1x106 PC3 with control or CTH knockdown were orthotopically injected into mouse prostate for 60 days . ( B ) , volume ( C ) were compared . Data are presented as means ± ( n = 10 mice per group ) . Data information : Student ' s t - was used for statistical ( * P < 0 . 05 ) .

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

1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. The tumor weight (B), and tumor volume (C) were compared. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


1x106 PC3 with control or CTH knockdown were orthotopically injected into mouse prostate for 60 days . Incidences of paraaortic lymph nodes metastasis ( D ) bone metastasis ( E ) are shown . Data are presented as means ± ( n = 10 mice per group ) . Data information : Student ' s t - was used for statistical ( * P < 0 . 05 ) .

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

1x106 PC3 cells with control or CTH knockdown were orthotopically injected into the mouse prostate for 60 days. Incidences of paraaortic lymph nodes metastasis (D) and bone metastasis (E) are shown. Data are presented as means ± SEM (n = 10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


( F ) Real - Time RT - PCR for CTH mRNA in PC3 orthotopic tumors from shCon or shCTH group . Data shown represent normalized means ± ( n = 10 mice per group ) . Data information : Student ' s t - was used for statistical ( * P < 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Real-Time RT-PCR analysis for CTH mRNA levels in PC3 orthotopic tumors from shCon or shCTH group. Data shown represent normalized means ± SEM (n=10 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Immunohistochemical of paraffin - embedded xenograft prostate using CD31 ( G ) antibody . total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± ( n = 4 - 5 mice per group ) . Data information : Student ' s t - was used for statistical ( * P < 0 . 05 ) .

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

Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using CD31 (G) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


Immunohistochemical of paraffin - embedded xenograft prostate using LYVE - 1 ( H ) antibody . total vessels number per mm2 was quantified . Scale bars : 0 . 1 mm . Data are presented as means ± ( n = 4 - 5 mice per group ) . Data information : Student ' s t - was used for statistical ( * P < 0 . 05 ) .

Immunohistochemical analysis of paraffin-embedded xenograft prostate tumor using LYVE-1 (H) antibody. The total vessels number per mm2 was quantified. Scale bars: 0.1 mm. Data are presented as means ± SEM (n=4-5 mice per group). Data information: Student's t-test was used for the statistical analysis (*P<0.05).


for apical marker CD13 on liver tissue sections from untreated mice or at indicated time points post PH . Shown are maximum projections of 50 µm z - stacks covering an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions of CV ( orange ) PV ( blue ) areas are shown as magnifications on left right , respectively .

Fluorescence staining for the apical marker CD13 on liver tissue sections from untreated mice or animals at indicated time points post PH. Shown are maximum projections of 50 µm z-stacks covering an entire CV-PV axis (CV, left; PV, right). Indicated regions of the CV (orange) and PV (blue) areas are shown as magnifications on the left and right, respectively.


of BC diameter within 18 zones along CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) in livers from untreated or mice at indicated timepoints post PH . diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in ( a ) . zones directly adjacent to CV PV were excluded from ( ~ 1 layer ) . Mean ± s . e . m , n = 3 - 6 mice per timepoint . BC diameter of untreated mice vs . 0 . 8 d , p = 1 . 62 * 10 - 11 ; untreated mice vs . 1 . 5 d , p = 1 . 18 * 10 - 12 ; untreated mice vs . 2 . 0 d , p = 5 . 69 * 10 - 11 ; untreated mice vs . 3 . 0 d , p = 6 . 92 * 10 - 12 ; untreated mice vs . 5 . 0 d , p = 1 . 65 * 10 - 7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) in livers from untreated animals or mice at indicated timepoints post PH. The diameter was measured from 3D BC network reconstructions of IF image stacks of CD13 as shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=3-6 mice per timepoint. BC diameter of untreated mice vs. 0.8 d, p=1.62*10-11; untreated mice vs. 1.5 d, p=1.18*10-12; untreated mice vs. 2.0 d, p=5.69*10-11; untreated mice vs. 3.0 d, p=6.92*10-12; untreated mice vs. 5.0 d, p=1.65*10-7.


EM of BC on liver tissue sections of untreated mice ( left ) 1 . 8 d post sham OP ( middle ) or PH ( right ) .

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EM images of BC on liver tissue sections of untreated mice (left) and 1.8 d post sham OP (middle) or PH (right).


of BC perimeter ( d ) total BC ( e ) from EM as representatively shown in ( c ) . Box - whisker plot with median , 25 - 75 quartiles minimum / maximum error bars , n = 5 - 6 mice per condition . In ( d ) , BC perimeter of untreated vs . sham condition , p = 0 . 94 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 01 . In ( e ) , BC of untreated vs . sham condition , p = 0 . 86 ( n . s . ) ; Untreated vs . PH condition , p = 0 . 03 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 BC perimeter (d) and total BC membrane length (e) from EM images as representatively shown in (c). Box-whisker plot with median, 25-75 quartiles and minimum/maximum error bars, n=5-6 mice per condition. In (d), BC perimeter of untreated vs. sham condition, p=0.94 (n.s.); Untreated vs. PH condition, p=0.01. In (e), BC membrane length of untreated vs. sham condition, p=0.86 (n.s.); Untreated vs. PH condition, p=0.03.


IF stainings of apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL . Shown are maximum projections of 50 µm z - stacks in CV ( left , orange ) PV ( right , blue ) region . in ( a ) ( f ) are background - subtracted . Scale bars , 50 µm ( a ) , 1 µm ( c ) , 5 µm ( f ) .

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

IF stainings of the apical marker CD13 on liver tissue sections from livers at 1 d post sham OP or BDL. Shown are maximum projections of 50 µm z-stacks in the CV (left, orange) and PV (right, blue) region. Images in (a) and (f) are background-subtracted. Scale bars, 50 µm (a), 1 µm (c), 5 µm (f).


stainings for F - actin apical marker CD13 in PV on liver tissue sections from untreated mice or at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm of apical F - actin from as representatively shown in ( a , as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid ) or sham OP ( dashed ) . Data is normalized to untreated ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 ( b ) mice per timepoint .

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

Fluorescence stainings for F-actin and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of apical F-actin intensity from images as representatively shown in (a, as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 (b) mice per timepoint.


stainings for pMLC apical marker CD13 in PV on liver tissue sections from untreated mice or at indicated time points post PH . Arrows indicate BC . Scale bar , 5 µm of pMLC ( d ) from as representatively shown in ( a , c ) as well as Appendix Fig . S2A , B , at indicated time points post PH ( solid ) or sham OP ( dashed ) . Data is normalized to untreated ( timepoint 0 ) . Mean ± s . e . m , n = 2 - 5 ( d ) mice per timepoint .

Fluorescence stainings for pMLC and the apical marker CD13 in the PV area on liver tissue sections from untreated mice or animals at indicated time points post PH. Arrows indicate BC. Scale bar, 5 µm Quantification of pMLC (d) intensity from images as representatively shown in (a,c) as well as Appendix Fig. S2A,B, at indicated time points post PH (solid line) or sham OP (dashed line). Data is normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=2-5 (d) mice per timepoint.


stainings for YAP , PCNA with marker DAPI on liver tissue sections from untreated mice at indicated time points post PH . show an entire CV - PV axis ( CV , left ; PV , right ) . Note , bright YAP in PV from cholangiocytes of bile .

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

Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.


stainings for YAP , PCNA with marker DAPI on liver tissue sections from untreated mice at indicated time points post PH . show an entire CV - PV axis ( CV , left ; PV , right ) . Indicated regions ( orange squares ) are shown as magnifications in ( b ) . Note , bright YAP in PV from cholangiocytes of bile .

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

Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice and animals at indicated time points post PH. Images show an entire CV-PV axis (CV, left; PV, right). Indicated regions (orange squares) are shown as magnifications in (b). Note, the bright YAP fluorescence in the PV area stem from cholangiocytes of the bile duct.


of mean YAP ( green ) PCNA ( magenta ) from of liver tissue sections at indicated timepoints post PH ( solid ) sham OP ( dashed ) as representatively shown in ( a ) Appendix Fig . S4A . Data was normalized to untreated ( timepoint 0 ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . YAP of PH vs . sham time course , p = 2 . 97 * 10 - 5 . PCNA of PH vs . sham time course , p = 7 . 51 * 10 - 6 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 mean nuclear YAP (green) and PCNA (magenta) intensity from images of liver tissue sections at indicated timepoints post PH (solid line) and sham OP (dashed line) as representatively shown in (a) and Appendix Fig. S4A. Data was normalized to untreated animals (timepoint 0). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of PH vs. sham time course, p=2.97*10-5. Nuclear PCNA intensity of PH vs. sham time course, p=7.51*10-6.


Spatial of mean YAP from as representatively shown in ( a ) Appendix Fig . S4A , at indicated time points post PH ( solid ) or sham OP ( dashed ) in 10 zones within CV - PV axis ( zone 1 , CV ; zone 10 , PV ) . Data is normalized to untreated ( not shown ) . Mean ± s . e . m , n = 3 - 5 mice per timepoint . YAP of sham vs . PH mice at 0 . 8d , p = 5 . 35 * 10 - 5 ; 1 . 5d , p = 3 . 95 * 10 - 5 ; 2d , p = 5 . 99 * 10 - 5 ; 3d , p = 2 . 16 * 10 - 5 ; 5d , p = 0 . 01 .

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

Spatial analysis of the mean nuclear YAP intensity from images as representatively shown in (a) and Appendix Fig. S4A, at indicated time points post PH (solid line) or sham OP (dashed line) in 10 zones within the CV-PV axis (zone 1, CV area; zone 10, PV area). Data is normalized to untreated animals (not shown). Mean ± s.e.m, n=3-5 mice per timepoint. Nuclear YAP intensity of sham vs. PH mice at 0.8d, p=5.35*10-5; 1.5d, p=3.95*10-5; 2d, p=5.99*10-5; 3d, p=2.16*10-5; 5d, p=0.01.


stainings for YAP , CD13 with marker DAPI on liver tissue sections from an untreated mouse or at indicated time points post PH . Arrows indicate BC . Note enrichment of apical YAP at 0 . 5 1 . 5d post PH .

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

Fluorescence stainings for YAP, CD13 and with the nuclear marker DAPI on liver tissue sections from an untreated mouse or animals at indicated time points post PH. Arrows indicate BC. Note the enrichment of apical YAP at 0.5 and 1.5d post PH.


Fractionation of liver tissue at 2d post PH or sham ( Sh ) OP untreated ( UT ) mice for ( Nuc ) , ( Cyto ) ( Mem ) fraction . TRF2 , transferrin receptor2 ( marker ) ; Histone H3 ( marker ) ; GAPDH , Glyceraldehyde 3 - phosphate dehydrogenase ( marker ) . of YAP in , fraction from immunoblots as representatively shown in ( f ) . YAP signal is normalized to fraction marker ( GAPDH , cytoplasm ; Histon H3 , nuclei ; TRF2 , ) to untreated condition ( not shown ) . n = 3 ( sham ) or 4 ( PH ) ; data is shown as mean + / - s . e . m . Differences between sham PH conditions for , fractions are not significant .

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

Fractionation of liver tissue at 2d post PH or sham (Sh) OP and untreated (UT) mice for nuclear (Nuc), cytoplasmic (Cyto) and membrane (Mem) fraction. TRF2, transferrin receptor2 (membrane marker); Histone H3 (nuclear marker); GAPDH, Glyceraldehyde 3-phosphate dehydrogenase (cytoplasmic marker). Quantification of YAP in the nuclear, cytoplasmic and membrane fraction from immunoblots as representatively shown in (f). YAP signal is normalized to the fraction marker (GAPDH, cytoplasm; Histon H3, nuclei; TRF2, membrane) and to the untreated condition (not shown). n = 3 (sham) or 4 (PH); data is shown as mean +/- s.e.m. Differences between sham and PH conditions for cytoplasmic, nuclear and membrane fractions are not significant.


EM image of immunogold - labelled YAP on liver tissue sections at 1 . 8 d post PH . Image ( A ) shows a BC in between two hepatocytes . Indicated regions ( black squares ) of apical ( a ' ) basolateral ( a ' ' ) of hepatocyte are shown as magnification . Arrows indicate gold particles .

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

EM image of immunogold-labelled YAP on liver tissue sections at 1.8 d post PH. Image (A) shows a BC in between two hepatocytes. Indicated regions (black squares) of the apical (a') and the basolateral (a'') membrane of the hepatocyte are shown as magnification. Arrows indicate gold particles.


Correlative electron microcopy of F - actin ( B ) YAP ( C ) on a liver tissue section at 1 . 8 d post PH . YAP was detected by immunogold - labelling , F - actin by with phalloidin . Indicated BC regions with high ( c ' , c ' ' ) or low ( c ' ' ' ) F - actin are shown as magnifications . Arrows indicate gold particles .

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

Correlative light and electron microcopy of F-actin (B) and YAP (C) on a liver tissue section at 1.8 d post PH. YAP was detected by immunogold-labelling, F-actin by fluorescence staining with phalloidin. Indicated BC regions with high (c', c'') or low (c''') F-actin levels are shown as magnifications. Arrows indicate gold particles.


stainings for YAP ( green ) , F - actin ( magenta ) with marker DAPI ( blue ) of hepatocyte cultures treated with DMSO ( control ) , Y27 , deoxycholic acid ( DCA ) or DCA + Y27 for ~ 18 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets .

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

Fluorescence stainings for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue) of primary hepatocyte cultures treated with DMSO (control), Y27, deoxycholic acid (DCA) or DCA+Y27 for ~18 h. Indicated areas (dashed rectangle) are shown as magnifications in insets.


of mean YAP from of hepatocytes treated with DMSO ( control ) , Y27 , DCA or DCA + Y27 for ~ 18 h as representatively shown in ( A ) . Data is normalized to untreated ( not shown ) . Mean ± s . e . m . , n = 7 ; DCA vs . DMSO p = 8 . 98 * 10 - 5 ; DCA + Y27 vs . DMSO , p = 0 . 05 ; Y27 vs . DMSO , p = 0 . 55 ( n . s . ) ; DCA + Y27 vs . DCA p = 0 .

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

Quantification of the mean nuclear YAP intensity from fluorescence images of primary hepatocytes treated with DMSO (control), Y27, DCA or DCA+Y27 for ~18 h as representatively shown in (A). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n = 7; DCA vs. DMSO p=8.98*10-5; DCA+Y27 vs. DMSO, p=0.05; Y27 vs. DMSO, p=0.55 (n.s.); DCA+Y27 vs. DCA p = 0.05


Western blot of pMLC GAPDH ( loading control ) of hepatocyte culture lysates . were untreated or incubated for 18 h with indicated

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Western blot of pMLC and GAPDH (loading control) of primary hepatocyte culture lysates. Cells were untreated or incubated for 18 h with the indicated compounds


stainings of hepatocytes for YAP ( green ) , F - actin ( magenta ) with marker DAPI ( blue ) . were treated with DMSO ( control ) , Y27 , SMIFH2 or Cytochalasin D ( CytoD ) for 6 h . Indicated areas ( dashed rectangle ) are shown as magnifications in insets . Note dilation of canaliculi ( asterisks ) fragmentation of F - actin ( arrows ) upon CytoD treatment .

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

Fluorescence stainings of primary hepatocytes for YAP (green), F-actin (magenta) and with the nuclear marker DAPI (blue). Cells were treated with DMSO (control), Y27, SMIFH2 or Cytochalasin D (CytoD) for 6 h. Indicated areas (dashed rectangle) are shown as magnifications in insets. Note the dilation of canaliculi (asterisks) and fragmentation of F-actin (arrows) upon CytoD treatment.


of mean YAP from of hepatocytes treated with DMSO , Saline , Y27 , Fasudil , SMIFH2 , CK666 , CytoD or Latrunculin A ( LatA ) for 6 h . Saline serves as control for Fasudil , DMSO serves as control for other conditions . Inhibitors affecting similar actin processes are displayed in same grey level . Saline , Fasudil , CK666 LatA conditions are not shown in ( d ) . Data is normalized to untreated ( not shown ) . Mean ± s . e . m . , n = 3 - 5 ; DMSO vs . Y27 , p = 0 . 002 ; DMSO vs . CytoD , p = 5 . 11 * 10 - 7 ; DMSO vs . LatA , p = 0 . 01 ; DMSO vs . CK666 , p = 0 . 15 ( n . s . ) ; DMSO vs . SMIFH2 , p = 0 . 11 ( n . s . ) ; Saline vs . Fasudil , p = 0 . 92 .

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

Quantification of the mean nuclear YAP intensity from images of primary hepatocytes treated with DMSO, Saline, Y27, Fasudil, SMIFH2, CK666, CytoD or Latrunculin A (LatA) for 6 h. Saline serves as control for Fasudil, DMSO serves as control for all other conditions. Inhibitors affecting similar actin processes are displayed in the same grey level. Saline, Fasudil, CK666 and LatA conditions are not shown in (d). Data is normalized to untreated cells (not shown). Mean ± s.e.m., n=3-5; DMSO vs. Y27, p=0.002; DMSO vs. CytoD, p=5.11*10-7; DMSO vs. LatA, p=0.01; DMSO vs. CK666, p=0.15 (n.s.); DMSO vs. SMIFH2, p= 0.11 (n.s.); Saline vs. Fasudil, p= 0.92.


Western blot of pMLC GAPDH ( loading control ) in untreated or actin inhibitor - treated hepatocyte culture lysates . Inhibitor treatments are same as in ( E ) . Scale bars , 20 µm ( A , D ) .

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

Western blot of pMLC and GAPDH (loading control) in untreated or actin inhibitor-treated primary hepatocyte culture lysates. Inhibitor treatments are the same as in (E). Scale bars, 20 µm (A, D).


stainings for apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH , treated with saline ( control ) or Rho kinase inhibitor Fasudil for 1 h . Shown are maximum projections of 50 µm stacks in PV . of BC diameter within 18 zones along CV - PV axis ( zone 1 , peri - central ; zone 18 , peri - portal ) from mice at 2 d post sham OP ( green ) or PH ( red ) , treated with saline ( control , solid ) or Fasudil ( dashed ) . Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in ( a ) . zones directly adjacent to CV PV were excluded from ( ~ 1 layer ) . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . BC diameter of sham operated mice treated with saline vs . Fasudil , p = 6 . 59 * 10 - 8 ; BC diameter of PH mice treated with saline vs . Fasudil , p = 2 . 31 * 10 - 14 .

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

Fluorescence stainings for the apical marker CD13 on liver tissue sections from mice at 2 d post sham OP or PH, treated with saline (control) or the Rho kinase inhibitor Fasudil for 1 h. Shown are maximum projections of 50 µm stacks in the PV area. Quantification of BC diameter within 18 zones along the CV-PV axis (zone 1, peri-central; zone 18, peri-portal) from mice at 2 d post sham OP (green) or PH (red), treated with saline (control, solid line) or Fasudil (dashed line). Diameter was measured from 3D BC network reconstructions of image stacks as representatively shown in (a). The zones directly adjacent to the CV and PV were excluded from the analysis (~ 1 cell layer). Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. BC diameter of sham operated mice treated with saline vs. Fasudil, p= 6.59*10-8; BC diameter of PH mice treated with saline vs. Fasudil, p=2.31*10-14.


stainings for YAP , PCNA with marker DAPI on liver tissue sections from untreated mice or at 2 d post sham OP or PH , treated with saline ( control ) or Fasudil for 1 h . Indicated regions ( dashed rectangle ) are shown as magnifications in insets . of mean YAP within 10 zones along CV - PV axis , ( zone 1 , peri - central ; zone 10 , peri - portal ) from IF as representatively shown in ( c ) . Data was normalized to untreated . Mean ± s . e . m , n = 7 - 8 mice per condition from 3 independent experiments . YAP of sham operated mice treated with saline vs . Fasudil , p = 0 . 13 ( n . s . ) ; YAP of PH mice treated with saline vs . Fasudil , p = 0 . 0004 .

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Fluorescence stainings for YAP, PCNA and with the nuclear marker DAPI on liver tissue sections from untreated mice or animals at 2 d post sham OP or PH, treated with saline (control) or Fasudil for 1 h. Indicated regions (dashed rectangle) are shown as magnifications in insets. Quantification of the mean nuclear YAP intensity within 10 zones along the CV-PV axis, (zone 1, peri-central; zone 10, peri-portal) from IF images as representatively shown in (c). Data was normalized to untreated animals. Mean ± s.e.m, n=7-8 mice per condition from 3 independent experiments. Nuclear YAP intensity of sham operated mice treated with saline vs. Fasudil, p=0.13 (n.s.) ; nuclear YAP intensity of PH mice treated with saline vs. Fasudil, p=0.0004.


b ) Caco - 2 were infected with SARS - CoV - 2 as per Figure 1A . were washed , fixed stained with DAPI ( blue ) to visualise nuclei incubated with anti - N ( red ) to visualise SARS - CoV - 2 infected . Scale bar denotes 10 µM .

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b) Caco-2 cells were infected with SARS-CoV-2 as per Figure 1A. Cells were washed, fixed and stained with DAPI (blue) to visualise cell nuclei and incubated with anti-N protein (red) to visualise SARS-CoV-2 infected cells. Scale bar denotes 10 µM.


c ) Infection rates were calculated using automated imaging software ( see materials methods ) calculated from six fields of view per time point per biological replicate . Data point indicates sample means error bars standard error of mean ( ) ( n = 3 ) . d ) SARS - CoV - 2 detected plotted as a function of time where each data point denotes a different replicate ( n = 3 ) .

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c) Infection rates were calculated using automated imaging software (see materials and methods) calculated from six fields of view per time point per biological replicate. Data point indicates the sample means and the error bars the standard error of the mean (SEM) (n=3). d) SARS-CoV-2 detected proteins plotted as a function of time where each data point denotes a different replicate (n=3).


a ) Total number of significantly up - or down - regulated ( | z - score | > 1 . 96 , q - value < 0 . 05 ) per time point of SARS - CoV - 2 infected Caco - 2 as described in 1A . that were significantly changed in abundance ( left ) stability ( right ) are sub - grouped according to either down - regulation / destabilization ( red ) or up - regulation / stabilisation ( turquoise ) .

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a) Total number of significantly up- or down-regulated proteins (|z-score| >1.96, q-value <0.05) per time point of SARS-CoV-2 infected Caco-2 cells as described in 1A. Proteins that were significantly changed in abundance (left) and stability (right) are sub-grouped according to either down-regulation/destabilization (red) or up-regulation/stabilisation (turquoise).


c ) SAFB phosphopeptide abundance changes during SARS - CoV - 2 infection . Vero phosphopeptide data from ( Bouhaddou et al , 2020 ) . Data previously acquired from Vero infected with SARS - CoV - 2 ( Bouhaddou et al , 2020 ) . S604 phosphosite that temporally coincides with SAFB thermal destabilisation ( Potel et al , 2020 ) . Shown above plot are thermostability abundance profiles of SAFB in SARS - CoV - 2 infected Caco - 2 as described in 1A ( n = 3 ) . Asterisk denotes significant regulation ( see methods ) .

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c) SAFB phosphopeptide abundance changes during SARS-CoV-2 infection. Vero cell phosphopeptide data from(Bouhaddou et al, 2020). Data previously acquired from Vero cells infected with SARS-CoV-2 (Bouhaddou et al, 2020). S604 phosphosite that temporally coincides with SAFB thermal destabilisation (Potel et al, 2020). Shown above the line plot are the thermostability and abundance profiles of SAFB in SARS-CoV-2 infected Caco-2 cells as described in 1A (n=3). Asterisk denotes significant regulation (see methods).


D ) Thermal stability profile for unmodified SAFB peptides ( green ) phosphorylated S604 ( pS604 ) ( purple ) in HeLa . Data from ( Potel et al , 2020 ) .

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D) Thermal stability profile for all unmodified SAFB peptides (green) and the phosphorylated S604 (pS604) (purple) in HeLa cells. Data from (Potel et al, 2020).


b ) corresponding HSP90 inhibitor , Tanespimycin , effectively suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Calu - 3 were pre - incubated with compound , followed by addition of SARS - CoV - 2 subsequent co - incubation in presence of compound for 20 - 24 hours . Viral load was quantified in parallel using antibody - detection of double stranded ( orange ) . Compound toxicity in Calu - 3 was assessed ( purple ) treated with compound alone for 20 - 24 hours .

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b) The corresponding HSP90 inhibitor, Tanespimycin, effectively suppressed SARS-CoV-2 proliferation (n=2). Calu-3 cells were pre-incubated with the compound, followed by the addition of SARS-CoV-2 and subsequent co-incubation in the presence of the compound for 20-24 hours. Viral load was quantified in parallel using antibody-based detection of double stranded RNA (orange). Compound toxicity in Calu-3 cells was assessed (purple) treated with the compound alone for 20-24 hours.


d ) Cyp1a1 inhibitor suppressed SARS - CoV - 2 proliferation ( n = 2 ) . Dose response curves were performed as described in 4A . See Appendix Figure S7 for additional tested drugs . Data displayed as described in 5B .

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d) The Cyp1a1 inhibitor suppressed SARS-CoV-2 proliferation (n=2). Dose response curves were performed as described in 4A. See Appendix Figure S7 for additional tested drugs. Data displayed as described in 5B.


A . NS1 dimer with one subunit in grey other colored by domain ( green , β - roll ; orange , wing with magenta connector subdomain ; blue , central β - ladder ) . N - linked glycosylation sites are shown in sticks , glycans are shown in spheres . B . Topology diagram for NS1 monomer , colored as pattern in ( A ) . Glycosylation sites are indicated with red hexagons disulfide bonds with yellow circles . C . Side views of NS1 dimer from wing ( left ) end of β - ladder ( right ) . β - roll ( green ) , connector subdomain ( orange ) intertwined loop ( yellow ) of wing domain form a discontinuous protrusion on one face of β - ladder with spaghetti loop ( cyan ) on other face . wing domain is omitted from left image for clarity .

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A. NS1 dimer with one subunit in grey and the other colored by domain (green, β-roll; orange, wing with magenta connector subdomain; blue, central β-ladder). N-linked glycosylation sites are shown in sticks, and the glycans are shown in spheres.B. Topology diagram for the NS1 monomer, colored as the pattern in (A). Glycosylation sites are indicated with red hexagons and disulfide bonds with yellow circles.C. Side views of the NS1 dimer from the wing (left) and the end of the β-ladder (right). The β-roll (green), connector subdomain (orange) and the intertwined loop (yellow) of the wing domain form a discontinuous protrusion on one face of the β-ladder with the spaghetti loop (cyan) on the other face. The wing domain is omitted from the left image for clarity.


ZIKV NS1 hexamer is modeled by using full - DENV2 NS1 structure ( PDB code : 4O6B ) ( Akey et al , 2014 ) . Each monomer is colored by domains ( green , β - roll ; orange , wing ; magenta , connector subdomain ; blue , central β - ladder ) . intertwined loop " spike " of ZIKV NS1 contributing to association is colored in red indicated by dotted lines .

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The ZIKV NS1 hexamer is modeled by using the full-length DENV2 NS1 structure (PDB code: 4O6B) (Akey et al, 2014). Each monomer is colored by domains (green, β-roll; orange, wing; magenta, connector subdomain; blue, central β-ladder). The intertwined loop "spike" of ZIKV NS1 contributing to membrane association is colored in red and indicated by dotted lines.


ABC . Electrostatic surface views of NS1 from DENV2 ( PDB : 4O6B , A ) WNV ( PDB : 4O6C , B ) ( Akey et al , 2014 ) , as well as ZIKV © , showing diverse characteristics on certain regions . Surfaces are colored by electrostatic potential at neutral pH from - 2 kT ( red ) to + 2 kT ( blue ) using PyMOL ( http : / / pymol . org / ) .

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ABC. Electrostatic surface views of NS1 from DENV2 (PDB: 4O6B, A) and WNV (PDB: 4O6C, B) (Akey et al, 2014), as well as ZIKV©, showing diverse characteristics on certain regions. Surfaces are colored by electrostatic potential at neutral pH from -2 kT (red) to +2 kT (blue) using PyMOL (http://pymol.org/).


A . NS1 surface is colored using ConSurf server ( Ashkenazy et al , 2016 ) according to conservation from most conserved ( magenta ) to most divergent ( cyan ) on an alignment of NS1 sequences from 73 flaviviruses .

A. The NS1 surface is colored using the ConSurf server (Ashkenazy et al, 2016) according to sequence conservation from the most conserved (dark magenta) to the most divergent (dark cyan) based on an alignment of NS1 sequences from 73 flaviviruses.


B . Models of intertwined loop " spike " interacting with . disordered " spike " regions are indicated by dash lines in DENV2 WNV NS1 structures , which consist of hydrophobic or positively charged .

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B. Models of intertwined loop "spike" interacting with the cellular membrane. The disordered "spike" regions are indicated by dash lines in DENV2 and WNV NS1 structures, which consist of hydrophobic or positively charged amino acids.


A . B - factor putty representation of intertwined loop structure . Shades of blue a narrower tube indicate regions with lower B - factors , whereas orange to red a wider tube indicate regions with higher B - factors . figure was prepared using PyMOL ( http : / / pymol . org / ) . B . 2Fo - Fc electron density map for intertwined loop ( yellow ) " spike " region residues contoured at 1 . 0 sigma is represented in blue . C . Detailed interactions of highly conserved residues W115 W118 with neighboring residues to stabilize conformation of intertwined loop . W115 forms a hydrogen - bonding interaction with E74 from α2 helix . W118 inserts into hydrophobic interspace between α1 α2 helix , formed by P36 , L42 , V71 , L75 I78 , W118 also interact with P36 D37 by hydrogen - bonding interactions .

A. B-factor putty representation of the intertwined loop structure. Shades of blue and a narrower tube indicate regions with lower B-factors, whereas orange to red and a wider tube indicate regions with higher B-factors. The figure was prepared using PyMOL (http://pymol.org/).B. The 2Fo-Fc electron density map for the intertwined loop (yellow) "spike" region residues contoured at 1.0 sigma is represented in blue.C. Detailed interactions of the highly conserved residues W115 and W118 with the neighboring residues to stabilize the conformation of the intertwined loop. W115 forms a hydrogen-bonding interaction with E74 from α2 helix. W118 inserts into the hydrophobic interspace between α1 and α2 helix, formed by P36, L42, V71, L75 and I78, and W118 also interact with P36 and D37 by hydrogen-bonding interactions.


( a ) Ten - month - old adult mice 20 - month - old aged mice were treated with an intraperitoneal injection of 5 mg kg - 1 CCCP their hearts were collected 12 h later . Representative electron of hearts from CCCP - treated adult aged mice ; original magnification × 5 , 000 ; scale bar , 2 μm . magnified photograph represents an autophagic vacuole containing mitochondria ; original magnification × 12 , 000 ; scale bar , 500 nm . Mitochondria incorporated into an autophagic vacuole in baseline CCCP - treated conditions were quantified blindly from 8 - 10 from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Ten-month-old adult mice and 20-month-old aged mice were treated with an intraperitoneal injection of 5 mg kg-1 CCCP and their hearts were collected 12 h later. Representative electron micrographs of hearts from CCCP-treated adult and aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents an autophagic vacuole containing mitochondria; original magnification × 12,000; scale bar, 500 nm. Mitochondria incorporated into an autophagic vacuole in baseline and CCCP-treated conditions were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( b , c ) CCCP - induced recruitment of Parkin p62 to mitochondria was determined by immunoblotting of heart mitochondria - rich fraction in adult aged mice ( b ) , DOX - treated mice ( c ) . Representative immunoblots are shown from four independent experiments .

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(b,c) CCCP-induced recruitment of Parkin and p62 to mitochondria was determined by immunoblotting of the heart mitochondria-rich fraction in adult and aged mice (b), and DOX-treated mice (c). Representative immunoblots are shown from four independent experiments.


( d ) Parkin was overexpressed in young senescent MEFs using adenovirus - mediated transduction for in vitro - bioassay to assess ability to clear damaged mitochondria . Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM . Results from four independent experiments performed in duplicate are shown . MEFs at passage 3 passage 9 were used as young senescent MEFs , respectively .

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(d) Parkin was overexpressed in young and senescent MEFs using adenovirus-mediated transduction for the in vitro cell-based bioassay to assess the ability to clear damaged mitochondria. Mitochondrial content after 24 h of treatment with 20 μM CCCP was assessed by flow cytometry for MitoTracker Green FM. Results from four independent experiments performed in duplicate are shown. MEFs at passage 3 and passage 9 were used as young and senescent MEFs, respectively.


( e ) Mitochondrial content was assessed by real - time PCR . Results are shown from six - independent experiments .

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(e) Mitochondrial DNA content was assessed by real-time PCR. Results are shown from six-independent experiments.


( f ) Mitochondrial content was assessed by immunoblotting for mitochondrial chaperone , GRP75 ( matrix ) I subunit , NDUFA9 ( inner ) . MEFs were treated with 100 nM bafilomycin - A1 , ( Baf - A1 , an inhibitor of vacuolar - type proton ATPase ) to inhibit autophagy . Representative immunoblots are shown from three independent experiments .

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(f) Mitochondrial content was assessed by immunoblotting for the mitochondrial chaperone, GRP75 (matrix protein) and complex I subunit, NDUFA9 (inner membrane protein). MEFs were treated with 100 nM bafilomycin-A1, (Baf-A1, an inhibitor of the vacuolar-type proton ATPase) to inhibit autophagy. Representative immunoblots are shown from three independent experiments.


( g ) Representative of GFP - LC3 expressing MEFs treated with 20 μM CCCP for 10 h before immunostaining of mitochondria with anti - TOM20 ( red ) ; original magnification , × 400 × 1 , 000 ; scale bar , 100 μm scale bar , 15 μm . scans below indicate colocalization between LC3 ( green ) mitochondria ( red ) correlate to arrows drawn in . WHL indicates whole - heart lysate . Data are shown as means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - unpaired Student ' s t - ) .

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(g) Representative images of GFP-LC3 expressing MEFs treated with 20 μM CCCP for 10 h before the immunostaining of mitochondria with anti-TOM20 (red); original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm. Line scans below the images indicate colocalization between LC3 (green) and mitochondria (red) and correlate to the arrows drawn in the images. WHL indicates whole-heart lysate. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) senescence - associated molecules p53 , p21 , p16 , H - Ras 12V Rb were overexpressed in MEFs by retrovirus - mediated transduction . After puromycin selection , mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus - mediated Parkin expression . Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM . Results are shown from four independent experiments performed in duplicate .

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(a) The senescence-associated molecules p53, p21, p16, H-Ras 12V and Rb were overexpressed in MEFs by retrovirus-mediated transduction. After puromycin selection, mitophagy induction was performed with treatment of 20 μM CCCP 24 h after adenovirus-mediated Parkin expression. Mitochondrial content 24 h after mitophagy induction was assessed by flow cytometry for MitoTracker Green FM. Results are shown from four independent experiments performed in duplicate.


( b ) Representative of MEFs infected with GFP - LC3 adenovirus stained with anti - TOM20 ( red ) to visualize mitochondria 10 h after mitophagy induction ; original magnification , × 400 × 1 , 000 ; scale bar , 100 μm scale bar , 15 μm .

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(b) Representative images of MEFs infected with the GFP-LC3 adenovirus and stained with anti-TOM20 (red) to visualize mitochondria 10 h after mitophagy induction; original magnification, × 400 and × 1,000; scale bar, 100 μm and scale bar, 15 μm.


( c ) Mitochondrial content of H - Ras 12 V expressing WT , p53 − / − p21 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(c) Mitochondrial content of H-Ras 12 V expressing WT, p53−/− and p21−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( d ) Mitochondrial content of WT p53 − / − MEFs was assessed by flow cytometry 24 h after mitophagy induction . WT p53 − / − MEFs were transfected with control or p53 - specific siRNAs or cultured in complete medium for 24 h in presence or absence of 20 μM pifithrin - α before mitophagy induction . Results are shown from four independent experiments performed in duplicate .

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(d) Mitochondrial content of WT and p53−/− MEFs was assessed by flow cytometry 24 h after mitophagy induction. WT and p53−/− MEFs were transfected with control or p53-specific siRNAs or cultured in complete medium for 24 h in the presence or absence of 20 μM pifithrin-α before mitophagy induction. Results are shown from four independent experiments performed in duplicate.


( e ) Murine HL - 1 cardiac myocytes were infected with retrovirus vectors encoding p53 , p21 or p16 after puromycin selection , underwent mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown .

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(e) Murine HL-1 cardiac myocytes were infected with retrovirus vectors encoding p53, p21 or p16 and after puromycin selection, underwent mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate are shown.


( f ) HL - 1 were transfected with control , p53 , p21 or p16 - specific siRNAs , followed by treatment with 0 . 02 μM DOX mitophagy induction . Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate .

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(f) HL-1 cells were transfected with control, p53, p21 or p16-specific siRNAs, followed by treatment with 0.02 μM DOX and mitophagy induction. Mitochondrial content is assessed by flow cytometry from four independent experiments performed in duplicate.


( g ) CCCP - induced recruitment of Parkin expression of Mfn1 Drp1 in DOX - treated WT , p53 − / − p21 − / − mice were determined by immunoblotting of heartmitochondria - rich fraction . Representative immunoblots are shown from four independent experiments . Data are shown as means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - unpaired Student ' s t - ) .

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(g) CCCP-induced recruitment of Parkin and expression of Mfn1 and Drp1 in DOX-treated WT, p53−/− and p21−/− mice were determined by immunoblotting of the heartmitochondria-rich fraction. Representative immunoblots are shown from four independent experiments. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


( a ) Whole - lysates of HL - 1 treated with 0 . 02 μM DOX transfected with FLAG - tagged Parkin or FLAG - empty vector were immunoprecipitated with anti - Flag - M2 antibody blotted with anti - p53 Flag antibodies .

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(a) Whole-cell lysates of HL-1 cells treated with 0.02 μM DOX and transfected with FLAG-tagged Parkin or the FLAG-empty vector were immunoprecipitated with the anti-Flag-M2 antibody and blotted with anti-p53 and Flag antibodies.


( b ) Whole - lysates of rat neonatal cardiomyocytes treated with indicated concentrations of H2O2 DOX for 24 h were immunoprecipitated with anti - Parkin antibody .

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(b) Whole-cell lysates of rat neonatal cardiomyocytes treated with the indicated concentrations of H2O2 and DOX for 24 h were immunoprecipitated with the anti-Parkin antibody.


( c ) heart lysates of adult aged mice were immunoprecipitated with anti - Parkin , p53 control IgG antibodies .

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(c) Cytosolic heart lysates of adult and aged mice were immunoprecipitated with anti-Parkin, p53 and control IgG antibodies.


( d ) Schematic representation of N - terminally FLAG - tagged WT Parkin various mutants of Parkin . ( e ) association of endogenous p53 with various Parkin mutants in DOX - treated HeLa . immunoprecipitation revealed that RING0 domain was essential for interaction with p53 .

(d) Schematic representation of N-terminally FLAG-tagged WT Parkin and various mutants of Parkin. (e) The association of endogenous p53 with various Parkin mutants in DOX-treated HeLa cells. The immunoprecipitation assay revealed that the RING0 domain was essential for the interaction with p53.


( f ) Schematic representation of GST - p53 fusion . ( g ) GST pull - down assays using GST - p53 fusion in vitro transcribed / translated FLAG - Parkin . Residues 81 - 160 of p53 were sufficient to bind Parkin .

(f) Schematic representation of the GST-p53 fusion proteins. (g) GST pull-down assays using GST-p53 fusion proteins and in vitro transcribed/translated FLAG-Parkin. Residues 81-160 of p53 were sufficient to bind Parkin.


( a ) HL - 1 transfected with control or p53 - specific siRNAs no . 1 cultured in complete medium for 24 h in presence or absence of 0 . 02 μM DOX . After treatment with 20 μM CCCP for 6 h , fractionation experiment was performed . Endogenous Parkin translocation to mitochondria ubiquitination were assessed by immunoblotting . Representative immunoblots are shown from three independent experiments .

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(a) HL-1 cells transfected with control or p53-specific siRNAs no.1 and cultured in complete medium for 24 h in the presence or absence of 0.02 μM DOX. After treatment with 20 μM CCCP for 6 h, the fractionation experiment was performed. Endogenous Parkin translocation to mitochondria and ubiquitination were assessed by immunoblotting. Representative immunoblots are shown from three independent experiments.


( b ) These samples were subjected to immunoprecipitation with anti - VDAC antibody to evaluate ubiquitination . Representative immunoblots are shown from three independent experiments .

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(b) These samples were subjected to immunoprecipitation with the anti-VDAC antibody to evaluate ubiquitination. Representative immunoblots are shown from three independent experiments.


( c , d ) Representative of YFP - Parkin overexpressing p53 − / − HCT116 re - transfected with WT p53 various mutants of p53 treated with 60 μM CCCP for 8h ( c ) 36 h ( d ) before immunostaining of mitochondria with anti - TOM20 ( red ) endogenous poly - ubiquitin with a specific antibody FK - 2 ( ) ; original magnification , × 630 ; scale bar , 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 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

(c,d) Representative images of YFP-Parkin overexpressing p53−/− HCT116 cells re-transfected with WT p53 and various mutants of p53 and treated with 60 μM CCCP for 8h (c) and 36 h (d) before the immunostaining of mitochondria with anti-TOM20 (red) and endogenous poly-ubiquitin with a specific antibody FK-2 (white); original magnification, × 630; scale bar, 20 μm.


( e - g ) Parkin mitochondrial translocation ( e ) ubiquitination ( f ) 8 h after CCCP treatment mitochondrial clearance ( g ) 36 h after CCCP treatment were quantified . A minimum of 300 - 400 YFP - positive were scored in three independent experiments . single - letter - acid code is used . NES indicates export signal ; NLS , localization signal ; ER , endoplasmic reticulum ; Ub , ubiquitin . Data are shown as means ± s . d . * P0 . 05 , compared with corresponding control ( two - unpaired Student ' s t - ) .

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(e-g) Parkin mitochondrial translocation (e) and ubiquitination (f) 8 h after CCCP treatment and mitochondrial clearance (g) 36 h after CCCP treatment were quantified. A minimum of 300-400 YFP-positive cells were scored in three independent experiments. The single-letter amino-acid code is used. NES indicates nuclear export signal; NLS, nuclear localization signal; ER, endoplasmic reticulum; Ub, ubiquitin. Data are shown as the means±s.d. *P0.05, compared with the corresponding control (two-tailed unpaired Student's t-test).


( a ) Representative electron of DOX - treated hearts . Arrows indicate abnormal mitochondria defined by marked swelling , vacuolation ( loss of electron density ) or disrupted cristae ; original magnification × 5 , 000 ; scale bar , 2 μm . magnified photograph represents abnormal mitochondria ( blue ) an autophagic vacuole containing mitochondria ( red ) ; original magnification × 12 , 000 ; scale bar , 500 nm . ( b , c ) Mitochondria incorporated into an autophagic vacuole ( b ) abnormal mitochondria ( c ) were quantified blindly from 8 - 10 from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Representative electron micrographs of DOX-treated hearts. Arrows indicate abnormal mitochondria defined by marked swelling, vacuolation (loss of electron density) or disrupted cristae; original magnification × 5,000; scale bar, 2 μm. The magnified photograph represents abnormal mitochondria (blue line) and an autophagic vacuole containing mitochondria (red line); original magnification × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( d ) 8 - hydroxy - 2 ′ - deoxyguanosine ( 8 - OHdG ) , popular marker for oxidative damage , in mitochondria from DOX - treated hearts . Results are shown from 6 - 8 hearts analysed in duplicate .

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(d) The 8-hydroxy-2′-deoxyguanosine (8-OHdG), popular marker for oxidative DNA damage, in mitochondria DNA from DOX-treated hearts. Results are shown from 6-8 hearts analysed in duplicate.


( e ) Maximum dP / dt was examined using cardiac catheterization under graded dobutamine infusion in PBS - treated ( control ) DOX - treated mice ( n = 6 - 8 per group ) . DKO indicates double knockout . Data are shown as means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - unpaired Student ' s t - ) .

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(e) Maximum dP/dt was examined using cardiac catheterization under graded dobutamine infusion in PBS-treated (control) and DOX-treated mice (n=6-8 per group). DKO indicates double knockout. Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


We used 20 - month - old heterozygous surviving grossly free . ( a , b ) sequential state III oxygen consumption rate of heart isolated mitochondria ( 300 μg ) was monitored with a fibre - optic system . Substrates inhibitors were added sequentially at final concentrations indicated as follow . FCCP , 1 μM carbonylcyanide - p - trifluoromethoxyphenylhydrazone ; M / P , 5 mM malate + 5 mM pyruvate ; Rot , 100 nM rotenone ; Suc , 5 mM succinate ; AA , 50 nM antimycin A ; TMPD / Asc , 0 . 4 mM N , N , N ′ , N ′ - tetramethyl - p - phenylenediamine + 1 mM ascorbate ; KCN , 5 mM potassium cyanide . Representative results are shown ( a ) oxygen consumption by each was calculated ( n = 5 - 8 per group , b ) .

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

We used 20-month-old heterozygous littermates surviving grossly tumor free. (a,b) The sequential state III oxygen consumption rate of heart isolated mitochondria (300 μg) was monitored with a fibre-optic system. Substrates and inhibitors were added sequentially at the final concentrations indicated as follow. FCCP, 1 μM carbonylcyanide-p-trifluoromethoxyphenylhydrazone; M/P, 5 mM malate+5 mM pyruvate; Rot, 100 nM rotenone; Suc, 5 mM succinate; AA, 50 nM antimycin A; TMPD/Asc, 0.4 mM N,N,N′,N′-tetramethyl-p-phenylenediamine +1 mM ascorbate; KCN, 5 mM potassium cyanide. Representative results are shown (a) and oxygen consumption by each complex was calculated (n=5-8 per group, b).


( c , d ) Mitochondrial particle ( c ) potential ( d ) were determined using flow cytometry for TMRE in cardiac mitochondria preparations . Results are shown from 4 - 8 hearts analysed in duplicate .

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(c,d) Mitochondrial particle size (c) and membrane potential (d) were determined using flow cytometry for TMRE in cardiac mitochondria preparations. Results are shown from 4-8 hearts analysed in duplicate.


( e ) H2O2 production with 0 . 5 mM NADH , I - linked substrate , was determined in absence or presence of 2 μM I inhibitor rotenone using Amplex Red ( n = 5 - 8 per group ) .

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(e) H2O2 production with 0.5 mM NADH, complex I-linked substrate, was determined in the absence or presence of 2 μM complex I inhibitor rotenone using Amplex Red (n=5-8 per group).


( f ) Myocardial high - energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC ( n = 5 per group ) .

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(f) Myocardial high-energy phosphate metabolism under 8γ dobutamine loading was measured biochemically with HPLC (n=5 per group).


( g ) Maximum dP / dt was examined using cardiac catheterization with graded a dobutamine infusion ( n = 5 per group ) .

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(g) Maximum dP/dt was examined using cardiac catheterization with graded a dobutamine infusion (n=5 per group).


( h ) showing heart sections after SA - β - gal ; original magnification , × 200 ; scale bar , 40 μm . SA - β - gal - positive were quantified ( n = 4 per group ) . Five per mouse were taken on brightfield DAPI was used to total number per section . HT indicates heterozygous ; DHT double heterozygous . Data are shown as means ± s . d . * P0 . 05 ; * * P0 . 01 ; * * * P0 . 001 ( two - unpaired Student ' s t - )

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(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section. HT indicates heterozygous; DHT double heterozygous. Data are shown as the means±s.d. *P0.05; **P0.01; ***P0.001 (two-tailed unpaired Student's t-test)


( a ) Representative electron of hearts of 20 - month - old aged mice ; original magnification × 5 , 000 ; scale bar , 2 μm . magnified photograph shows an autophagic vacuole containing mitochondria ; original magnification , × 12 , 000 ; scale bar , 500 nm . ( b , c ) Mitochondria incorporated into an autophagic vacuole ( b ) abnormal mitochondria ( c ) were quantified blindly from 8 - 10 from different fields ( original magnification × 5 , 000 ) ( n = 3 per group ) .

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(a) Representative electron micrographs of the hearts of 20-month-old aged mice; original magnification × 5,000; scale bar, 2 μm. The magnified photograph shows an autophagic vacuole containing mitochondria; original magnification, × 12,000; scale bar, 500 nm. (b,c) Mitochondria incorporated into an autophagic vacuole (b) and abnormal mitochondria (c) were quantified blindly from 8-10 images from different fields (original magnification × 5,000) (n=3 per group).


( d , e ) sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using Seahorse XF24 Analyser . With initial presence of 5 μg mitochondria per well , 0 . 5 mM NADH 4 μM FCCP , injections were performed as follows : port A , 50 μl of 20 μM rotenone ( 2 μM final ) ; port B , 55 μl of 100 mM succinate ( 10 mM final ) ; port C , 60 μl of 40 μM antimycin A ( 4 μM final ) ; port D , 65 μl of 100 mM ascorbate plus 1 mM TMPD ( 10 μM 100 μM final , respectively ) . Representative results of aged are shown ( d ) oxygen consumption by each was calculated from four independent experiments , five replicates per sample , four samples per experiment ( n = 8 per group , e ) .

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(d,e) The sequential state III oxygen consumption rate of heart isolated mitochondria was assessed using the Seahorse XF24 Analyser. With the initial presence of 5 μg mitochondria per well, 0.5 mM NADH and 4 μM FCCP, injections were performed as follows: port A, 50 μl of 20 μM rotenone (2 μM final); port B, 55 μl of 100 mM succinate (10 mM final); port C, 60 μl of 40 μM antimycin A (4 μM final); port D, 65 μl of 100 mM ascorbate plus 1 mM TMPD (10 μM and 100 μM final, respectively). Representative results of aged littermates are shown (d) and oxygen consumption by each complex was calculated from four independent experiments, five replicates per sample, and four samples per experiment (n=8 per group, e).


( f ) NADH - driven H2O2 production was determined using Amplex Red . Results are shown from five hearts analysed in duplicate .

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(f) NADH-driven H2O2 production was determined using Amplex Red. Results are shown from five hearts analysed in duplicate.


( g ) Maximum dP / dt was examined using cardiac catheterization with graded dobutamine infusion in WT Parkin Tg aged ( n = 8 per group ) .

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(g) Maximum dP/dt was examined using cardiac catheterization with graded dobutamine infusion in WT and Parkin Tg aged littermates (n=8 per group).


( h ) showing heart sections after SA - β - gal ; original magnification , × 200 ; scale bar , 40 μm . SA - β - gal - positive were quantified ( n = 4 per group ) . Five per mouse were taken on brightfield DAPI was used to total number per section .

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(h) Photographs showing heart sections after SA-β-gal staining; original magnification, × 200; scale bar, 40 μm. SA-β-gal-positive cells were quantified (n=4 per group). Five images per mouse were taken on brightfield and DAPI staining was used to count the total cell number per section.


( i ) Real - time PCR assessing expression of cycle inhibitors proinflammatory cytokines ( n = 8 per group ) .

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(i) Real-time PCR assessing the expression of cell cycle inhibitors and proinflammatory cytokines (n=8 per group).


( j ) Representative immunoblots assessing expression of cycle inhibitors are shown ; Data are shown as means ± s . d . * P0 . 05 ; * * P0 . 01 ( two - unpaired Student ' s t - ) .

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(j) Representative immunoblots assessing the expression of cell cycle inhibitors are shown; Data are shown as the means±s.d. *P0.05; **P0.01 (two-tailed unpaired Student's t-test).


A - F Brain MRI of proband at 23 years . ( A ) Sagittal fast spin echo ( FSE ) T1 - weighted image shows thinning of corpus callosum , dilation of IV ventricle of cisterna magna reduced volume of vermis brainstem ; two lacunar lesions are evident in dorsal pons . ( B ) T2 - weighted axial fluid - attenuated inversion recovery ( FLAIR ) show hyperintense periventricular matter with several lacunar lesions also in basal ganglia thalami . ( C ) MR angiography 3D TOF ( time of flight ) reconstruction shows two saccular aneurisms in right M1 ( ø 4 mm ) left M2 ( ø 2 . 5 mm ) segments of middle cerebral arteries ( arrows ) . ( D ) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging ( DTI ) in right side of dorsal pons . Severe cavitations lateral ventricles dilation ( left > right ) on FLAIR ( B , E ) diffuse microbleeds as small hypointense foci on SWI are visible ( F ) in same slices of ( E ) . R = right , L = left .

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A-F Brain MRI of the proband at 23 years. (A) Sagittal fast spin echo (FSE) T1-weighted image shows thinning of the corpus callosum, dilation of the IV ventricle and of the cisterna magna and reduced volume of vermis and brainstem; two lacunar lesions are evident in the dorsal pons. (B) T2-weighted axial fluid-attenuated inversion recovery (FLAIR) images show hyperintense periventricular white matter with several lacunar lesions also in the basal ganglia and thalami. (C) MR angiography 3D TOF (time of flight) reconstruction shows two saccular aneurisms in the right M1 (ø 4 mm) and left M2 (ø 2.5 mm) segments of middle cerebral arteries (arrows). (D) A recent ischemic hyperintense lesion is detected on diffusion tensor imaging (DTI) in the right side of the dorsal pons. Severe cavitations and lateral ventricles dilation (left > right) on FLAIR images (B, E) and diffuse microbleeds as small hypointense foci on SWI are visible (F) in the same slices of (E). R = right, L = left.


G , H Brain MRI scans of asymptomatic parents of proband show multiple focal hyperintensities on T2 - weighted in periventricular subcortical cerebral matter , expression of gliosis secondary to chronic small vessel ischemic changes , more evident in father ( G ) , 56 years , than in mother ( H ) , 54 years .

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G, H Brain MRI scans of the asymptomatic parents of the proband show multiple focal hyperintensities on T2-weighted images in the periventricular and subcortical cerebral white matter, expression of gliosis secondary to chronic small vessel ischemic changes, more evident in the father (G), 56 years, than in the mother (H), 54 years.


Gene expression of NOTCH3 , HES1 , HEY1 , HEYL 17 recently identified targets were established by a real - time PCR panel in skeletal muscle of controls ( n = 6 ) , proband ( II : 1 ) , parents ( mother I : 1 , father I : 2 ) CADASIL ( n = 3 ) . Graph shows gene expression fold changes relative to controls normalized on GAPDH ( reference gene ) , expressed as mean of two experiments ± . Among 17 novel assayed target genes , RCAN2 , ANGPT4 , HP SORBS were not detected in any sample were therefore not reported . Green red lines indicate 2 . 0 0 . 5 fold changes , respectively . Statistical significances P - values are reported in Supplementary Table S1 .

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Gene expression of NOTCH3, HES1, HEY1, HEYL and the 17 recently identified targets were established by a real-time PCR panel assay in skeletal muscle of controls (n = 6), proband (II:1), parents (mother I:1, father I:2) and CADASIL patients (n = 3). Graph shows gene expression fold changes relative to controls and normalized on GAPDH (reference gene), expressed as mean of two experiments ± SEM. Among the 17 novel assayed target genes, RCAN2, ANGPT4, HP and SORBS were not detected in any sample and were therefore not reported. Green and red lines indicate 2.0 and 0.5 fold changes, respectively. Statistical significances and P-values are reported in Supplementary Table S1.


A - C Histopathological examination of skin ( A ) skeletal muscle biopsies ( B , C ) of control ( left ) , proband ( middle ) CADASIL ( right ) . ( A ) Collagen IV ( in green ) in skin vessels : Collagen wall appears compact in control , while it is disorganized in skin vessels of proband of CADASIL . Derangement of collagen wall in single collagen is more evident in proband than in CADASIL , where collagen wall is quite compact . Vessel ' s endothelium is delineated by ULEX ( in red ) . Collagen IV in skeletal muscle vessels ( B ) recapitulates skin picture . Smooth muscle actin immunostaining of skeletal muscle biopsies ( C ) shows attenuated SMCs in tunica media of both proband CADASIL , with foci characterized by a complete SMCs loss ( particularly in CADASIL ) thinning of vessel wall ( arrows ) .

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A-C Histopathological examination of skin (A) and skeletal muscle biopsies (B, C) of control (left), proband (middle) and CADASIL patient (right). (A) Collagen IV staining (in green) in skin vessels: Collagen wall appears compact in the control, while it is disorganized in the skin vessels of the proband and of the CADASIL patient. Derangement of the collagen wall in single collagen fibers is more evident in the proband than in the CADASIL patient, where collagen wall is quite compact. Vessel's endothelium is delineated by ULEX staining (in red). Collagen IV staining in skeletal muscle vessels (B) recapitulates the skin picture. Smooth muscle actin immunostaining of skeletal muscle biopsies (C) shows attenuated SMCs in the tunica media of both the proband and the CADASIL patient, with foci characterized by a complete SMCs loss (particularly in the CADASIL patient) and thinning of the vessel wall (arrows).


D , E Ultrastructural of skin biopsies . ( D ) Skin biopsy of proband : ( left panel ) At low magnification , tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal with a multilayered aspect ( arrows ) ; SMCs are identified by presence of " focal adhesions " ( square boxes ) ; ( middle panel ) collagen fibrils appear organized in a parallel pathway along SMC ( arrow ) , note " focal adhesions " ( square box ) ; ( right panel ) collagen fibrils ( " CF " ) are more represented in dense bundles than elastic ones ( " E " ) . No GOM is detected , in contrast to CADASIL ( E ) , showing deposits of granular osmiophilic material ( arrows ) located between SMC plasmalemma basal lamina .

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D, E Ultrastructural analysis of skin biopsies. (D) Skin biopsy of the proband: (left panel) At low magnification, the tunica media of a vessel shows irregular SMCs surrounded by a markedly thickened basal membrane with a multilayered aspect (arrows); SMCs are identified by the presence of "focal adhesions" (square boxes); (middle panel) collagen fibrils appear organized in a parallel pathway along SMC (arrow), note the "focal adhesions" (square box); (right panel) collagen fibrils ("CF") are more represented in dense bundles than elastic ones ("E"). No GOM is detected, in contrast to CADASIL patient (E), showing deposits of granular osmiophilic material (arrows) located between SMC plasmalemma and basal lamina.


F KCNA5 immunostaining of skeletal muscle biopsies of control ( left ) , proband ( middle ) CADASIL ( right ) . A global decrease in reactivity is evident in vessels of both proband CADASIL . Note multiple areas in which KCNA5 is barely detectable ( arrows ) .

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F KCNA5 immunostaining of skeletal muscle biopsies of control (left), proband (middle) and CADASIL patient (right). A global decrease in reactivity is evident in the vessels of both the proband and the CADASIL patient. Note multiple areas in which KCNA5 is barely detectable (arrows).


( b ) 2D - [ 1H ; 15N ] - TROSY spectrum of 400 µM 2H , 13C , 15N - labeled TREM2 - TMH ( residues R161 - H206 ) in DPC micelles at 37 ° C . Assigned resonances are labeled .

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(b) 2D-[1H;15N]-TROSY spectrum of 400 µM 2H,13C,15N-labeled TREM2-TMH (residues R161-H206) in DPC micelles at 37 °C. Assigned resonances are labeled.


( c ) Secondary chemical shifts plotted against residue number indicates an α - helical region between residues 173 198 . used TREM2 - TMH construct along with predicted transmembrane helix content ( aa 175 - 195 ) is also indicated .

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(c) Secondary chemical shifts plotted against the residue number indicates an α-helical region between residues 173 and 198. The used TREM2-TMH construct along with the predicted transmembrane helix content (aa 175-195) is also indicated.


( d ) Circular dichroism ( CD ) spectrum of TREM2 - TMH in DPC micelles indicates an α - helical secondary structure content of ~ 50 % .

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(d) Circular dichroism (CD) spectrum of TREM2-TMH in DPC micelles indicates an α-helical secondary structure content of ~50%.


( e ) 3D - 15N - edited NOESY spectra show that transmembrane helix is interrupted at residues 190 - 192 . Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α - helical secondary structure .

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(e) 3D-15N-edited NOESY spectra show that the transmembrane helix is interrupted at residues 190-192. Dashed red lines indicate sequential amide NOE contacts that are characteristic for an α-helical secondary structure.


( a , b ) Overlay of 2D - [ 1H , 15N ] - TROSY spectra of 2H , 15N - labeled TREM2 - TMH in DPC micelles ( black ) in with ( a ) unlabeled DAP12 ( red ) or ( b ) TREM2 - TMH lacking its positively charged lysine residue ( K186A , red ) .

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(a, b) Overlay of 2D-[1H,15N]-TROSY spectra of 2H,15N-labeled TREM2-TMH in DPC micelles (black) and in complex with (a) unlabeled DAP12 (red) or (b) TREM2-TMH lacking its positively charged lysine residue (K186A, red).


( c ) Chemical shift perturbations ( CSPs ) between two spectra in panels ( a ) or ( b ) . α - helical secondary structure as determined for wild - type TREM2 - TMH is indicated above position of K186A mutation is labeled in green .

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(c) Chemical shift perturbations (CSPs) between the two spectra in panels (a) or (b). The α-helical secondary structure as determined for wild-type TREM2-TMH is indicated above and the position of the K186A mutation is labeled in green.


( f ) For TREM2 - K186A - TMH a ~ 7 % increase in α - helical secondary structure can be detected by CD spectroscopy .

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(f) For TREM2-K186A-TMH a ~7% increase in α-helical secondary structure can be detected by CD spectroscopy.


( g ) A continuous α - helical secondary structure in kink region ( aa 189 - 192 ) was probed by sequential amide NOE contacts in TREM2 - K186A - TMH . Red dots indicate chemical shift of intra - residual ( i ) amide resonance red broken lines inter - residual connectivity ( i ± 1 ) .

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(g) A continuous α-helical secondary structure in the kink region (aa 189-192) was probed by sequential amide NOE contacts in TREM2-K186A-TMH. Red dots indicate the chemical shift of intra-residual (i) amide resonance and red broken lines the inter-residual connectivity (i±1).


( a , b ) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild - type TREM2 - TMH ( a ) K186A variant ( b ) , indicative of motions in nanosecond to picosecond time scale . S2 can adopt values between 1 ( rigid ) 0 ( unrestricted motion ) . Error bars have been obtained by a Monte - Carlo - simulation - error estimation motional model selection procedure implemented in software Relax

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(a,b) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild-type TREM2-TMH (a) and the K186A variant (b), indicative of motions in the nanosecond to picosecond time scale. S2 can adopt values between 1 (rigid) and 0 (unrestricted motion). Error bars have been obtained by a Monte-Carlo-simulation-based error estimation and motional model selection procedure implemented in the software Relax


( a , b ) Generalized order parameter S2 for backbone amide resonances derived from NMR relaxation experiments with wild - type TREM2 - TMH ( a ) K186A variant ( b ) , indicative of motions in nanosecond to picosecond time scale . S2 can adopt values between 1 ( rigid ) 0 ( unrestricted motion ) . Error bars have been obtained by a Monte - Carlo - simulation - error estimation motional model selection procedure implemented in software

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( a ) Paramagnetic relaxation enhancement ( PRE ) with a free - radical - modified stearic fatty acid in DPC micelles . Low values indicate location inside whereas values close to one suggest a location outside detergent micelle . Orange bars : TREM2 - TMH wt , green bars : K186A variant . N - terminal residues of TREM2 - TMH - wt are located deeper inside micelles as in K186A case , whereas C - terminal end is positioned at outside for wild - type inside for K186A variant . Negative bars indicate missing assignments .

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(a) Paramagnetic relaxation enhancement (PRE) with a free-radical-modified stearic fatty acid in DPC micelles. Low values indicate location inside whereas values close to one suggest a location outside the detergent micelle. Orange bars: TREM2-TMH wt, green bars: K186A variant. The N-terminal residues of TREM2-TMH-wt are located deeper inside the micelles as in the K186A case, whereas the C-terminal end is positioned at the outside for wild-type and inside for the K186A variant. Negative bars indicate missing assignments.


( b ) NOE - cross peaks between backbone amides of TREM2 - TMH wt ( top ) or K186A variant ( bottom ) selected regions of DPC ( see color coding of structure of DPC molecule on right ) . K186A variant shows strong contacts to hydrophobic aliphatic regions of detergent throughout transmembrane helix whereas no such contacts can be seen at kink site of wild - type helix , indicating a location outside of micelle for this part .

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(b) NOE-cross peaks between backbone amides of TREM2-TMH wt (top) or the K186A variant (bottom) and selected regions of DPC (see color coding of the structure of the DPC molecule on the right). The K186A variant shows strong contacts to the hydrophobic aliphatic regions of the detergent throughout the transmembrane helix whereas no such contacts can be seen at the kink site of the wild-type helix, indicating a location outside of the micelle for this part.


( b ) Immunoblot detection of ICD accumulation of TREM2 wildtype , K186A , K186P , K186L variants mediated by γ - secretase cleavage upon overnight incubation of preparations at 37 ° C . ICD formation is blocked by 1 µM of transition state analogue L - 685 , 458 . Note that inhibition of ICD formation from wt TREM2 was less efficient . Due to charge within TMH wt TREM2 is less efficiently transported to surface ( Lanier et al , 1998 ) therefore retained within endoplasmic reticulum , where other proteases not related to γ - secretase therefore not inhabitable by L - 685 , 458 may cleave . In vitro generation of ICD of APP ( AICD ) was used as a positive control . Immunoblot detection of mature nicastrin presenilin 1 NTF , which results from presenilin endoproteolysis necessary to activate γ - secretase ( Thinakaran et al , 1996 ) , proves comparable amounts of γ - secretase in assays .

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(b) Immunoblot detection of ICD accumulation of TREM2 wildtype, K186A, K186P, and K186L variants mediated by γ-secretase cleavage upon overnight incubation of membrane preparations at 37 °C. ICD formation is blocked by 1 µM of the transition state analogue L-685,458. Note that inhibition of ICD formation from wt TREM2 was less efficient. Due to the charge within the TMH wt TREM2 is less efficiently transported to the cell surface (Lanier et al, 1998) and therefore retained within the endoplasmic reticulum, where other proteases not related to γ-secretase and therefore not inhabitable by L-685,458 may cleave. In vitro generation of the ICD of APP (AICD) was used as a positive control. Immunoblot detection of mature nicastrin and the presenilin 1 NTF, which results from presenilin endoproteolysis necessary to activate γ-secretase (Thinakaran et al, 1996), proves comparable amounts of γ-secretase in the individual assays.


( c ) MALDI MS of ε - cleavage sites of wt mutant TREM2 C - terminal fragments . Mass spectrometry reveals ε - cleavage C - terminal of A192 for wt CTF . MS determination of ε - cleavage site of TREM2 CTF K186A reveals major minor ε - cleavage sites C - terminal of A195 L193 , respectively . exact same cleavage pattern is observed for K186L mutant . MS of cleavage pattern of K186P mutant reveals major minor ε - cleavage sites C - terminal of L193 S191 . Minor peaks that could not be assigned are indicated by an asterisk .

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(c) MALDI MS analysis of the ε-cleavage sites of wt and mutant TREM2 C-terminal fragments. Mass spectrometry reveals ε-cleavage C-terminal of A192 for the wt CTF. MS determination of the ε-cleavage site of TREM2 CTF K186A reveals major and minor ε-cleavage sites C-terminal of A195 and L193, respectively. The exact same cleavage pattern is observed for the K186L mutant. MS analysis of the cleavage pattern of the K186P mutant reveals major and minor ε-cleavage sites C-terminal of L193 and S191. Minor peaks that could not be assigned are indicated by an asterisk.


( d , e ) Chemically induced unfolding of TREM2 - TMH wild - type ( d ) K186A variant ( e ) demonstrates that less dynamic variant is more stable than wild - type .

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(d, e) Chemically induced unfolding of TREM2-TMH wild-type (d) and the K186A variant (e) demonstrates that the less dynamic variant is more stable than the wild-type protein.


( f ) Water exchange peak as observed in a 3D - 15N - edited - [ 1H , 1H ] - NOESY experiment which is only absent if amide moiety is present in a stable hydrogen bond .

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(f) Water exchange peak as observed in a 3D-15N-edited-[1H,1H]-NOESY experiment which is only absent if the amide moiety is present in a stable hydrogen bond.


Representative of of Mex3a mutant mice control at postnatal day ( P ) 15 . Scale bar , 1 cm . Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue ( right panels ) . Scale bars , 50 μm . offspring number ( n ) observed genotype frequencies ( % ) resulting from heterozygous crosses are indicated below .

Representative images of the size of Mex3a mutant mice and control littermates at postnatal day (P)15. Scale bar, 1 cm. Genotypes were confirmed by Mex3a mRNA ISH in intestinal tissue (right panels). Scale bars, 50 μm. The offspring number (n) and observed genotype frequencies (%) resulting from heterozygous crosses are indicated below.


Absolute of Mex3a KO mice control at different ages . Data is represented in a box whiskers plot as mean ( middle ) with minimum maximum distribution values . Each point depicts one ( WT : P1 , n = 15 ; P8 , n = 36 ; P15 , n = 43 ; P22 , n = 14 ; Adult , n = 14 ; Mex3a + / - : P1 , n = 24 ; P8 , n = 90 ; P15 , n = 90 ; P22 , n = 37 ; Adult , n = 21 ; Mex3a - / - : P1 , n = 9 ; P8 , n = 24 ; P15 , n = 26 ; P22 , n = 14 ; Adult , n = 8 ) * * * P = 0 . 0003 , * * * * P < 0 . 0001 , two - way ANOVA .

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Absolute weight of Mex3a KO mice and control littermates at different ages. Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one animal (WT: P1, n = 15; P8, n = 36; P15, n = 43; P22, n = 14; Adult, n = 14; Mex3a+/-: P1, n = 24; P8, n = 90; P15, n = 90; P22, n = 37; Adult, n = 21; Mex3a-/-: P1, n = 9; P8, n = 24; P15, n = 26; P22, n = 14; Adult, n = 8) ***P = 0.0003, ****P < 0.0001, two-way ANOVA test.


Kaplan - Meier survival curves for different Mex3a genotypes ( n = 80 for each genotype ) * * * * P < 0 . 0001 , Log - rank ( Mantel - Cox ) .

Kaplan-Meier survival curves for the different Mex3a genotypes (n = 80 for each genotype) ****P < 0.0001, Log-rank (Mantel-Cox) test.


Macroscopic assessment of gastrointestinal tract of Mex3a KO WT mice . These are representative of phenotype observed in mutant euthanized at different postnatal days . Boxed areas depict distal small intestinal section ( ileum ) used for subsequent immunohistochemical analyses .

Macroscopic assessment of the gastrointestinal tract of Mex3a KO and WT mice. These images are representative of the phenotype observed in mutant animals euthanized at different postnatal days. Boxed areas depict the distal small intestinal section (ileum) used for subsequent immunohistochemical analyses.


H & E of a Mex3a KO WT littermate at P19 . Inserts depict high magnification of boxed areas .

H&E staining of a Mex3a KO and WT littermate at P19. Inserts depict high magnification of the boxed areas.


Average crypt depth ( C ) of WT ( n = 3 , > 40 crypts counted per ) Mex3a mutants ( n = 6 , > 20 crypts counted per ) . Also shown is average villi height ( D ) of WT ( n = 3 , > 25 villi counted per ) Mex3a mutants ( n = 6 , > 15 villi counted per ) . Data is represented as mean ± standard error between P16 P18 . * P = 0 . 03 , * * * P = 0 . 0006 , Student ` s t .

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Average crypt depth (C) of WT animals (n = 3, > 40 crypts counted per animal) and Mex3a mutants (n = 6, > 20 crypts counted per animal). Also shown is the average villi height (D) of WT animals (n = 3, > 25 villi counted per animal) and Mex3a mutants (n = 6, > 15 villi counted per animal). Data is represented as mean ± standard error between P16 and P18. *P = 0.03, ***P = 0.0006, Student`s t test.


Immunohistochemistry for ( E ) tuft marker DCLK1 ( black arrowheads indicate positive ) , ( F ) Paneth marker

Immunohistochemistry staining for (E) the tuft cell marker DCLK1 (black arrowheads indicate positive cells), (F) the Paneth cell marker LYZ1


Immunohistochemistry for proliferation marker KI67 .

Immunohistochemistry staining for the proliferation marker KI67.


mRNA ISH for markers ( H ) Lgr5 , ( I ) Olfm4 , ( J ) Lrig1 , ( K ) Hopx in ileal sections of Mex3a mutants littermate controls at P19 . Inserts depict high magnification of boxed areas .

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mRNA ISH staining for the stem cell markers (H) Lgr5, (I) Olfm4, (J) Lrig1, and (K) Hopx in ileal sections of Mex3a mutants and littermate controls at P19. Inserts depict high magnification of the boxed areas.


qPCR of expression level of indicated markers in freshly isolated crypt fractions from Mex3a KO WT ( n = 3 of each genotype ) . Data is represented as mean fold - change plus standard error in Mex3a KO mice relatively to WT ( dashed ) . * P = 0 . 0208 , * * P < 0 . 01 , * * * P = 0 . 0007 , Student ` s t .

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qPCR analysis of the expression level of the indicated stem cell markers in freshly isolated crypt fractions from Mex3a KO and WT animals (n = 3 of each genotype). Data is represented as mean fold-change plus standard error in Mex3a KO mice relatively to WT animals (dashed line). *P = 0.0208, **P < 0.01, ***P = 0.0007, Student`s t test.


of ( diameter ) of Mex3a KO WT organoid - initiating structures 2 days after crypt isolation ( n = 3 for each genotype , > 150 structures counted in total ) . Data is represented in a box whiskers plot as mean ( middle ) with minimum maximum distribution values . Each point depicts one organoid - initiating structure . * * * * P < 0 . 0001 , Student ` s t . Phase contrast microscopy of intestinal organoids generated by Mex3a - / - WT crypts 6 days after crypt isolation . Scale bars , 100 μm .

Quantification of the size (diameter length) of Mex3a KO and WT organoid-initiating structures 2 days after crypt isolation (n = 3 for each genotype, > 150 structures counted in total). Data is represented in a box and whiskers plot as mean (middle line) with the minimum and maximum distribution values. Each point depicts one organoid-initiating structure. ****P < 0.0001, Student`s t test. Phase contrast microscopy images of intestinal organoids generated by Mex3a-/- and WT crypts 6 days after crypt isolation. Scale bars, 100 μm.


Representative phase contrast microscopy of Mex3a KO WT intestinal organoids at days 2 4 of culture after 10 weekly passages . Scale bars , 100 μm .

Representative phase contrast microscopy images of Mex3a KO and WT intestinal organoids at days 2 and 4 of culture after 10 weekly passages. Scale bars, 100 μm.


qPCR of crypt lineage marker genes in organoid cultures at day 2 . Data is presented as mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed ) . * P < 0 . 05 , Student ` s t .

qPCR analysis of crypt lineage marker genes in organoid cultures at day 2. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line). *P < 0.05, Student`s t test.


Immunohistochemistry for Paneth marker LYZ1 in Mex3a KO WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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Immunohistochemistry staining for the Paneth cell marker LYZ1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


qPCR of crypt lineage marker genes in organoid cultures at day 4 . Data is presented as mean fold - change plus standard error ( n = 4 for each genotype ) for target gene expression in Mex3a KO intestinal organoids relative to WT ( dashed ) .

qPCR analysis of crypt lineage marker genes in organoid cultures at day 4. Data is presented as the mean fold-change plus standard error (n = 4 for each genotype) for target gene expression in Mex3a KO intestinal organoids relative to WT (dashed line).


mRNA ISH for markers Mex3a , Lgr5 , Hopx Lrig1 in Mex3a KO WT intestinal organoid sections at day 2 of culture . Scale bars , 50 μm .

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mRNA ISH staining for the stem cell markers Mex3a, Lgr5, Hopx and Lrig1 in Mex3a KO and WT intestinal organoid sections at day 2 of culture. Scale bars, 50 μm.


Representative of immunofluorescence for proliferation marker KI67 BrdU incorporation in Mex3a KO WT . Mice were injected at P17 sacrificed at P20 ( n = 3 for each genotype ) . Scale bars , 50 μm .

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Representative images of immunofluorescence staining for the proliferation marker KI67 and BrdU incorporation in Mex3a KO and WT animals. Mice were injected at P17 and sacrificed at P20 (n = 3 for each genotype). Scale bars, 50 μm.


Hierarchical clustering of samples ( n = 3 for each genotype ) of differentially expressed genes between Mex3a - / - WT mice . red colour represents over - expression , while blue colour under - expression .

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Hierarchical clustering of samples (n = 3 for each genotype) and heatmap of differentially expressed genes between the Mex3a-/- and WT mice. The red colour represents over-expression, while the blue colour under-expression.


GSEA pre - ranked comparing Mex3a - / - transcriptional profile against ( E )

GSEA pre-ranked analysis comparing the Mex3a-/- transcriptional profile against (E) the Mex3ahigh