneous measurement with digital calipers; P-values: 9.21 × 10−3 at day 15, 6.02 × 10−4 at day 18, 2.49 × 10−4 at day 20; two-way ANOVA with Bonferroni post-test. (B) Luminescence of tumors after intraperitoneal injection of firefly d-luciferin; P = 0.0133 at day 20; two-way ANOVA with Bonferroni post-test. (C) Tumor mass at necropsy; P = 0.0299, one-sided Welch's t-test. (D) Luminescence images of one representative mouse in each group.E-G Panc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection; n = 4 animals in shNC+GEM group, n = 5 animals in all other groups; significantly different (P < 0.05) between groups: shNC vs. shNC+GEM (*), shNC vs. shTBL1 (#), shNC vs. shTBL1+GEM ($), shNC+GEM vs. shTBL1 (%), shNC+GEM vs. shTBL+GEM (†); shTBL1 vs. shTBL1+GEM (‡); two-way ANOVA with Bonferroni post-test. (E) Tumor volume determined by transcutaneous measurement with digital calipers. P-values: day 17: 8.48 × 10−3 (#), 1.07 × 10−3 ($), day 20: 3.07 × 10−3 (*), 9.37 × 10−6 (#), 1.56 × 10−8 ($), 0.0239 (†). (F) Tumor volume relative to the volume at day 7 post-implantation; P-values: day 17: 3.94 × 10−3 ($), day 20: 1.09 × 10−3 (*), 1.39 × 10−7 ($), 0.01195 (%), 3.12 × 10−6 (‡). (G) Area under curve for relative tumor volume; P = 0.0195 (‡).


Figure 5TBL1 - depletion leads to reduction in PI3 kinase p110α and cell cycle - associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6 / Nmice . Seven days later , mice were treated with 20 mg / kg gemcitabine delivered by intraperitoneal injection . After 21 days , mice were sacrificed , tumors were removed and proteins were extracted and immunoblotted .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5TBL1-depletion leads to reduction in PI3 kinase p110α and cell cycle-associated proteinsPanc02 cells with stable expression of shRNA were implanted subcutaneously into C57Bl6/Nmice. Seven days later, mice were treated with 20 mg/kg gemcitabine delivered by intraperitoneal injection. After 21 days, mice were sacrificed, tumors were removed and proteins were extracted and immunoblotted.


Figure 2Mabs - laden wet - spun yarns showing progressive myogenic differentiation over culturing time . As can be noted , Mabs morphology evolves from a round shape ( single cells at early time points ) into a more elongated one ( generally within the first 3 - 5 days of culture ) , eventually forming long - range myotubes . For the sake of clarity , samples used for bright field investigation were prepared out of few layers of fibers to favor the recognition of cell organization .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Mabs-laden wet-spun yarns showing progressive myogenic differentiation over culturing time. As can be noted, Mabs morphology evolves from a round shape (single cells at early time points) into a more elongated one (generally within the first 3-5 days of culture), eventually forming long-range myotubes. For the sake of clarity, samples used for bright field investigation were prepared out of few layers of fibers to favor the recognition of cell organization.


Figure 4A ) Immunocompromised mouse leg showing ablated TA ( control ) and implanted TA ( graft ) at day 0 ; inset showing muscle mass removed next to the implanted Mabs loaded constructs shaped to fill the TA lodge . B ) Implanted myo - substitute 20 days after grafting . D ) Graphical representation of the scored TA weight ( n = 6 ) . The dashed area indicates the range of native TA mass depending primarily on mouse weight and age , statistical significance was analyzed by Anova test ( P < 0 . 05 was considered significant : * = 0 , 0306 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A) Immunocompromised mouse leg showing ablated TA (control) and implanted TA (graft) at day 0; inset showing muscle mass removed next to the implanted Mabs loaded constructs shaped to fill the TA lodge. B) Implanted myo-substitute 20 days after grafting.D) Graphical representation of the scored TA weight (n=6). The dashed area indicates the range of native TA mass depending primarily on mouse weight and age, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0306).


Figure 5Cross and longitudinal sections of ablated ( control ) and implanted ( graft ) TA were marked with LacZ and immunoassayed for MHC ( red ) , LAM ( green ) . Cross - and longitudinal sections were immunostained against MHC and LAM and overlaid to LacZ labeled images . Dashed area indicates the enlarged view displaying sarcomeres ( asterisks ) in combination with LacZ positive nuclei ( arrowheads ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Cross and longitudinal sections of ablated (control) and implanted (graft) TA were marked with LacZ and immunoassayed for MHC (red), LAM (green). Cross- and longitudinal sections were immunostained against MHC and LAM and overlaid to LacZ labeled images. Dashed area indicates the enlarged view displaying sarcomeres (asterisks) in combination with LacZ positive nuclei (arrowheads).


Figure 6D , E ) Vessel density assessment by means of fluorescence , statistical significance was analyzed by Anova test ( P < 0 . 05 was considered significant : * = 0 , 0019 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6D, E) Vessel density assessment by means of fluorescence, statistical significance was analyzed by Anova test (P < 0.05 was considered significant: *=0,0019).


Figure 7A ) Immunofluorescence against NeuF ( green ) and MHC ( red ) revealing reconstructed muscle innervation ( asterisks ) . B ) Superimposed image in ( a ) over LacZ stained image . C ) Syn and BTX immunolabelled sections showing neuromuscular junction formation ( arrowheads ) , inset enlarged view of dashed box .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O

Figure 7A) Immunofluorescence against NeuF (green) and MHC (red) revealing reconstructed muscle innervation (asterisks). B) Superimposed image in (a) over LacZ stained image.C) Syn and BTX immunolabelled sections showing neuromuscular junction formation (arrowheads), inset enlarged view of dashed box.


Figure 8Wet - spun yarn containing human myogenic precursors showed progressive cell differentiation over time ( arrows in day 3 ) ; dashed box enlargement reveals still undifferentiated cells after 15 days of culture ( Upper panel ) . Middle and lower panel showing MHC immunostaining ( red ) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture , revealing a significant difference in terms of myotube organization between the two hydrogel systems , with the wet - spun constructs greatly outperforming the bulk gels . Such difference is quantified in the polar plots . Nuclei were counterstained by DAPI ( Blue ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8Wet-spun yarn containing human myogenic precursors showed progressive cell differentiation over time (arrows in day 3); dashed box enlargement reveals still undifferentiated cells after 15 days of culture (Upper panel). Middle and lower panel showing MHC immunostaining (red) performed on microfiber yarns and bulk structure loaded with human myoblasts upon 15 days of culture, revealing a significant difference in terms of myotube organization between the two hydrogel systems, with the wet-spun constructs greatly outperforming the bulk gels. Such difference is quantified in the polar plots. Nuclei were counterstained by DAPI (Blue).


Figure 1 ( B ) Detection of soluble , cleaved NrCAM ( sNrCAM ) and full - length , mature NrCAM ( mNrCAM ) and soluble APPα ( sAPPα ) in neuronal supernatants and lysates ( prepared from E16 neurons ) , treated with GI254023x ( 5 µM ) , or solvent for 48h . Densitometric quantifications of the Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - test n = 6 - 8 ) . ( C ) Detection of sAPPα , sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro ( DIV7 ) . The neurons prepared from floxed ADAM10 ( ADAM10 fl / fl ) mice were infected with a lentivirus encoding improved Cre recombinase ( iCre ) or a control GFP lentivirus at DIV2 . Conditioned media were collected for 48 h . Densitometric quantifications of the Western Blots are shown ( * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - test n = 6 - 8 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Detection of soluble, cleaved NrCAM (sNrCAM) and full-length, mature NrCAM (mNrCAM) and soluble APPα (sAPPα) in neuronal supernatants and lysates (prepared from E16 neurons), treated with GI254023x (5 µM), or solvent for 48h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).(C) Detection of sAPPα, sNrCAM and mNrCAM in ADAM10 cKO neuronal supernatants and lysates at 7 days in vitro (DIV7). The neurons prepared from floxed ADAM10 (ADAM10 fl/fl) mice were infected with a lentivirus encoding improved Cre recombinase (iCre) or a control GFP lentivirus at DIV2. Conditioned media were collected for 48 h. Densitometric quantifications of the Western Blots are shown (** p < 0.01; *** p < 0.001; **** p < 0.0001, two-sided students t-test n = 6-8).


Figure 2HEK 293 cells were transfected with a C - terminally VSV - tagged NrCAM construct or empty vector . Cells were then treated with DAPT ( 1 µM ) , GI254023x ( 5 µM ) , either substances or solvent for 24 h . The C - terminal NrCAM - fragment was detected with an antibody against the VSV - tag . Representative Western Blots are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2HEK 293 cells were transfected with a C-terminally VSV-tagged NrCAM construct or empty vector. Cells were then treated with DAPT (1 µM), GI254023x (5 µM), either substances or solvent for 24 h. The C-terminal NrCAM-fragment was detected with an antibody against the VSV-tag. Representative Western Blots are shown.


Figure 3 ( A ) ADAM10 fl / fl neurons were treated with an iCre ( to induce an ADAM10 KO ) , or a control lentivirus at DIV2 . At DIV7 , cell surface proteins were labeled with biotin and enriched by streptavidin pull - down . The biotinylated proteins were detected by immunoblotting . Total lysates were analyzed to compare mNrCAM ' s surface / lysate levels . Densitometric quantifications of the Western Blots are shown . Two - sided students t - test ( * * * * p < 0 . 0001 , n = 7 ) . Given are mean + / - the standard error of them mean . The mean levels of solvent treated cells were set to 1 . ( B ) Primary murine neurons were treated with GI254023x ( 5 µM ) or solvent , for 48h . The cell surface proteins were enriched like in A . Densitometric quantifications of the Western Blots are shown . Two - sided students t - test ( * * * * p < 0 . 0001 , n = 11 ) . Given are mean + / - the standard error of them mean . The mean levels of solvent treated cells were set to 1 . Representative Western Blots are shown . ( C ) Workflow of the neurite outgrowth assay . ( D ) Representative pictures of single neurites at time - point 0h ( DIV3 ) and 24h ( DIV4 ) . Neurons were infected 4h after plating with lentiviruses encoding GFP ( to visualize the neurons ) together with shRNA expression cassettes ( sh1 and sh2 ) targeting either NrCAM or carrying a scrambled ( scr ) control construct . Images of neurites were taken at three days in vitro ( DIV3 ) and 24h later at DIV4 . In order to study the effect of ADAM10 on neurite outgrowth , neurons were treated with the ADAM10 inhibitor GI254023x , or vehicle ( control ) , at DIV3 , after taking the first pictures with an epifluorescent microscope . The differences in neurite length were calculated as absolute values ( neurite length at 24h minus neurite length of 0h ) for individual neurites passing through the middle channels of the chambers . Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered . The red arrows indicate the start and the end of the respective length measurements . The scale bar indicates 40 µm . ( E ) One - way ANOVA with post hoc Dunnett ' s test . Given are mean + / - the standard error of them mean ( * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 , n - numbers for each condition are shown in the graph in 3E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) ADAM10 fl/fl neurons were treated with an iCre (to induce an ADAM10 KO), or a control lentivirus at DIV2. At DIV7, cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAM's surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1.(B) Primary murine neurons were treated with GI254023x (5 µM) or solvent, for 48h. The cell surface proteins were enriched like in A. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (**** p < 0.0001, n = 11). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.(C) Workflow of the neurite outgrowth assay. (D) Representative pictures of single neurites at time-point 0h (DIV3) and 24h (DIV4). Neurons were infected 4h after plating with lentiviruses encoding GFP (to visualize the neurons) together with shRNA expression cassettes (sh1 and sh2) targeting either NrCAM or carrying a scrambled (scr) control construct. Images of neurites were taken at three days in vitro (DIV3) and 24h later at DIV4. In order to study the effect of ADAM10 on neurite outgrowth, neurons were treated with the ADAM10 inhibitor GI254023x, or vehicle (control), at DIV3, after taking the first pictures with an epifluorescent microscope. The differences in neurite length were calculated as absolute values (neurite length at 24h minus neurite length of 0h) for individual neurites passing through the middle channels of the chambers. Only neurites that had already entered the main channel at 0h and had not yet left those channels at 0h were considered. The red arrows indicate the start and the end of the respective length measurements. The scale bar indicates 40 µm. (E) One-way ANOVA with post hoc Dunnett's test. Given are mean +/- the standard error of them mean (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001, n-numbers for each condition are shown in the graph in 3E).


Figure 4 ( A ) Wt neurons were kept in culture until DIV5 , then the cells were treated with acitretin ( 4 µM ) or vehicle control for 48h , as described earlier ( Tippmann et al , 2009 ) . ( B ) Densitometric quantifications of the Western Blots are shown . Given are mean + / - the standard error of the mean ( * * * p < 0 . 001 ; * * * * p < 0 . 0001 , two - sided students t - test n = 9 - 20 ) . The mean levels of solvent treated cells were set to 1 . ( C ) Detection of sNrCAM and human serum albumin ( hSA ) in AD patients CSF that had been treated with acitretin ( 30 mg / d ) or placebo for 30 days ( n = 9 ) ( Endres et al , 2014 ) . ( D ) Densitometric quantification of the Western Blots in ( C ) . sNrCAM / hSA ratios were calculated , and then the treatment to baseline ratios ( t / b ) for every patient were calculated . Two - sided student ' s t - test ( n = 9 ) . Given are mean + / - the standard error of them mean . Representative Western Blots are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Wt neurons were kept in culture until DIV5, then the cells were treated with acitretin (4 µM) or vehicle control for 48h, as described earlier (Tippmann et al, 2009). (B) Densitometric quantifications of the Western Blots are shown. Given are mean +/- the standard error of the mean (*** p < 0.001; **** p < 0. 0001, two-sided students t-test n = 9-20). The mean levels of solvent treated cells were set to 1.(C) Detection of sNrCAM and human serum albumin (hSA) in AD patients CSF that had been treated with acitretin (30 mg/d) or placebo for 30 days (n = 9) (Endres et al, 2014). (D) Densitometric quantification of the Western Blots in (C). sNrCAM/hSA ratios were calculated, and then the treatment to baseline ratios (t/b) for every patient were calculated. Two-sided student's t-test (n = 9). Given are mean +/- the standard error of them mean. Representative Western Blots are shown.


Figure 5 ( A ) To ensure proper synapse formation , primary neurons were kept in culture for ten days prior to the treatment ( Wan et al , 2012 ) . Then , cells were treated with NMDA ( 50 µM ) , NMDA ( 50 µM ) and GI254023x ( 5 µM ) , GI254023x ( 5 µM ) alone or vehicle for 30 minutes . Total cellular mADAM10 levels remained unchanged by the treatment , which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes ( Marcello et al , 2007 ) . Densitometric quantifications of the Western Blots are shown . One - way ANOVA with post hoc Dunnett ' s test ( * * * p < 0 . 001 , n = 7 ) . Given are mean + / - the standard error of them mean . The mean levels of solvent treated cells were set to 1 . The membrane was reprobed with the different indicated antibodies . ( B ) Wt neurons were kept in culture until DIV10 , then the cells were treated with NMDA ( 50 µM ) , or vehicle for 30 minutes . Cell surface proteins were labeled with biotin and enriched by streptavidin pull - down . The biotinylated proteins were detected by immunoblotting . Total lysates were analyzed to compare mNrCAMs surface / lysate levels . Densitometric quantifications of the Western Blots are shown . Two - sided students t - test ( * * p < 0 . 01 , n = 6 ) . Given are mean + / - the standard error of them mean . The mean levels of solvent treated cells were set to 1 . Representative Western Blots are shown .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) To ensure proper synapse formation, primary neurons were kept in culture for ten days prior to the treatment (Wan et al, 2012). Then, cells were treated with NMDA (50 µM), NMDA (50 µM) and GI254023x (5 µM), GI254023x (5 µM) alone or vehicle for 30 minutes. Total cellular mADAM10 levels remained unchanged by the treatment, which is in line with NMDA altering mainly the intracellular localization of ADAM10 by driving the protein to the synaptic membranes (Marcello et al, 2007). Densitometric quantifications of the Western Blots are shown. One-way ANOVA with post hoc Dunnett's test (*** p < 0.001, n = 7). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. The membrane was reprobed with the different indicated antibodies.(B) Wt neurons were kept in culture until DIV10, then the cells were treated with NMDA (50 µM), or vehicle for 30 minutes. Cell surface proteins were labeled with biotin and enriched by streptavidin pull-down. The biotinylated proteins were detected by immunoblotting. Total lysates were analyzed to compare mNrCAMs surface/lysate levels. Densitometric quantifications of the Western Blots are shown. Two-sided students t-test (** p < 0.01, n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


Figure 6Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days . Every circle represents one protein . The log10 transformed t - test p - values ( again treatment / baseline for every patient ) are plotted against the log2 transformed label - free quantification intensity ratios of acitretin and placebo CSF . ADAM10 substrates are indicated in bold writing . In total , more than 50 ADAM10 substrates were identified , but only the significantly altered substrates as well as NrCAM and APP are indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Volcano plot of the proteomic analysis of CSF from AD patients treated with acitretin or placebo for 30 days. Every circle represents one protein. The log10 transformed t-test p-values (again treatment/baseline for every patient) are plotted against the log2 transformed label-free quantification intensity ratios of acitretin and placebo CSF. ADAM10 substrates are indicated in bold writing. In total, more than 50 ADAM10 substrates were identified, but only the significantly altered substrates as well as NrCAM and APP are indicated.


Figure 7HEK293 cells were transiently transfected with hNrCAM , FLAG - tagged hTSPAN15 , either plasmids or the empty vector . Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate . Densitometric quantification of the Western Blots are shown ( * * p < 0 . 01 ) , ( * * * p < 0 . 001 ) , ( * * * * p < 0 . 0001 ) , two - sided students t - test , or one - way ANOVA with post hoc Dunnett ' s test ( n = 6 ) . Given are mean + / - the standard error of them mean . The mean levels of solvent treated cells were set to 1 . Representative Western Blots are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7HEK293 cells were transiently transfected with hNrCAM, FLAG-tagged hTSPAN15, either plasmids or the empty vector. Detection of sAPPα and sNrCAM in the conditioned media and ADAM10 in the cell lysate. Densitometric quantification of the Western Blots are shown (** p < 0.01), (*** p < 0.001), (**** p < 0.0001), two-sided students t-test, or one-way ANOVA with post hoc Dunnett's test (n = 6). Given are mean +/- the standard error of them mean. The mean levels of solvent treated cells were set to 1. Representative Western Blots are shown.


Figure 1 ( b ) Kinematics of the embryo surface . A kymograph of the embryo surface ( φ axis ) from a Tg ( β - actin : m - GFP ) embryo shows the continuous displacement of the EVL / yolk margin , which accelerates after 50 % epiboly , while the yolk cell membrane does not show any shift ( see Movie EV1 ) . ( c ) Theoretical kymograph from 50 % to 70 % epiboly superimposed , between the red lines , on the experimental kymograph ( b ) . Membrane signal intensity is color - coded ( blue to red ) . White arrowheads point to flow lines ( black ) . ( d ) AV velocities , normalized to EVL / yolk margin velocity , were measured by PIV of time - lapse recordings of the surface of lectin - soaked embryos . The plots correspond to three different epiboly stages ( 30 % , blue ; 60 % , red ; and 90 % , green ) . Blue and red arrows point to the width of the E - YSL at 30 and 60 % epiboly respectively ( from Appendix Figure S1 ) . ( e ) The yolk cell membrane is highly convoluted ( white brackets ) ahead of the EVL leading cells and condenses over time . Tg ( β - actin : m - GFP ) embryo at 30 % ( left ) and 60 % ( right ) epiboly . Scale bar 25 μm . Inset shows a high magnification of the wrinkled area ( arrowhead ) . Scale bar 10 μm . ( f ) Actin accumulates within wrinkles at the E - YSL . The yolk cell membrane of LifeAct - GFP injected embryos ( actin - green ) was stained with lectin - TRITC ( red ) ( top ) . Orthogonal projection of the wrinkled area ( bottom ) . Wrinkles are outlined with a white dotted line . Scale bar 2 μm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(b) Kinematics of the embryo surface. A kymograph of the embryo surface (φ axis) from a Tg (β-actin:m-GFP) embryo shows the continuous displacement of the EVL / yolk margin, which accelerates after 50% epiboly, while the yolk cell membrane does not show any shift (see Movie EV1). (c) Theoretical kymograph from 50 % to 70 % epiboly superimposed, between the red lines, on the experimental kymograph(b). Membrane signal intensity is color-coded (blue to red). White arrowheads point to flow lines (black).(d) AV velocities, normalized to EVL / yolk margin velocity, were measured by PIV of time-lapse recordings of the surface of lectin-soaked embryos. The plots correspond to three different epiboly stages (30 %, blue; 60 %, red; and 90 %, green). Blue and red arrows point to the width of the E-YSL at 30 and 60 % epiboly respectively (from Appendix Figure S1).(e) The yolk cell membrane is highly convoluted (white brackets) ahead of the EVL leading cells and condenses over time. Tg (β-actin:m-GFP) embryo at 30 % (left) and 60 % (right) epiboly. Scale bar 25 μm. Inset shows a high magnification of the wrinkled area (arrowhead). Scale bar 10 μm.(f) Actin accumulates within wrinkles at the E-YSL. The yolk cell membrane of LifeAct-GFP injected embryos (actin - green) was stained with lectin-TRITC (red) (top). Orthogonal projection of the wrinkled area (bottom). Wrinkles are outlined with a white dotted line. Scale bar 2 μm.


Figure 2 ( a ) Yolk granules flow in stereotyped patterns as epiboly progresses . PIV of time - lapse snapshots imaged by two - photon microscopy of a Tg ( β - actin : m - GFP ) embryo ( from Movie EV2 ) . Upon epiboly onset , yolk granules flow towards the animal pole displaying two internal vortices near the E - YSL . At 50 % , the movement of the yolk granules halts , but resumes afterwards until the end of epiboly . Animal ( A ) and vegetal ( V ) poles are indicated . Scale bar 100 μm . ( b ) Internal yolk content speed ( μm / min ) at the center of the embryo , over time ( min ) . Inset : Spherical coordinates . The main axis of the sphere is represented by z , from the animal to the vegetal pole . The polar angle is represented by φ and the radial coordinate is denoted ρ . ( c ) Vortex midpoints distance from the center ( ρ ) , over time ( min ) . ( d ) Vortex midpoints position in relation to the φ axis , over time ( min ) . Black lines represent individual measurements . Red lines represent averages . ( e ) Correlation between vortex displacement ( blue , average of n = 3 embryos ) vs . EVL margin displacement ( red , average of n = 4 embryos ) along the main axis ( z ) , over time ( min ) . Standard Errors are displayed . Arrow points to 50 % epiboly .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2(a) Yolk granules flow in stereotyped patterns as epiboly progresses. PIV of time-lapse snapshots imaged by two-photon microscopy of a Tg (β-actin:m-GFP) embryo (from Movie EV2). Upon epiboly onset, yolk granules flow towards the animal pole displaying two internal vortices near the E-YSL. At 50 %, the movement of the yolk granules halts, but resumes afterwards until the end of epiboly. Animal (A) and vegetal (V) poles are indicated. Scale bar 100 μm. (b) Internal yolk content speed (μm/min) at the center of the embryo, over time (min). Inset: Spherical coordinates. The main axis of the sphere is represented by z, from the animal to the vegetal pole. The polar angle is represented by φ and the radial coordinate is denoted ρ. (c) Vortex midpoints distance from the center (ρ), over time (min). (d) Vortex midpoints position in relation to the φ axis, over time (min). Black lines represent individual measurements. Red lines represent averages. (e) Correlation between vortex displacement (blue, average of n=3 embryos) vs. EVL margin displacement (red, average of n = 4 embryos) along the main axis (z), over time (min). Standard Errors are displayed. Arrow points to 50 % epiboly.


Figure 4 ( a ) Yolk microrheology . Mean squared displacement ( MSD ) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos . Solid and dashed black lines are Mean ± Standard Error computed from 99 particles . Red line is the fit of the Stokes - Einstein equation . MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag , Δt , consistent with a Newtonian liquid behavior with a viscosity of 129 mPa · s . A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila ( He et al . , 2014 ) . Interestingly , the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C . elegansembryos ( Daniels et al . , 2006 ) and of the cytoplasm of several cell types ( Wirtz , 2009 ) . ( b ) Viscoelasticity of the yolk cortex . Effective complex modulus ( g * ) measured by indenting the surface of zebrafishembryos with AFM . Red symbols are the elastic modulus ( g ' ) and yellow symbols are the viscous modulus ( g ' ' ) , respectively . Standard Errors are displayed . In all the measured frequencies cortex rheology is dominated by a solid - like behavior with the viscous modulus 5 times lower that the elastic modulus . No significant frequency dependence was found in the elastic ( ANOVA , p = 0 . 123 ) and viscous modulus ( ANOVA , p = 0 . 719 ) . ( c ) Flow lines from snapshots of the PIV measurements ( at 40 , 60 and 80 % epiboly ) of a two - photon video time - lapse ( from Movie EV4 ) of a Tg ( β - actin : m - GFP ) embryo ( top ) . Fitted flow lines using HR ar shown at the bottom . The relative mean square errors ( RMSE ) of the fitting are displayed as a percentage at the bottom left of each panel . Scale bar 100 μm . ( d ) Mechanical power maps over time obtained by HR of the same embryos as in ( c ) ( from Movie EV5 ) . The RMSE of the power is shown as a percentage at the bottom left of each panel . The mechanical work to produce doming ( 40 % epiboly ) arises mainly at the blastoderm ( left ) ; at 60 % epiboly ( middle ) DCs undergo ingression ( red ) while the E - YSL resists constriction ( blue ) . At 80 % epiboly ( right ) , the active work leading to the active movement of the EVL / yolk margin mainly maps at the E - YSL ( red ) while the blastoderm undergoes elastic resistance ( blue ) . White arrows point to the main observed events . Scale bar 100 μm . ( e ) AV ( red ) and CC ( green ) stresses and their differences ( blue ) normalized values in arbitrary units from the regression analysis ( Movie EV6 ) were plotted as a function of the φ angle ( 0 - 1 ) along the cortex from animal to vegetal . The equator - dotted yellow line - , yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed ( 40 % ; 60 % ; 80 % epiboly ) . The CC stress steeps up from animal to vegetal and the AV shows a more complex profile . Their difference changes senses around the EVL / yolk margin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(a) Yolk microrheology. Mean squared displacement (MSD) of thermal fluctuations of fluorescent nanoparticles embedded in the yolk of zebrafishembryos. Solid and dashed black lines are Mean ± Standard Error computed from 99 particles. Red line is the fit of the Stokes-Einstein equation. MSD of nanoparticles embedded in the yolk exhibits a proportional dependence on time lag, Δt, consistent with a Newtonian liquid behavior with a viscosity of 129 mPa·s. A linear time dependence of MSD with similar values of yolk viscosity has been recently reported in Drosophila (He et al., 2014). Interestingly, the viscosity of the zebrafish yolk is two orders of magnitude lower than that of C. elegansembryos (Daniels et al., 2006) and of the cytoplasm of several cell types (Wirtz, 2009).(b) Viscoelasticity of the yolk cortex. Effective complex modulus (g*) measured by indenting the surface of zebrafishembryos with AFM. Red symbols are the elastic modulus (g') and yellow symbols are the viscous modulus (g''), respectively. Standard Errors are displayed. In all the measured frequencies cortex rheology is dominated by a solid-like behavior with the viscous modulus 5 times lower that the elastic modulus. No significant frequency dependence was found in the elastic (ANOVA, p = 0.123) and viscous modulus (ANOVA, p = 0.719).(c) Flow lines from snapshots of the PIV measurements (at 40, 60 and 80 % epiboly) of a two-photon video time-lapse (from Movie EV4) of a Tg (β-actin:m-GFP) embryo (top). Fitted flow lines using HR ar shown at the bottom. The relative mean square errors (RMSE) of the fitting are displayed as a percentage at the bottom left of each panel. Scale bar 100 μm.(d) Mechanical power maps over time obtained by HR of the same embryos as in (c) (from Movie EV5). The RMSE of the power is shown as a percentage at the bottom left of each panel. The mechanical work to produce doming (40 % epiboly) arises mainly at the blastoderm (left); at 60 % epiboly (middle) DCs undergo ingression (red) while the E-YSL resists constriction (blue). At 80 % epiboly (right), the active work leading to the active movement of the EVL / yolk margin mainly maps at the E-YSL (red) while the blastoderm undergoes elastic resistance (blue). White arrows point to the main observed events. Scale bar 100 μm.(e) AV (red) and CC (green) stresses and their differences (blue) normalized values in arbitrary units from the regression analysis (Movie EV6) were plotted as a function of the φ angle (0-1) along the cortex from animal to vegetal. The equator - dotted yellow line -, yolk surface - purple shadow - and the RMSE of the mechanical pressure as a percentage for each time point are displayed (40 %; 60 %; 80 % epiboly). The CC stress steeps up from animal to vegetal and the AV shows a more complex profile. Their difference changes senses around the EVL / yolk margin.


Figure 5 ( a ) Correlation between the differential tension ( cyan ) represented by the recoil velocities differences ( m / s ) between CC and AV laser cuts and their difference ( blue ) calculated by regression analysis at the E - YSL during epiboly ( % ) . The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points . ( b ) AV tension ( averaged recoil velocity in m / s ) in the yolk cortex at different distances from the EVL ( 0 , 10 , 20 , 40 and 60 m ) and at different epiboly stages ( 50 % , blue ; 60 - 70 % , red ; 80 % epiboly , green ) . Significance changes are highlighted and Standard Errors are displayed . ( c ) Probing mechanical parameters of living embryos with AFM . Force - indentation curves recorded at cantilever velocity of 10 m / s ( blue ) fitted with a " liquid balloon " model ( red ) . Inset : AFM force - indentation description . ( d ) Averaged values of mean surface tension ( pN / μm ) on the yolk obtained with AFM at different epiboly stages ( left ) , and the significant difference of tension at 60 - 70 % epiboly ( p = 0 . 0245 ) between the E - YSL and the vegetal pole ( right ) . Standard Errors are displayed .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(a) Correlation between the differential tension (cyan) represented by the recoil velocities differences (m/s) between CC and AV laser cuts and their difference (blue) calculated by regression analysis at the E-YSL during epiboly (%). The difference values in normalized relative units were fitted applying a constant adjustment ratio at all time points.(b) AV tension (averaged recoil velocity in m/s) in the yolk cortex at different distances from the EVL (0, 10, 20, 40 and 60 m) and at different epiboly stages (50 %, blue; 60-70 %, red; 80 % epiboly, green). Significance changes are highlighted and Standard Errors are displayed.(c) Probing mechanical parameters of living embryos with AFM. Force-indentation curves recorded at cantilever velocity of 10 m/s (blue) fitted with a "liquid balloon" model (red). Inset: AFM force-indentation description.(d) Averaged values of mean surface tension (pN / μm) on the yolk obtained with AFM at different epiboly stages (left), and the significant difference of tension at 60-70 % epiboly (p = 0.0245) between the E-YSL and the vegetal pole (right). Standard Errors are displayed.


Figure 6 ( a ) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut ( red line time point ) in the E - YSL of a Myosin - GFP transgenic embryo [ Tg ( β - actin : myl 12 . 1 - e - GFP ) ] . After a rapid opening , an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched ( elastic ) than the vegetal yolk cellmembrane / cortex . When the gap is closed , the cut center returns to its initial position and the elastic equilibrium is restored . Scale bar 10 μm . ( d ) Dynamic simulation of AV and CC stresses normalized values in arbitrary units ( from Movie EV9 - 40 % ; 65 % ; 80 % epiboly ) represented as in Figure 1a . The AV and CC stresses are equal at the poles , as the epiboly simulation progresses , the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(a) Kymograph showing the deformation of the cortex along a segment perpendicular to a laser cut (red line time point) in the E-YSL of a Myosin-GFP transgenic embryo [Tg (β-actin:myl 12.1-e-GFP)]. After a rapid opening, an active contraction displaces the cut centroid vegetalward indicating that the EVL is more easily stretched (elastic) than the vegetal yolk cellmembrane/cortex. When the gap is closed, the cut center returns to its initial position and the elastic equilibrium is restored. Scale bar 10 μm.(d) Dynamic simulation of AV and CC stresses normalized values in arbitrary units (from Movie EV9 - 40 %; 65 %; 80 % epiboly) represented as in Figure 1a. The AV and CC stresses are equal at the poles, as the epiboly simulation progresses, the CC stress steeps up from animal to vegetal and the difference between CC and AV stresses change sense at the EVL / yolk margin.