C . Representative images ( top panels ) and corresponding H & amp ; amp ; E micrographs ( bottom panels ) of the liver from control ( NaCl - injected , left panels ) or Tie2 - GFP mice 25 and 35 days ( center and right panels , respectively ) post - intrasplenic injection of 5x103 CT26 . Large metastatic foci in the liver of mice that received CT26 cells are indicated ( arrowheads ) . The dashed line in the H & amp ; amp ; E panels identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm .

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

C. Representative images (top panels) and corresponding H&amp;E micrographs (bottom panels) of the liver from control (NaCl-injected, left panels) or Tie2-GFP mice 25 and 35 days (center and right panels, respectively) post-intrasplenic injection of 5x103 CT26. Large metastatic foci in the liver of mice that received CT26 cells are indicated (arrowheads). The dashed line in the H&amp;E panels identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm.


D . Liver RNA was extracted from mice transplanted and injected as described in B ( NaCl n = 4 , day 25 n = 7 , day 35 n = 4 ) . The average GFP expression value of NaCl injected mice estimated by quantitative real - time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney 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

D. Liver RNA was extracted from mice transplanted and injected as described in B (NaCl n=4, day 25 n=7, day 35 n=4). The average GFP expression value of NaCl injected mice estimated by quantitative real-time PCR was set to 1 and utilized as reference to calculate the fold increase values of CT26 injected mice; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice that were injected intrasplenically with either NaCl ( upper panels ) or 5x103 CT26 ( bottom panels , 25 days post - injection ) . Note that TEMs ( identified as GFP + , macrophage mannose receptor [ MMR ] + and F4 / 80 + cells ) gather in the proximity of CRC metastatic foci ( identified by the dashed lines ) ; ✱ = CRC metastatic area ; scale bars = 100μm .

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

E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice that were injected intrasplenically with either NaCl (upper panels) or 5x103 CT26 (bottom panels, 25 days post-injection). Note that TEMs (identified as GFP+, macrophage mannose receptor [MMR]+ and F4/80+ cells) gather in the proximity of CRC metastatic foci (identified by the dashed lines); ✱=CRC metastatic area; scale bars=100μm.


A . Contrast - enhanced magnetic resonance imaging ( MRI ) of the liver from representative Tie2 - GFP mice ( red frame ) or Tie2 - IFNα ( blue frame ) that were intrasplenically injected with 5x103 CT26 . Red arrows identify CRC liver metastases of representative z - sections . Tumors were characterized as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Each panel refers to a single mouse analyzed at different time points ; n . a . = not assessed , refers to a mouse euthanized before the specified time point ; scale bars = 5mm . B . Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis . Mice were treated as described in A . Mock / Tie2 - GFP n = 11 , Tie2 - IFNα n = 9 ; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point ; p - values were calculated by Fisher ' s exact test . C . Tumor volume quantification measured by MRI analysis of the same mice described in B . Each symbol corresponds to the same mouse analyzed at different time points ; horizontal bars = mean values ; note that Mock / Tie2 - GFP mice were euthanized or died before day 54 ; p - values were calculated by Mann - Whitney 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

A. Contrast-enhanced magnetic resonance imaging (MRI) of the liver from representative Tie2-GFP mice (red frame) or Tie2-IFNα (blue frame) that were intrasplenically injected with 5x103 CT26. Red arrows identify CRC liver metastases of representative z-sections. Tumors were characterized as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Each panel refers to a single mouse analyzed at different time points; n.a.=not assessed, refers to a mouse euthanized before the specified time point; scale bars=5mm.B. Percentage of mice bearing at least one CRC liver metastasis estimated by MRI analysis. Mice were treated as described in A. Mock/Tie2-GFP n=11, Tie2-IFNα n=9; the oblique black line pattern within the red columns depicts the percentage of mice euthanized or that died before the indicated time point; p-values were calculated by Fisher's exact test.C. Tumor volume quantification measured by MRI analysis of the same mice described in B. Each symbol corresponds to the same mouse analyzed at different time points; horizontal bars=mean values; note that Mock/Tie2-GFP mice were euthanized or died before day 54; p-values were calculated by Mann-Whitney test.


D , E . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 ( D ) and Irf7 ( E ) within brain ( n = 4 , n = 2 ) , kidney ( n = 4 , n = 3 ) and metastatic liver ( n = 4 , n = 7 ) of Tie2 - GFP or Tie2 - IFNα mice euthanized 25 days post - intrasplenic injection of 5x103 CT26 . The basal expression of Oas1 and Irf7 estimated in brain of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values depicted . Mean values are shown ; error bars = S . E . M . ; p - value was calculated by Mann - Whitney 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

D, E. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Oas1 (D) and Irf7 (E) within brain (n=4, n=2), kidney (n=4, n=3) and metastatic liver (n=4, n=7) of Tie2-GFP or Tie2-IFNα mice euthanized 25 days post-intrasplenic injection of 5x103 CT26. The basal expression of Oas1 and Irf7 estimated in brain of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values depicted. Mean values are shown; error bars=S.E.M.; p-value was calculated by Mann-Whitney test.


F . Eighty - four or 54 days post - NaCl or 5x103 CT26 intrasplenic injection , Sham / CTRL ( n = 5 ) or Tie2 - IFNα mice ( n = 7 ) were infected with LCMV ( Armstrong strain , 200 pfu intraperitoneally injected ) . Eight days post - infection white blood cells were isolated and analyzed for LCMV specific CD8 + T cell response . Left panel : percentage of total CD8 + T cells ; middle panel : percentage of CD8 + / NP118 + T cells ( NP118 = recombinant dimeric H - 2d / Ig fusion protein complexed with the immune - dominant H - 2d - restricted LCMV NP118 - 126 peptide ) ; right panel : percentage of CD8 + / IFNγ + T cells after in vitro stimulation with virus - specific H - 2d - restricted peptide ( NP118 - 126 ) ; data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; differences were not statistically significant ( by unpaired Student ' s 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

F. Eighty-four or 54 days post-NaCl or 5x103 CT26 intrasplenic injection, Sham/CTRL (n=5) or Tie2-IFNα mice (n=7) were infected with LCMV (Armstrong strain, 200 pfu intraperitoneally injected). Eight days post-infection white blood cells were isolated and analyzed for LCMV specific CD8+ T cell response. Left panel: percentage of total CD8+ T cells; middle panel: percentage of CD8+/NP118+ T cells (NP118=recombinant dimeric H-2d/Ig fusion protein complexed with the immune-dominant H-2d-restricted LCMV NP118-126 peptide); right panel: percentage of CD8+/IFNγ+ T cells after in vitro stimulation with virus-specific H-2d-restricted peptide (NP118-126); data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; differences were not statistically significant (by unpaired Student's t-Test).


G . Kaplan - Meier survival curves of the indicated groups of mice described in A . Sham ( n = 3 ) , Mock ( wt ) / Tie2 - GFP ( n = 11 ) ; Tie2 - IFNα ( n = 9 ) ; data pooled from three independent experiments ; p = 0 . 001 by log - rank / Mantel - Cox test . The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal ( red frame : Mock / Tie2 - GFP , day 35 post - CT26 injection ) opposed to the lack of lesions in Tie2 - IFNα treated mice at later time points ( blue frame : day 450 post - CT26 injection ) .

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

G. Kaplan-Meier survival curves of the indicated groups of mice described in A. Sham (n=3), Mock(wt)/Tie2-GFP (n=11); Tie2-IFNα (n=9); data pooled from three independent experiments; p=0.001 by log-rank/Mantel-Cox test. The inset images show representative macroscopic photographs of the metastatic progression in the liver of a control animal (red frame: Mock/Tie2-GFP, day 35 post-CT26 injection) opposed to the lack of lesions in Tie2-IFNα treated mice at later time points (blue frame: day 450 post-CT26 injection).


A . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2 - GFP or Tie2 - IFNαmice that were intrasplenically injected with either NaCl ( n = 4 and n = 4 , respectively ) or 5x105CT26 - RFP and euthanized 5 minutes ( n = 9 , n = 5 ) , 3 days ( n = 6 , n = 3 ) and 7 days ( n = 5 , n = 3 ) thereafter . The basal expression of Irf7 estimated in control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed at the following time points post - injection . Data pooled from four independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increase in Irf7 expression levels from the liver of Tie2 - GFPmice was statistically significant ( p = 0 . 004 by one - way Anova test , not reported on graph ) .

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

A. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible gene Irf7within the liver of Tie2-GFP or Tie2-IFNαmice that were intrasplenically injected with either NaCl (n=4 and n=4, respectively) or 5x105CT26-RFP and euthanized 5 minutes (n=9, n=5), 3 days (n=6, n=3) and 7 days (n=5, n=3) thereafter. The basal expression of Irf7 estimated in control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed at the following time points post-injection. Data pooled from four independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increase in Irf7 expression levels from the liver of Tie2-GFPmice was statistically significant (p=0.004 by one-way Anova test, not reported on graph).


B . Representative RFP immunostaining from the liver of Tie2 - GFP mice ( left panels ) or Tie2 - IFNα mice ( right panels ) at the indicated time points after 5x105 CT26 - RFP intrasplenic injection . Arrowheads highlight single or clustered RFP positive cells ; scale bars = 100μm . C . Immunostaining quantification of hepatic CT26 - RFP arrival ( 5 minutes post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 2 ) and expansion ( day 3 post - injection : Tie2 - GFP n = 6 , Tie2 - IFNα n = 3 ; day 7 post - injection : Tie2 - GFP n = 5 , Tie2 - IFNα n = 3 ) in the liver of Tie2 - GFP mice or Tie2 - IFNα mice that were injected with 5x105 CT26 - RFP as described in B . Data pooled from two independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test . The increased percent of RFP + areas in the liver of Tie2 - GFP mice was statistically significant ( p = 0 . 0009 by one - way Anova test , not reported on graph ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative RFP immunostaining from the liver of Tie2-GFP mice (left panels) or Tie2-IFNα mice (right panels) at the indicated time points after 5x105 CT26-RFP intrasplenic injection. Arrowheads highlight single or clustered RFP positive cells; scale bars=100μm.C. Immunostaining quantification of hepatic CT26-RFP arrival (5 minutes post-injection: Tie2-GFP n=6, Tie2-IFNα n=2) and expansion (day 3 post-injection: Tie2-GFP n=6, Tie2-IFNα n=3; day 7 post-injection: Tie2-GFP n=5, Tie2-IFNα n=3) in the liver of Tie2-GFP mice or Tie2-IFNα mice that were injected with 5x105 CT26-RFP as described in B. Data pooled from two independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test. The increased percent of RFP+ areas in the liver of Tie2-GFP mice was statistically significant (p=0.0009 by one-way Anova test, not reported on graph).


A . Representative contrast - enhanced MRI panels depicting the liver of Mock / Tie2 - GFP ( red frame ) or Tie2 - IFNα ( blue frame ) chimeric mice . Each frame shows the metastatic progression 14 and 21 days post - intrasplenic injection of 5x104 MC38 within the indicated chimeric group . BM donors and recipient mouse strains are indicated on the left of each frame ; red arrows or dashed red lines identify CRC liver metastases of representative z - sections . Tumors were identified as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences respectively ; scale bars = 5mm . B . Tumor volume quantification ( based on MRI analyses ) of lesions detected in the same livers described in A . Chimeric groups and number of mice analyzed are listed ; data pooled from three independent experiments ; mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney 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

A. Representative contrast-enhanced MRI panels depicting the liver of Mock/Tie2-GFP (red frame) or Tie2-IFNα (blue frame) chimeric mice. Each frame shows the metastatic progression 14 and 21 days post-intrasplenic injection of 5x104 MC38 within the indicated chimeric group. BM donors and recipient mouse strains are indicated on the left of each frame; red arrows or dashed red lines identify CRC liver metastases of representative z-sections. Tumors were identified as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences respectively; scale bars=5mm.B. Tumor volume quantification (based on MRI analyses) of lesions detected in the same livers described in A. Chimeric groups and number of mice analyzed are listed; data pooled from three independent experiments; mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


B . Tumor volume quantification measured by MRI analysis of Tie2 - GFP ( n = 6 ) or Tie2 - IFNα mice ( n = 9 ) at the indicated time points post - transplant , one additional Tie2 - IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs ' test and not further analyzed ; mean values are shown ; error bars = S . E . M . ; p = 0 . 051 , by Mann - Whitney 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

B. Tumor volume quantification measured by MRI analysis of Tie2-GFP (n=6) or Tie2-IFNα mice (n=9) at the indicated time points post-transplant, one additional Tie2-IFNα mouse showed a tumor volume at day 21 of more than 820 mm3 and was statistically rejected by the Grubbs' test and not further analyzed; mean values are shown; error bars=S.E.M.; p=0.051, by Mann-Whitney test.


C . Images ( top panels ) and corresponding contrast - enhanced MRI ( middle and bottom panels ) of the liver from representative Tie2 - GFP mice or Tie2 - IFNα mice that were intrahepatically injected with 5x103 CT26 . White dashed lines identify macroscopic lesions . Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z - stacks . Tumors detected by MRI analysis appeared as hypointense and slightly - hyperintense regions in T1 and T2 weighted sequences , respectively . Scale bars = 5mm .

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

C. Images (top panels) and corresponding contrast-enhanced MRI (middle and bottom panels) of the liver from representative Tie2-GFP mice or Tie2-IFNα mice that were intrahepatically injected with 5x103 CT26. White dashed lines identify macroscopic lesions. Red and green dashed lines identify hepatic or extrahepatic CRC metastases respectively from representative MRI z-stacks. Tumors detected by MRI analysis appeared as hypointense and slightly-hyperintense regions in T1 and T2 weighted sequences, respectively. Scale bars=5mm.


D . Percentage of mice with peritoneal carcinomatosis ( defined by the presence of multiple intra - peritoneal lesions ) determined by MRI analysis from the same mice described in B . Note that while intrahepatic number of lesions remained similar between time points , extrahepatic tumor spreading was significantly reduced in Tie2 - IFNα mice . P - values were calculated by Fisher ' s exact 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

D. Percentage of mice with peritoneal carcinomatosis (defined by the presence of multiple intra-peritoneal lesions) determined by MRI analysis from the same mice described in B. Note that while intrahepatic number of lesions remained similar between time points, extrahepatic tumor spreading was significantly reduced in Tie2-IFNα mice. P-values were calculated by Fisher's exact test.


E . Confocal immunofluorescence images of representative liver sections from Tie2 - GFP mice at distal ( upper panels ) or proximal ( bottom panels ) areas to CRC liver metastasis , 30 days post HSPC transplant . Note that TEMs were identified as GFP + cells and by the concomitant expression of different levels of the myeloid cell marker CD11b + as highlighted in the inset ( merge panels , white arrows ) ; the dashed line identifies the metastasis margin ; ✱ = CRC metastatic area ; scale bars = 100μm ; insets depict the corresponding areas identified by dashed squares in the merge panels , magnified 1 . 5 - folds .

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

E. Confocal immunofluorescence images of representative liver sections from Tie2-GFP mice at distal (upper panels) or proximal (bottom panels) areas to CRC liver metastasis, 30 days post HSPC transplant. Note that TEMs were identified as GFP+ cells and by the concomitant expression of different levels of the myeloid cell marker CD11b+ as highlighted in the inset (merge panels, white arrows); the dashed line identifies the metastasis margin; ✱=CRC metastatic area; scale bars=100μm; insets depict the corresponding areas identified by dashed squares in the merge panels, magnified 1.5-folds.


F , G . Quantitative real - time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2 - GFP ( n = 4 ) or Tie2 - IFNα ( n = 4 ) mice euthanized 30 days post - transplant . The basal expression of Irf7 estimated in liver of control mice ( i . e . Tie2 - GFP injected with saline ) , was set to 1 and utilized to calculate the fold increase values observed . Mean values are shown ; error bars = S . E . M . ; p - values were calculated by Mann - Whitney test .

F, G. Quantitative real-time PCR analyses of the relative expression levels of the interferon inducible genes Irf7 and Oas1 within liver of Tie2-GFP (n=4) or Tie2-IFNα (n=4) mice euthanized 30 days post-transplant. The basal expression of Irf7 estimated in liver of control mice (i.e. Tie2-GFP injected with saline), was set to 1 and utilized to calculate the fold increase values observed. Mean values are shown; error bars=S.E.M.; p-values were calculated by Mann-Whitney test.


H . Kaplan - Meier survival curves of the indicated groups of mice . Tie2 - GFP ( n = 3 ) ; Tie2 - IFNα ( n = 5 ) ; p = 0 . 007 by log - rank / Mantel - Cox 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

H. Kaplan-Meier survival curves of the indicated groups of mice. Tie2-GFP (n=3); Tie2-IFNα (n=5); p=0.007 by log-rank/Mantel-Cox test.


A . IHL and TEM characterization in the liver of patients described in Appendix Fig S6A . Liver samples from surgical resections ( n = 9 ) were weighted and IHLs were isolated , counted and normalized over tissue weight ( left panel ) . Flow cytometry analysis revealed that TEMs , identified as 7AAD - / CD45 + / CD11b + / CD14 + / Tie2 + cells , are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions ( right panel ) . Control = hepatic hemangioma ; Distal = distal to CRC liver metastasis ( > 1 cm from the lesion ) ; Proximal = proximal to CRC liver metastasis ( < 1 cm from the lesion ) ; p - values were calculated by Wilcoxon matched pairs 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

A. IHL and TEM characterization in the liver of patients described in Appendix Fig S6A. Liver samples from surgical resections (n=9) were weighted and IHLs were isolated, counted and normalized over tissue weight (left panel). Flow cytometry analysis revealed that TEMs, identified as 7AAD-/CD45+/CD11b+/CD14+/Tie2+ cells, are detectable within the total IHL populations and accumulate preferentially in the proximity of the metastatic lesions (right panel). Control=hepatic hemangioma; Distal=distal to CRC liver metastasis (>1 cm from the lesion); Proximal=proximal to CRC liver metastasis (<1 cm from the lesion); p-values were calculated by Wilcoxon matched pairs Test.


B - C . Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal ( > 1 cm ; left panels ) or proximal ( < 1 cm ; right panels ) sites from the CRC liver lesion . C . Higher magnification of the panels identified by a rectangle in panel B . Negative control ( bottom panels ) is obtained by omitting primary anti - TIE2 antibodies . TIE2immunostaining , indicates that sites that are distal from the lesion contain only TIE2 + cells with an apparent endothelial morphology ( characterized by an elongated appearance , arrows ) while sites that are proximal to the lesion also contain additional TIE2 + cells with an apparent monocyte - like morphology ( characterized by a round appearance , arrowheads ) . The dashed lines identifies the metastasis margin ; ✱ = CRC metastatic area . Scale bars of upper panels = 50μm ; middle and bottom panels = 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C. Representative TIE2 immunohistochemistry staining from the liver of a patient with liver CRC metastases from distal (>1 cm; left panels) or proximal (<1 cm; right panels) sites from the CRC liver lesion. C. Higher magnification of the panels identified by a rectangle in panel B. Negative control (bottom panels) is obtained by omitting primary anti-TIE2 antibodies. TIE2immunostaining, indicates that sites that are distal from the lesion contain only TIE2+ cells with an apparent endothelial morphology (characterized by an elongated appearance, arrows) while sites that are proximal to the lesion also contain additional TIE2+ cells with an apparent monocyte-like morphology (characterized by a round appearance, arrowheads). The dashed lines identifies the metastasis margin; ✱=CRC metastatic area. Scale bars of upper panels=50μm; middle and bottom panels=20 μm.


( B ) Cells expressing fluorescently - tagged α2 - tubulin ( atb2 ) were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs recorded within the final 1 . 1 µm of the cell for each strain . Red bars signify the

(B) Cells expressing fluorescently-tagged α2-tubulin (atb2) were imaged every 5 seconds and the dwell time of ~100 individual iMTs recorded within the final 1.1 µm of the cell for each strain. Red bars signify the mean


( C ) Kymographs showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged microtubules ( MT ) in the presence ( top panels ) and absence ( bottom panels ) of Mcp1 . Banding on MT , caused by unequal incorporation of fluorescence , illustrates the force exerted on the MT by continued growth into the cell cortex . Dashed yellow line indicates the cell

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(C) Kymographs showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged microtubules (MT) in the presence (top panels) and absence (bottom panels) of Mcp1. Banding on MT, caused by unequal incorporation of fluorescence, illustrates the force exerted on the MT by continued growth into the cell cortex. Dashed yellow line indicates the cell end


( D ) MT growth speed was calculated from kymographs from control ( n = 16 ) and ∆ mcp1 cells ( n = 11 ) , Klp5 / Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control ( n = 44 ) and ∆ mcp1 cells ( n = 32 )

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(D) MT growth speed was calculated from kymographs from control (n=16) and ∆mcp1 cells (n=11), Klp5/Klp6 walk speed was calculated from multiple individual runs on the MT lattice in control (n=44) and ∆mcp1 cells (n=32)


( E ) Average intensity of Klp5 / Klp6 at the plus ends of iMTs from multiple kymographs of control ( n = 19 ) or ∆ mcp1 cells ( n = 14 )

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(E) Average intensity of Klp5/Klp6 at the plus ends of iMTs from multiple kymographs of control (n=19) or ∆mcp1 cells (n=14)


( F ) Mixing experiment to compare fluorescently - tagged Klp5 / Klp6 levels between cells either expressing ( blue , closed arrowheads ) or deleted ( red , open arrowhead ) for Mcp1 distinguished by the absence of fluorescently - tagged nuclear envelope protein Cut11 ( left panel ) . Scale bar , 5µm . Box plot ( right panel ) shows quantitated fluorescence values for nuclear levels of Klp5 / Klp6 in control ( n = 44 ) and ∆ mcp1 cells ( n = 45 ) and at the MT plus end in control ( n = 64 ) and ∆ mcp1 cells ( n = 35 ) prior to

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(F) Mixing experiment to compare fluorescently-tagged Klp5/Klp6 levels between cells either expressing (blue, closed arrowheads) or deleted (red, open arrowhead) for Mcp1 distinguished by the absence of fluorescently-tagged nuclear envelope protein Cut11 (left panel). Scale bar, 5µm. Box plot (right panel) shows quantitated fluorescence values for nuclear levels of Klp5/Klp6 in control (n=44) and ∆mcp1 cells (n=45) and at the MT plus end in control (n=64) and ∆mcp1 cells (n=35) prior to shrinkage


( A ) Log phase cells expressing fluorescently - tagged MTs were imaged and the levels of either fluorescently - tagged Klp5 / Klp6 or Tea2 at the plus ends of growing MTs determined . Measurements ( klp5 - mNG klp6 - mNG , n = 305 ; tea2 - GFP , n = 359 ) were plotted against microtubule ( MT ) length and third order polynomial curves fitted to the

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(A) Log phase cells expressing fluorescently-tagged MTs were imaged and the levels of either fluorescently-tagged Klp5/Klp6 or Tea2 at the plus ends of growing MTs determined. Measurements (klp5-mNG klp6-mNG, n=305; tea2-GFP, n=359) were plotted against microtubule (MT) length and third order polynomial curves fitted to the data


( B ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Tea2 kinesin in control cells ( top panels ) or in the absence of Klp6 ( middle panels ) or Mcp1 ( bottom panels ) . Dashed yellow line indicates the cell

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(B) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Tea2 kinesin in control cells (top panels) or in the absence of Klp6 (middle panels) or Mcp1 (bottom panels). Dashed yellow line indicates the cell end


( C ) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in ( B ) for control ( n = 16 ) , ∆ klp6 ( n = 13 ) or ∆ mcp1 ( n = 14 ) cells

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(C) Intensity of Tea2 at plus ends quantitated from multiple kymographs of the type in (B) for control (n=16), ∆klp6 (n=13) or ∆mcp1 (n=14) cells


( D ) Kymographs showing fluorescently - tagged MTs co - imaged with fluorescently - tagged Klp5 / Klp6 in control ( top panels , repeated from 1C ) or ∆ tea2 cells ( bottom panels ) . Dashed yellow line indicates the cell

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(D) Kymographs showing fluorescently-tagged MTs co-imaged with fluorescently-tagged Klp5/Klp6 in control (top panels, repeated from 1C) or ∆tea2 cells (bottom panels). Dashed yellow line indicates the cell end


( E ) Cells expressing fluorescently - tagged tubulin were imaged every 5 seconds and the dwell time of ~ 100 individual iMTs for each condition recorded within the final 1 . 1 µm of the cell . Red bars signify the

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(E) Cells expressing fluorescently-tagged tubulin were imaged every 5 seconds and the dwell time of ~100 individual iMTs for each condition recorded within the final 1.1 µm of the cell. Red bars signify the mean


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Tea2 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the position of the cell end . Plots ( bottom left panels ) show both the relative fluorescence intensity and position for Tea2 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar 2µm . Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 ( green ) and Tip1 ( magenta ) over time ( top right ) and the distance of these pixels from the cell end ( bottom right )

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(A) Kymograph (top left panels) showing fluorescently-tagged Tea2 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the position of the cell end. Plots (bottom left panels) show both the relative fluorescence intensity and position for Tea2 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar 2µm. Data quantitated from this kymograph by extracting the maximal intensity pixel value at the MT plus end for Tea2 (green) and Tip1 (magenta) over time (top right) and the distance of these pixels from the cell end (bottom right)


( A ) Kymograph ( top left panels ) showing fluorescently - tagged Klp5 / Klp6 ( Kinesin ) co - imaged with fluorescently - tagged Tip1 ( Tip1 - Tdtom ) . Dashed yellow line indicates the cell end . Plots ( bottom left panels ) show both the fluorescence intensity and position of Klp5 / Klp6 ( green ) and Tip1 ( magenta ) corresponding to the numbered sections of the kymograph . Scale bar , 2µm . Intensity of the maximal value pixel at the plus end ( top right panel ) and its position relative to the cell end ( bottom right panel ) for both Klp5 / Klp6 ( green ) and Tip1 ( magenta ) plotted relative to MT shrinkage for this kymograph

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(A) Kymograph (top left panels) showing fluorescently-tagged Klp5/Klp6 (Kinesin) co-imaged with fluorescently-tagged Tip1 (Tip1-Tdtom). Dashed yellow line indicates the cell end. Plots (bottom left panels) show both the fluorescence intensity and position of Klp5/Klp6 (green) and Tip1 (magenta) corresponding to the numbered sections of the kymograph. Scale bar, 2µm. Intensity of the maximal value pixel at the plus end (top right panel) and its position relative to the cell end (bottom right panel) for both Klp5/Klp6 (green) and Tip1 (magenta) plotted relative to MT shrinkage for this kymograph


( A ) Time - dependent killing of a panel of bacterial isolates in the presence of meropenem ( MEM10 , 10 µg / ml ) . Cells were grown overnight and sub - cultured for 30 minutes at 37 ° C before being exposed to meropenem at 10 µg / mL and moved to a 37 ° C stationary incubator . Samples were taken at each indicated time point and spot - titered onto LB medium to quantify colony forming units ( CFU / mL ) throughout meropenem treatment . ( B ) images of cells corresponding to the time points in ( A ) were taken after labeling with membrane stain FM4 - 64 . E . cloacae , Enterobacter cloacae ; K . aerogenes , Klebsiella aerogenes ; K . pneumoniae , Klebsiella pneumoniae ; E . coli , Escherichia coli

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(A) Time-dependent killing of a panel of bacterial isolates in the presence of meropenem (MEM10, 10 µg/ml). Cells were grown overnight and sub-cultured for 30 minutes at 37°C before being exposed to meropenem at 10 µg/mL and moved to a 37°C stationary incubator. Samples were taken at each indicated time point and spot-titered onto LB medium to quantify colony forming units (CFU/mL) throughout meropenem treatment. (B) images of cells corresponding to the time points in (A) were taken after labeling with membrane stain FM4-64. E. cloacae, Enterobacter cloacae; K. aerogenes, Klebsiella aerogenes; K. pneumoniae, Klebsiella pneumoniae; E. coli, Escherichia coli


( D ) Regulatory modes of representative VxrB - controlled genes in response to PenG . Upper plot shows VxrB binding patterns ( assessed via ChIP - Seq ) , lower panel shows transcriptional response upon overexpression of VxrB D78E ( assessed via RNA - Seq ) . RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG ( 100 µg / ml for 3 hours ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(D) Regulatory modes of representative VxrB-controlled genes in response to PenG. Upper plot shows VxrB binding patterns (assessed via ChIP-Seq), lower panel shows transcriptional response upon overexpression of VxrB D78E (assessed via RNA-Seq). RNA expression was confirmed by S1 nuclease mapping assay in wild type and ΔvxrAB mutant strains after exposure to PenG (100 µg/ml for 3 hours). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


Reduction of cell wall synthesis gene expression causes a tolerance defect . ( C ) Survival of a ∆ pbp1b ∆ pbp1a Piptg : pbp1a strain after 6 hours of PenG exposure . Pup indicates conditions of high IPTG concentrations ( 100 µM ) , Pdown low IPTG concentration ( 2 µM ) . Survival fraction is cfu / ml of PenG - treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration .

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Reduction of cell wall synthesis gene expression causes a tolerance defect. (C) Survival of a ∆pbp1b ∆pbp1a Piptg:pbp1a strain after 6 hours of PenG exposure. Pup indicates conditions of high IPTG concentrations (100 µM), Pdown low IPTG concentration (2 µM). Survival fraction is cfu/ml of PenG-treated cultures normalized to untreated cultures grown in and plated on medium containing the same IPTG concentration.


( D ) Time - dependent killing experiment in the presence of PenG ( 100 µg / ml , 10 x MIC ) . All data are means ( + / - standard error ) of 3 independent biological replicates .

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(D) Time-dependent killing experiment in the presence of PenG (100 µg/ml, 10 x MIC). All data are means (+/- standard error) of 3 independent biological replicates.


( C ) Time - course of iron accumulation in PenG - treated WT and ∆ vxrAB cells as measured by ICP - MS . Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ P = 0 . 001 ; ∗ ∗ ∗ P < 0 . 0003 ( paired t - test ) .

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(C) Time-course of iron accumulation in PenG-treated WT and ∆vxrAB cells as measured by ICP-MS. Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗P=0.001; ∗∗∗P<0.0003 (paired t-test).


( B ) . To test PenG - mediated induction of Fur - regulated genes , S1 nuclease mapping assays were performed on the small RNA ryhB , the RyhB target sodB , hutA / B , coding for heme transport systems and vc2212 , which encodes a component of a ferric citrate uptake system ) . Cells were grown to mid - exponential phase followed by exposure to H2O2 or PenG for the indicated duration . Numbers represent expression levels normalized to untreated control ( averages from three independent replicates ) . White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes . Source data is available as supplemental material .

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(B). To test PenG-mediated induction of Fur-regulated genes, S1 nuclease mapping assays were performed on the small RNA ryhB, the RyhB target sodB, hutA/B, coding for heme transport systems and vc2212, which encodes a component of a ferric citrate uptake system). Cells were grown to mid-exponential phase followed by exposure to H2O2 or PenG for the indicated duration. Numbers represent expression levels normalized to untreated control (averages from three independent replicates). White demarcation line between time points indicates that these data points were not adjacent in the original experiment but were cropped for presentation purposes. Source data is available as supplemental material.


( B ) and ( C ) Iron uptake mutants restore ∆ vxrAB growth on 10 % sucrose . The indicated strains were grown to exponential phase ( OD600 ~ 0 . 5 ) in LB medium and spot - plated on 10 % Sucrose plates with or without additional iron sulfate . An example is shown in ( B ) , quantification of cfu is shown in ( C ) . Data information : Data are average of 3 independent biological replicates , error bars represent standard deviation . ∗ ∗ ∗ P < 0 . 0003 ; ∗ ∗ ∗ ∗ P < 0 . 0001 ( paired t - test ) .

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(B) and (C) Iron uptake mutants restore ∆vxrAB growth on 10% sucrose. The indicated strains were grown to exponential phase (OD600~0.5) in LB medium and spot-plated on 10% Sucrose plates with or without additional iron sulfate. An example is shown in (B), quantification of cfu is shown in (C). Data information: Data are average of 3 independent biological replicates, error bars represent standard deviation. ∗∗∗ P<0.0003; ∗∗∗∗ P<0.0001 (paired t-test).


( B ) Excretion of V . cholerae . Fecal samples were collected every two hours and assessed for their viable cell content ( Colony Forming Units , CFU ) to monitor the level of V . cholerae excretion . Each symbol represents fecal samples from an individual mouse ( n = 4 ) . The number of bacterial CFU was normalized per milligram of feces ( CFU / mg ) . Error bars represent standard deviation . ∗ ∗ P < 0 . 005 ; ∗ ∗ ∗ P < 0 . 001 ( two - way ANOVA ) .

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(B) Excretion of V. cholerae. Fecal samples were collected every two hours and assessed for their viable cell content (Colony Forming Units, CFU) to monitor the level of V. cholerae excretion. Each symbol represents fecal samples from an individual mouse (n=4). The number of bacterial CFU was normalized per milligram of feces (CFU/mg). Error bars represent standard deviation. ∗∗ P<0.005; ∗∗∗ P<0.001 (two-way ANOVA).


( E ) Time - dependent killing of respiratory chain mutants ( ∆ nqrA , encoding NADH dehydrogenase and ∆ ubiA , encoding an early step of ubiquinone synthesis ) in the presence of PenG ( 100 µg / ml , 10xMIC ) . All strains were grown in LB 0 . 2 % glucose . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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(E) Time-dependent killing of respiratory chain mutants (∆nqrA, encoding NADH dehydrogenase and ∆ubiA, encoding an early step of ubiquinone synthesis) in the presence of PenG (100 µg/ml, 10xMIC). All strains were grown in LB 0.2 % glucose. Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


( F ) Growth - phase dependent killing assay of ROS detoxification systems ( ∆ fur , ∆ sodB , and ∆ oxyR1 ∆ katG ∆ katB ) . At designated time - points , a 5 ml aliquot was withdrawn and exposed to 100 µg / ml PenG for 3 h . CFU / mL for each time point was assessed before ( solid lines ) and after addition of PenG ( dotted lines ) . Data information : Data are mean of three independent biological replicates and error bars represent standard deviation .

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(F) Growth-phase dependent killing assay of ROS detoxification systems (∆fur, ∆sodB, and ∆oxyR1∆katG∆katB). At designated time-points, a 5 ml aliquot was withdrawn and exposed to 100 µg/ml PenG for 3 h. CFU/mL for each time point was assessed before (solid lines) and after addition of PenG (dotted lines). Data information: Data are mean of three independent biological replicates and error bars represent standard deviation.


Possible correlation between AR - and UPR - associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort ( n = 190 ) ( http : / / www . cbioportal . org / public - portal / index . do ) . Tumors were stratified according to AR status into three groups , that is ARlow ( n = 60 ) , ARmedium ( n = 70 ) , and ARhigh ( n = 60 ) . The levels of UPR gene expression in the three groups were compared using Pearson ' s correlation analysis by the R software and presented as a heatmap . There were significant differences between the three groups ( Supplementary Table S2 ) .

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Possible correlation between AR- and UPR-associated gene expression was assessed in the global gene expression data available in the TCGA Prostate Adenocarcinoma cohort (n = 190) (http://www.cbioportal.org/public-portal/index.do). Tumors were stratified according to AR status into three groups, that is ARlow (n = 60), ARmedium (n = 70), and ARhigh (n = 60). The levels of UPR gene expression in the three groups were compared using Pearson's correlation analysis by the R software and presented as a heatmap. There were significant differences between the three groups (Supplementary Table S2).


The expression profiles of some prominent UPR genes from the data in ( A ) , including ERN1 ( IRE1 ) , EDEM1 , ATF6 , and DNAJC3 ( P58IPK ) , are presented . P - values of the different genes are given .

The expression profiles of some prominent UPR genes from the data in (A), including ERN1 (IRE1), EDEM1, ATF6, and DNAJC3 (P58IPK), are presented. P-values of the different genes are given.


A , B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR ( qPCR ) . Controls were treated with vehicle for 84 h and set to 100 . Data represent the mean of three independent experiments in triplicate , and bars represent SE . P - values ranged between 1 . 66 × 10 − 5 and 0 . 025 , and IRE1α expression in R1881 48 h was * P = 0 . 013 with respect to vehicle - treated cells using unpaired Student ' s t - test .

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A, B mRNA expression levels in LNCaP cells for the indicated genes were investigated using quantitative PCR (qPCR). Controls were treated with vehicle for 84 h and set to 100. Data represent the mean of three independent experiments in triplicate, and bars represent SE. P-values ranged between 1.66 × 10−5 and 0.025, and IRE1α expression in R1881 48 h was *P = 0.013 with respect to vehicle-treated cells using unpaired Student's t-test.


C - E IRE1α , XBP - 1S , and XBP - 1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration . The value at t = 0 was set to 1 . Columns represent the mean of at least three independent tumors for each time point , and bars represent SE . P - values ranged between 5 . 7 × 10 − 10 and 0 . 0002 . IRE1α expression at 48 h post - castration was 3 . 17 × 10 − 6 with respect to t = 0 using unpaired Student ' s t - test .

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C-E IRE1α, XBP-1S, and XBP-1U mRNA in CWR22xenografts grown in nude mice and collected at the indicated times after castration. The value at t = 0 was set to 1. Columns represent the mean of at least three independent tumors for each time point, and bars represent SE. P-values ranged between 5.7  ×  10−10 and 0.0002. IRE1α expression at 48 h post-castration was 3.17 × 10−6 with respect to t = 0 using unpaired Student's t-test.


F , G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis . Data presented are representative of three independent experiments .

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F, G Protein expression in LNCaP cells upon treatment with R1881 for the indicated times by Western blot analysis. Data presented are representative of three independent experiments.


After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells . Expression in cells transfected with CTRL siRNA was set to 1 . Bars represent SE with * P = 0 . 001 indicating significant difference between AR siRNA - and control siRNA - transfected cells using paired Student ' s t - test .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.A Expression level of AR mRNA upon siRNA treatment for 48 h assessed by qPCR in LNCaP cells. Expression in cells transfected with CTRL siRNA was set to 1. Bars represent SE with *P = 0.001 indicating significant difference between AR siRNA- and control siRNA-transfected cells using paired Student's t-test.


After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . B - E Same as in ( A ) , but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation . Expression in cells transfected with CTRL siRNA was set to 100 . Bars represent SE . P - values are shown indicating significant difference between AR siRNA - and control siRNA - transfected cells using unpaired Student ' s t - test .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.B-E Same as in (A), but mRNA expression levels of the indicated UPR genes were determined by qPCR at indicated time points after R1881 stimulation. Expression in cells transfected with CTRL siRNA was set to 100. Bars represent SE. P-values are shown indicating significant difference between AR siRNA- and control siRNA-transfected cells using unpaired Student's t-test.


After starvation , LNCaP cells were transfected with control ( CTRL ) siRNA or AR siRNA . Cells were then treated with R1881 for the indicated times . Controls were treated with vehicle for 48 h . F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis . Representative blots for three independent experiments are shown .

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After starvation, LNCaP cells were transfected with control (CTRL) siRNA or AR siRNA. Cells were then treated with R1881 for the indicated times. Controls were treated with vehicle for 48 h.F Expression of the indicated proteins under conditions indicated on the top label was determined by Western blot analysis. Representative blots for three independent experiments are shown.


A , B LNCaP ( A ) or VCaP cells ( B ) were cultured and treated with vehicle ( C ) for 48 h or R1881 for 24 and 48 h . The cells were then fixed , and ChIP assay was performed as described in Materials and Methods using AR antibody . The data shown are representative of one experiment in duplicate . Error bars represent SE . * P < 0 . 01 for LNCaP and * P < 0 . 04 for VCaP indicate significant difference between C ( control ) and R1881 using unpaired Student ' s t - test .

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A, B LNCaP (A) or VCaP cells (B) were cultured and treated with vehicle (C) for 48 h or R1881 for 24 and 48 h. The cells were then fixed, and ChIP assay was performed as described in Materials and Methods using AR antibody. The data shown are representative of one experiment in duplicate. Error bars represent SE. *P < 0.01 for LNCaP and *P < 0.04 for VCaP indicate significant difference between C (control) and R1881 using unpaired Student's t-test.


A Knockdown of IRE1α or XBP - 1 leads to a decrease in cell survival . LNCaP cells were transfected with siRNA targeting either IRE1α or XBP - 1 ( 5 nM ) and starved in 2 % CT - FCS medium for 3 days before cell viability was measured using the CCK - 8 assay . The graph is representative of one experiment in triplicate and was repeated three times with similar results . Error bars represent SD with * P = 6 . 6 × 10 − 5 and 4 . 5 × 10 − 5 for comparison between Ctrl and siRNA against IRE1α and XBP - 1 , respectively , using paired Student ' s t - test .

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A Knockdown of IRE1α or XBP-1 leads to a decrease in cell survival. LNCaP cells were transfected with siRNA targeting either IRE1α or XBP-1 (5 nM) and starved in 2% CT-FCS medium for 3 days before cell viability was measured using the CCK-8 assay. The graph is representative of one experiment in triplicate and was repeated three times with similar results. Error bars represent SD with *P = 6.6 × 10−5 and 4.5 × 10−5 for comparison between Ctrl and siRNA against IRE1α and XBP-1, respectively, using paired Student's t-test.


B XBP - 1 rescues the growth defect of siIRE1α - transfected LNCaP cells . LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent . One day after siRNA transfection , the cells were transfected with either vector control ( Empty ) or Flag - XBP - 1S ( XBP - 1S ) . Three days after transfection , cells were harvested for western blot analysis or cultured for three more days before being applied to cell proliferation assay using the CCK - 8 reagent . The data are representative of two experiments in triplicate . Error bars represent SE . * P = 0 . 02 , * * P = 8 . 54 × 10 − 7 using paired Student ' s t - test .

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B XBP-1 rescues the growth defect of siIRE1α-transfected LNCaP cells. LNCaP cells were transfected with 5 nM of indicated siRNA using Lipofectamine RNAiMax reagent. One day after siRNA transfection, the cells were transfected with either vector control (Empty) or Flag-XBP-1S (XBP-1S). Three days after transfection, cells were harvested for western blot analysis or cultured for three more days before being applied to cell proliferation assay using the CCK-8 reagent. The data are representative of two experiments in triplicate. Error bars represent SE. *P = 0.02, **P = 8.54 × 10−7 using paired Student's t-test.


C , D IRE1α and XBP - 1 knockdown inhibits clonogenic capacity of LNCaP cells . Control LN - Scr ( Scr ) , LN - shIRE1 ( shIRE1 ) , or LN - shXBP1 ( shXBP - 1 ) cells were cultured for 3 weeks . The colonies formed were stained with crystal violet and photographed . The extent of IRE1α and XBP - 1 knockdown was determined by Western blot analysis . The area covered by colonies was quantified using the Gene Tools software ( SynGene ) . The data are representative of three experiments in triplicate . Error bars represent SEM . * P = 4 . 38 × 10 − 7 and * * P = 3 . 39 × 10 − 20 using paired Student ' s t - test .

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C, D IRE1α and XBP-1 knockdown inhibits clonogenic capacity of LNCaP cells. Control LN-Scr (Scr), LN-shIRE1 (shIRE1), or LN-shXBP1 (shXBP-1) cells were cultured for 3 weeks. The colonies formed were stained with crystal violet and photographed. The extent of IRE1α and XBP-1 knockdown was determined by Western blot analysis. The area covered by colonies was quantified using the Gene Tools software (SynGene). The data are representative of three experiments in triplicate. Error bars represent SEM. *P = 4.38 × 10−7 and **P = 3.39 × 10−20 using paired Student's t-test.


E Growth analysis of xenograftedLNCaPtumors in nude mice . LNCaP cells expressing shRNA against IRE1α ( LN - shIRE1 ) , XBP1 ( LN - shXBP - 1 ) , or control shRNA ( LN - Scr ) were subcutaneously implanted into both flanks of male nude mice ( 6 mice per group ) . Tumor size was measured at the indicated time points . Representative pictures of the tumors at harvest are shown . Error bars indicate SEM . * P = 0 . 03 for shIRE1 at week 7 , P = 0 . 02 for shXBP - 1 at week 7 , * * P = 0 . 01 for both shIRE1 and shXBP - 1 at week 8 using unpaired Student ' s t - test .

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E Growth analysis of xenograftedLNCaPtumors in nude mice. LNCaP cells expressing shRNA against IRE1α (LN-shIRE1), XBP1 (LN-shXBP-1), or control shRNA (LN-Scr) were subcutaneously implanted into both flanks of male nude mice (6 mice per group). Tumor size was measured at the indicated time points. Representative pictures of the tumors at harvest are shown. Error bars indicate SEM. *P = 0.03 for shIRE1 at week 7, P = 0.02 for shXBP-1 at week 7, **P = 0.01 for both shIRE1 and shXBP-1 at week 8 using unpaired Student's t-test.


F PCNA immunostaining in tumors from animals bearing LN - shIRE1 , LN - shXBP - 1 , or LN - Scr tumors . Scale bars : 100 μm .

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F PCNA immunostaining in tumors from animals bearing LN-shIRE1, LN-shXBP-1, or LN-Scr tumors. Scale bars: 100 μm.


LNCaP xenografts were grown in nude mice until palpable . Mice were then intraperitoneally injected with 0 . 5 mg / kg toyocamycin or saline ( Vehicle ) ( tumor numbers : n = 15 , orn = 10 , respectively ) twice weekly . Tumor sizes were measured weekly with calipers . Error bars indicate SEM . * P = 0 . 04 ; * * P = 0 . 0004 ; * * * P = 0 . 002 using unpaired Student ' s t - test . Same procedure was repeated on VCaP xenografts , with similar findings as in LNCaP xenografts ( tumor numbers : n = 9 , n = 11 ) . Error bars indicate SEM . * P = 0 . 02 and * * P = 0 . 0006 using unpaired Student ' s t - test .

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LNCaP xenografts were grown in nude mice until palpable. Mice were then intraperitoneally injected with 0.5 mg/kg toyocamycin or saline (Vehicle) (tumor numbers:n = 15, orn = 10, respectively) twice weekly.Tumor sizes were measured weekly with calipers. Error bars indicate SEM. *P = 0.04; **P = 0.0004; ***P = 0.002 using unpaired Student's t-test.Same procedure was repeated on VCaP xenografts, with similar findings as in LNCaP xenografts (tumor numbers: n = 9, n = 11). Error bars indicate SEM. *P = 0.02 and **P = 0.0006 using unpaired Student's t-test.


XBP - 1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle . Tumors were harvested , RNA - extracted , and qPCR performed . Error bars indicate SEM . * P = 0 . 04 for LNCaP and * * P = 0 . 02 for VCaP using unpaired Student ' s t - test .

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XBP-1S mRNA levels in LNCaP xenografts from animals treated with toyocamycin or vehicle. Tumors were harvested, RNA-extracted, and qPCR performed. Error bars indicate SEM. *P = 0.04 for LNCaP and **P = 0.02 for VCaP using unpaired Student's t-test.


PCNA staining in tumors from animals treated with either toyocamycin or saline . Scale bar : 100 μm .

PCNA staining in tumors from animals treated with either toyocamycin or saline. Scale bar: 100 μm.


XBP - 1S expression was determined by IHC in two different cohorts of human prostatectomy samples . Representative pictures of benign and tumor samples .

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XBP-1S expression was determined by IHC in two different cohorts of human prostatectomy samples.Representative pictures of benign and tumor samples.


Tissue microarrays ( Wang et al , 2010 ) containing 25 benign and 283 tumor samples were stained with a XBP - 1S specific antiserum and scored by a pathologist . The P - value indicates the difference between XBP - 1S staining ( strong and moderate ) in normal vs cancer cells using Mann - Whitney test .

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Tissue microarrays (Wang et al, 2010) containing 25 benign and 283 tumor samples were stained with a XBP-1S specific antiserum and scored by a pathologist. The P-value indicates the difference between XBP-1S staining (strong and moderate) in normal vs cancer cells using Mann-Whitney test.


XBP - 1S expression was determined by IHC of a neoadjuvant hormone therapy ( NHT ) tissue microarray containing samples from hormone naïve ( untreated ) ( n = 25 ) , patients that received NHT for 1 - 6 months ( n = 33 ) , and patients that received NHT for 7 - 12 months ( n = 50 ) , as indicated . Representative images are shown . Scale bars : 100 μm . Quantitative presentation of the data from ( C ) . * P = 0 . 006 in unpaired Student ' s t - test .

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XBP-1S expression was determined by IHC of a neoadjuvant hormone therapy (NHT) tissue microarray containing samples from hormone naïve (untreated) (n = 25), patients that received NHT for 1-6 months (n = 33), and patients that received NHT for 7-12 months (n = 50), as indicated. Representative images are shown. Scale bars: 100 μm.Quantitative presentation of the data from (C). *P = 0.006 in unpaired Student's t-test.


2 - NBD - glucose uptake in murine pancreatic endothelial cells ( MS1 ) and primary human umbilical vein endothelial cells ( HUVEC ) treated for 2 h with VEGF - B167 ( B167 ) or VEGF - B186 ( B186 ) compared to control treated cells . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . Scale bar , 20 μm .

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2-NBD-glucose uptake in murine pancreatic endothelial cells (MS1) and primary human umbilical vein endothelial cells (HUVEC) treated for 2 h with VEGF-B167 (B167) or VEGF-B186 (B186) compared to control treated cells. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). Scale bar, 20 μm.


Trans - cellular 2 - NBD - glucose transport kinetics in MS1 cells treated for 2 h with VEGF - B186 . Data presented as mean ± SEM from three individual experiments . Statistical evaluation using t - test , p - value : * * < 0 . 01 ( compared to untreated control ) .

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Trans-cellular 2-NBD-glucose transport kinetics in MS1 cells treated for 2 h with VEGF-B186. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using t-test, p-value: ** <0.01 (compared to untreated control).


Cardiac glycogen content in 17 weeks old male Vegfb + / + ( n = 6 ) , Vegfb + / - ( n = 6 ) and Vegfb - / - mice ( n = 5 ) presented as mean ± SEM . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 ( compared to Vegfb + / + ) .

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Cardiac glycogen content in 17 weeks old male Vegfb+/+ (n=6), Vegfb+/- (n=6) and Vegfb-/- mice (n=5) presented as mean ± SEM. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05 (compared to Vegfb+/+).


Glucose uptake and accumulation ( 18F - DG PET ) in hearts of HFD fed wild - type ( WT ) mice pre - treated for 1 week with 2H10 antibody ( n = 4 ) compared to control antibody treated mice ( n = 4 ) . Data presented as mean ± SEM . Statistical evaluation using two - way ANOVA , p - value : * * * * < 0 . 0001 .

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Glucose uptake and accumulation (18F-DG PET) in hearts of HFD fed wild-type (WT) mice pre-treated for 1 week with 2H10 antibody (n=4) compared to control antibody treated mice (n=4). Data presented as mean ± SEM. Statistical evaluation using two-way ANOVA, p-value: **** <0.0001.


Linear regression analysis of the 18F - DG PET data in D assessing differences in initial ( T = 0 . 9 - 6 . 5 min ) glucose uptake rate . Data presented as mean curve slope ( m ) ± SEM . Statistical evaluation comparing slope differences using analysis of covariance ( ANCOVA ) , p - value : * < 0 . 05 .

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Linear regression analysis of the 18F-DG PET data in D assessing differences in initial (T=0.9-6.5 min) glucose uptake rate. Data presented as mean curve slope (m) ± SEM. Statistical evaluation comparing slope differences using analysis of covariance (ANCOVA), p-value: * <0.05.


Relative mRNA expression of the Slc2a glucose transporter ( GLUT ) family members ( 1 - 13 ) in cardiac ECs derived from Vegfb + / + ( n = 4 ) and Vegfb - / - ( n = 4 ) mice . Data represent normalized PLIER values ( mean ± SEM ) from microarray analysis performed on n = 4 + 4 RNA samples ( one RNA sample / mouse ) .

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Relative mRNA expression of the Slc2a glucose transporter (GLUT) family members (1-13) in cardiac ECs derived from Vegfb+/+ (n=4) and Vegfb-/- (n=4) mice. Data represent normalized PLIER values (mean ± SEM) from microarray analysis performed on n=4+4 RNA samples (one RNA sample/mouse).


mRNA expression levels of the SLC2A transporter ( GLUT ) family members ( 1 - 14 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression levels of the SLC2A transporter (GLUT) family members (1-14) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


mRNA expression levels of VEGFB , SLC2A1 , NRP1 and FLT1 ( VEGFR1 ) in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression levels of VEGFB, SLC2A1, NRP1 and FLT1 (VEGFR1) in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


2 - NBD - glucose uptake in response to 2 h treatment with VEGF - B186 ( B186 ) in HUVECs exhibiting siRNA - mediated knock - down of FLT1 , NRP1 , SCL2A1 , KDR ( VEGFR2 ) or control siRNA . Data presented as mean ± SEM from three individual experiments . Statistical evaluation using one - way ANOVA and Sidak ´ s multiple comparisons test , p - value : * < 0 . 05 ( comparisons made to respective untreated control ) .

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2-NBD-glucose uptake in response to 2 h treatment with VEGF-B186 (B186) in HUVECs exhibiting siRNA-mediated knock-down of FLT1, NRP1, SCL2A1, KDR (VEGFR2) or control siRNA. Data presented as mean ± SEM from three individual experiments. Statistical evaluation using one-way ANOVA and Sidak´s multiple comparisons test, p-value: * <0.05 (comparisons made to respective untreated control).


Representative images of GLUT1 ( left panels ) and CD31 ( right panels ) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts . Scale bar , 100 μm .

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Representative images of GLUT1 (left panels) and CD31 (right panels) immunohistochemistry performed on consecutive sections from paraffin embedded mouse hearts. Scale bar, 100 μm.


mRNA expression analysis from enriched cardiac endothelial cells ( open columns , n = 4 mice ) or whole heart ( closed columns , n = 3 mice ) . Data presented as mean ± StDev relative to Rpl19 expression . Enrichment factor is calculated as mean cardiac EC / mean whole heart and presented in the box .

mRNA expression analysis from enriched cardiac endothelial cells (open columns, n=4 mice) or whole heart (closed columns, n= 3 mice). Data presented as mean ± StDev relative to Rpl19 expression. Enrichment factor is calculated as mean cardiac EC/mean whole heart and presented in the box.


Immunofluorescence labeling of GLUT1 ( white ) , early endosomes ( EEA1 , green ) and nuclei ( blue ) in HUVECs treated with VEGF - B186 for 10 min , 30 min or 2 h . Scale bar , 10 µm .

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Immunofluorescence labeling of GLUT1 (white), early endosomes (EEA1, green) and nuclei (blue) in HUVECs treated with VEGF-B186 for 10 min, 30 min or 2 h. Scale bar, 10 µm.


GLUT1 protein expression in HUVECs treated with VEGF - B186 for 15 min or 2 h . Data presented as mean ± StDev of 3 - 4 independent experiments relative to β - Actin expression . Statistical evaluation using t - test revealed no significant changes . Western blots shown in right panel .

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GLUT1 protein expression in HUVECs treated with VEGF-B186 for 15 min or 2 h. Data presented as mean ± StDev of 3-4 independent experiments relative to β-Actin expression. Statistical evaluation using t-test revealed no significant changes. Western blots shown in right panel.


Sucrose gradient ( 15 - 50 % sucrose ) fractions of HUVEC cellular lysates after 2 h VEGF - B186 stimulation were analyzed with Western blots shown in Appendix Fig S2 . Different cellular sub - compartments were identified by accumulation of specific protein markers : EEA1 for early endosomes , LIMPII for late endosomes / lysosomes , flotillin for plasma membrane / lipid rafts and calnexin for endoplasmic reticulum ( ER ) ( left panels ) . Amount and sub - cellular distribution of VEGFR1 , NRP1 and GLUT1 protein in control or VEGF - B186 stimulated HUVECs shown in panels to the right . Data represent % protein relative to total protein of interest measured from a representative experiment .

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Sucrose gradient (15-50% sucrose) fractions of HUVEC cellular lysates after 2 h VEGF-B186 stimulation were analyzed with Western blots shown in Appendix Fig S2. Different cellular sub-compartments were identified by accumulation of specific protein markers: EEA1 for early endosomes, LIMPII for late endosomes/lysosomes, flotillin for plasma membrane/lipid rafts and calnexin for endoplasmic reticulum (ER) (left panels). Amount and sub-cellular distribution of VEGFR1, NRP1 and GLUT1 protein in control or VEGF-B186 stimulated HUVECs shown in panels to the right. Data represent % protein relative to total protein of interest measured from a representative experiment.


Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF - B186 stimulation in different sucrose fractions ( upper panel ) . Data from two independent experiments are shown . Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF - B186 stimulation . Changes in GLUT1 content in response to VEGF - B treatment are shown in the table ( bottom panel ) .

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Quantification of GLUT1 protein in % relative to total measured protein in HUVECs after 2 h VEGF-B186 stimulation in different sucrose fractions (upper panel). Data from two independent experiments are shown. Green arrows mark fractions with increased GLUT1 content and red arrows mark fractions with decreased GLUT1 content after VEGF-B186 stimulation. Changes in GLUT1 content in response to VEGF-B treatment are shown in the table (bottom panel).


Representative confocal ( grey ) and epifluorescence ( blue ) images of filipin staining in HUVECs in response to 2 h VEGF - B186 stimulation ( left panels ) . Cellular cholesterol content measured by quantification of fluorescent filipin staining . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 . Scale bars , 10 µm .

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Representative confocal (grey) and epifluorescence (blue) images of filipin staining in HUVECs in response to 2 h VEGF-B186 stimulation (left panels). Cellular cholesterol content measured by quantification of fluorescent filipin staining. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05. Scale bars, 10 µm.


Measurement of cholesterol content ( B ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

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Measurement of cholesterol content (B) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


Measurement of glucose uptake ( C ) and FA uptake ( D ) in HUVECs after VEGF - B186 stimulation for different time - points . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using t - test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

O O B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Measurement of glucose uptake (C) and FA uptake (D) in HUVECs after VEGF-B186 stimulation for different time-points. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using t-test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


Colorimetric quantification of non - esterified cholesterol extracted from HUVECs stimulated with VEGF - B186 for 20 min . Data are presented as mean ± SEM of three individual experiments . Statistical evaluation using t - test , p - value : * < 0 . 05 .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Colorimetric quantification of non-esterified cholesterol extracted from HUVECs stimulated with VEGF-B186 for 20 min. Data are presented as mean ± SEM of three individual experiments. Statistical evaluation using t-test, p-value: * <0.05.


Uptake of a fluorescent cholesterol tracer ( TopFluor ) in HUVECs after VEGF - B186 stimulation for 20 min or 2 h . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 , * * * < 0 . 001 ( compared to respective untreated control ) .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Uptake of a fluorescent cholesterol tracer (TopFluor) in HUVECs after VEGF-B186 stimulation for 20 min or 2 h. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01, *** <0.001 (compared to respective untreated control).


Quantification of the major cholesteryl ester ( CE ) species in HUVECs after VEGF - B186 stimulation for 20 min , analyzed by lipid mass spectrometry . Data presented as mean ± StDev of an experiment performed in triplicates .

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

Quantification of the major cholesteryl ester (CE) species in HUVECs after VEGF-B186 stimulation for 20 min, analyzed by lipid mass spectrometry. Data presented as mean ± StDev of an experiment performed in triplicates.


Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 1 h or after 15 min treatment with cholesterol - extracting methyl - beta - cyclodextrin ( MbCD ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to untreated control ) .

O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 1 h or after 15 min treatment with cholesterol-extracting methyl-beta-cyclodextrin (MbCD). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: *** <0.001 (compared to untreated control).


Measurement of cellular cholesterol content ( upper panel ) and glucose uptake ( lower panel ) in HUVECs treated with VEGF - B186 for 15 min alone or supplemented with soluble methyl - beta - cyclodextrin complexed with cholesterol ( 5 , 10 or 20 μg / ml cholesterol ) . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to control w / o cholesterol ) .

O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O

Measurement of cellular cholesterol content (upper panel) and glucose uptake (lower panel) in HUVECs treated with VEGF-B186 for 15 min alone or supplemented with soluble methyl-beta-cyclodextrin complexed with cholesterol (5, 10 or 20 μg/ml cholesterol). Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to control w/o cholesterol).


mRNA expression analysis of lipoprotein receptors in HUVECs . Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments .

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mRNA expression analysis of lipoprotein receptors in HUVECs. Data presented as mean ± StDev relative to RPL19 expression from 3 independent experiments.


Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cellular cholesterol content in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


glucose uptake ( C ) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 ( SCARB1 , SRBI ) followed by 15 min stimulation with VEGF - B186 . Data presented as mean ± SEM from representative experiments performed in triplicates . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to respective control ) .

B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

glucose uptake (C) in HUVECs treated with control siRNA or siRNA targeting LDLR or Scavenger receptor B1 (SCARB1, SRBI) followed by 15 min stimulation with VEGF-B186. Data presented as mean ± SEM from representative experiments performed in triplicates. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to respective control).


Cellular cholesterol content of ECs freshly isolated from Ldlr + / + and Ldlr - / - mice stimulated for 15 min with VEGF - B186 . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * * * < 0 . 001 ( compared to Ldlr + / + control ) .

O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cellular cholesterol content of ECs freshly isolated from Ldlr+/+ and Ldlr-/- mice stimulated for 15 min with VEGF-B186. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: *** <0.001 (compared to Ldlr+/+ control).


Cellular cholesterol content ( left panel ) and glucose uptake ( right panel ) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin . Data presented as mean ± SEM from a representative experiment performed in triplicates . Statistical evaluation using one - way ANOVA and Tukey ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 , * * * < 0 . 001 ( compared to untreated control ) . p - value : # < 0 . 05 , # # # < 0 . 001 ( compared to simvastatin control ) .

O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O B-MEASURED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O

Cellular cholesterol content (left panel) and glucose uptake (right panel) of HUVECs treated with de novo cholesterol synthesis inhibitor simvastatin. Data presented as mean ± SEM from a representative experiment performed in triplicates. Statistical evaluation using one-way ANOVA and Tukey´s multiple comparisons test, p-value: * <0.05, ** <0.01, *** <0.001 (compared to untreated control). p-value: # <0.05, ### <0.001 (compared to simvastatin control).


Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF - B167 or VEGF - B186 stimulation . Data presented as mean ± SEM from three independent experiments . Western blot shown below . Statistical evaluation using t - test , p - value : * < 0 . 05 ( compared to control ) .

O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Cell surface biotinylated LDLR protein levels in HUVECs after 20 min of VEGF-B167 or VEGF-B186 stimulation. Data presented as mean ± SEM from three independent experiments. Western blot shown below. Statistical evaluation using t-test, p-value: * <0.05 (compared to control).


Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non - permeabilized cells ( white , left panels ) , Dil - LDL cell surface binding ( evaluated at 4 ° C , middle panels ) and Dil - LDL uptake ( evaluated at 37 ° C , right panels ) in HUVECs in response to VEGF - B186 stimulation for 10 min . Scale bar , 10 µm .

O O 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-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative images of cell surface distribution of the LDLR by immunofluorescence staining of non-permeabilized cells (white, left panels), Dil-LDL cell surface binding (evaluated at 4 °C, middle panels) and Dil-LDL uptake (evaluated at 37 °C, right panels) in HUVECs in response to VEGF-B186 stimulation for 10 min. Scale bar, 10 µm.


Quantification of non - esterified cholesterol content by means of filipin staining in the blood vessel compartment ( podocalyxin + ) in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - mice ( n = 7 ) , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * < 0 . 01 ( compared to Vegfb + / + mice ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of non-esterified cholesterol content by means of filipin staining in the blood vessel compartment (podocalyxin+) in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- mice (n=7), normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, ** <0.01 (compared to Vegfb+/+ mice).


Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 15 - week old male C57BL / 6 Vegfb + / - ( n = 5 ) and Vegfb - / - ( n = 7 ) mice , normalized to wildtype ( Vegfb + / + ) mice ( n = 8 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * * < 0 . 01 ( compared to Vegfb + / + mice ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 15-week old male C57BL/6 Vegfb+/- (n=5) and Vegfb-/- (n=7) mice, normalized to wildtype (Vegfb+/+) mice (n=8). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: ** <0.01 (compared to Vegfb+/+ mice).


Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 25 - week old male CD1 Flt1 + / lacz ( n = 20 ) mice , normalized to wildtype CD1 ( Flt1 + / + ) mice ( n = 16 ) . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using t - test , p - value : * * < 0 . 01 .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 25-week old male CD1 Flt1+/lacz (n=20) mice, normalized to wildtype CD1 (Flt1+/+) mice (n=16). Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using t-test, p-value: ** <0.01.


Quantification of non - esterified cholesterol content by means of filipin staining in hearts of 12 to 16 - week old male C57BL / 6 wildtype ( Vegfb + / + ) ( n = 4 ) , BKS . C57BL / 6 db / + Vegfb + / + ( n = 8 ) , db / db Vegfb + / - ( n = 10 ) and db / db Vegfb - / - ( n = 4 ) mice , normalized to db / db Vegfb + / + ( n = 6 ) mice . Data presented as box - and - whisker plots . Boxes represents lower / upper quartiles with the median values indicated with a horizontal line . Whiskers represent min - max values . Statistical evaluation using one - way ANOVA and Dunnett ´ s multiple comparisons test , p - value : * < 0 . 05 , * * * < 0 . 001 ( compared to db / db Vegfb + / + mice ) .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of non-esterified cholesterol content by means of filipin staining in hearts of 12 to 16-week old male C57BL/6 wildtype (Vegfb+/+) (n=4), BKS.C57BL/6 db/+ Vegfb+/+ (n=8), db/db Vegfb+/- (n=10) and db/db Vegfb-/- (n=4) mice, normalized to db/db Vegfb+/+ (n=6) mice. Data presented as box-and-whisker plots. Boxes represents lower/upper quartiles with the median values indicated with a horizontal line. Whiskers represent min-max values. Statistical evaluation using one-way ANOVA and Dunnett´s multiple comparisons test, p-value: * <0.05, *** <0.001 (compared to db/db Vegfb+/+ mice).