F . BMDM were treated with 0 . 004 % DMSO or 100 nM MLi2 prior to live surface with Alexa - 594 conjugated rat anti - mouse CD11b antibody on ice for ten minutes . Three wells of from each condition were fixed imaged for initial surface CD11b binding ( shown in red as indicated ) . Six wells of from each condition were further incubated at 37 ° C to allow rapid internalization of CD11b - antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0 . 5 % acetic acid on ice for one min . Three of six wells were fixed surface - stained with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes remaining on surface ( shown in green as indicated ) . Internalized CD11b - antibody complexes are shown in red . other three wells of were further incubated at 37 ° C again for ten min to allow recycling of internalized CD11b - antibody followed by surface with Alexa - 488 conjugated goat anti - rat antibody to show CD11b - antibody complexes recycled back to surface . Representative from n = 3 biologically independent experiments are shown . Scale bars represent 30 μm . G - I . ( G ) initial surface CD11b , or ( H ) internalized CD11b , or ( I ) recycled CD11b were calculated from > 20 analyzed for each condition from n = 3 biologically independent experiments . Each dot represents mean value from one experiment . Error bars show ± with significance assessed by Mann - Whitney . * represents P < 0 . 05 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BMDM cells were treated with 0.004% DMSO or 100 nM MLi2 prior to live cell surface staining with Alexa-594 conjugated rat anti-mouse CD11b antibody on ice for ten minutes. Three wells of cells from each condition were fixed and imaged for initial surface CD11b binding (shown in red as indicated). Six wells of cells from each condition were further incubated at 37°C to allow rapid internalization of CD11b-antibody complexes for ten minutes prior to striping of surface CD11b antibody using 0.5% acetic acid on ice for one min. Three of the six wells were fixed and surface-stained with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes remaining on the cell surface (shown in green as indicated). Internalized CD11b-antibody complexes are shown in red. The other three wells of cells were further incubated at 37°C again for ten min to allow recycling of internalized CD11b-antibody complex followed by surface staining with Alexa-488 conjugated goat anti-rat antibody to show CD11b-antibody complexes recycled back to the surface. Representative images from n=3 biologically independent experiments are shown. Scale bars represent 30 μm. G-I. (G) The initial surface CD11b, or (H) internalized CD11b, or (I) recycled CD11b were calculated from >20 images analyzed for each condition from n=3 biologically independent experiments. Each dot represents the mean value from one experiment. Error bars show ± SEM with significance assessed by Mann-Whitney test. * represents P<0.05.


A . mouse bone marrow - derived macrophage ( BMDM ) from adult WT - mLRRK2 BAC transgenic mice were treated with DMSO , 1 μM Rab10 anti - sense oligonucleotide for 4 days , 100 nM MLi2 for 12 hours , as indicated , before stimulation with 100 nM CCL5 for indicated time ( hours ) . Representative immunoblots from n = 3 biologically independent experiments are shown . B . Relative pSer473 - Akt were normalized to total Akt calculated as fold of naïve ( without CCL5 stimulation ) . Data shown are group means ± from n = 3 biologically independent replicates , with significance assessed by one - way ANOVA with Tukey ' s post hoc with treated groups compared to matched control groups , with * * representing 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Primary mouse bone marrow-derived macrophage cells (BMDM) from adult male WT-mLRRK2 BAC transgenic mice were treated with DMSO, 1 μM Rab10 anti-sense oligonucleotide for 4 days, 100 nM MLi2 for 12 hours, as indicated, before stimulation with 100 nM CCL5 for the indicated time (hours). Representative immunoblots from n=3 biologically independent experiments are shown. B. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Data shown are group means ± SEM from n=3 biologically independent replicates, with significance assessed by one-way ANOVA with Tukey's post hoc test with treated groups compared to matched control groups, with ** representing P< 0.01.


C . BMDM , as described in panel ( A ) , were additionally treated with DMSO ( control ) , EIPA , or Akti - 1 / 2 ( Akt1 / 2 inhibitor ) as indicated , for 1 hour after CCL5 stimulation . Representative immunoblots from n = 3 biologically independent experiments are shown . D . Relative pSer473 - Akt were normalized to total Akt calculated as fold of naïve ( without CCL5 stimulation ) . Each dot represents one experiment , with error bars showing ± . Significance was assessed by one - way ANOVA with Tukey ' s post hoc , with * * * representing P < 0 . 0005 , * * * * P < 0 . 0001 .

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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BMDM cells, as described in panel (A), were additionally treated with DMSO (control), EIPA, or Akti-1/2(Akt1/2 inhibitor) as indicated, for 1 hour after CCL5 stimulation. Representative immunoblots from n=3 biologically independent experiments are shown. D. Relative pSer473-Akt levels were normalized to total Akt and calculated as fold of naïve cells (without CCL5 stimulation). Each dot represents one experiment, with error bars showing ± SEM. Significance was assessed by one-way ANOVA with Tukey's post hoc test, with *** representing P< 0.0005, ****P< 0.0001.


E . BMDM , as described in panels ( A C ) , were seeded in Transwell ( Sigma ) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers . After two hours , failing to migrate through matrices were removed with a cotton swab . Migrated were counted with DAPI , with representative shown at low magnification . Representative from n = 3 biologically independent experiments are shown . F . Relative chemotaxis activity was calculated as fold of naïve that migrated without any added CCL5 . Each dot represents mean of collected from one experiment . Data are group means ± , with significance assessed by multiple t . , with * representing 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. BMDM cells, as described in panels (A and C), were seeded in Transwell (Sigma) chemotaxis assays using CCL5 as a chemoattractant in bottom chambers. After two hours, the cells failing to migrate through the matrices were removed with a cotton swab. Migrated cells were counted with DAPI images, with representative images shown at low magnification. Representative images from n=3 biologically independent experiments are shown. F. Relative chemotaxis activity was calculated as fold of naïve cells that migrated without any added CCL5. Each dot represents the mean of images collected from one experiment. Data are group means ± SEM, with significance assessed by multiple t test., with* representing P<0.05.


( A ) Mean clinical scores ( ± ) of C57BL / 6 → C57BL / 6 controls treated with DTX CD11c - DTR / GFP → C57BL / 6 treated with DTX or PBS during whole observation period starting from day - 1 . received in vitro - generated 2d2 . tdRFP Th17 intravenously on day 0 . DC depletion in CD11c - DTR / GFP → C57BL / 6 reduces encephalitogenicity of adoptively transferred 2d2 . tdRFP Th17 . Mann - Whitney - U - was performed on mean clinical scores ( n = 11PBS / 13DTX ) ; * p < 0 . 05 , * * p < 0 . 01 ; see also Appendix Table S1 . Pooled data from two independent experiments .

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

(A) Mean clinical scores (±SEM) of C57BL/6→C57BL/6 controls treated with DTX and CD11c-DTR/GFP→C57BL/6 treated with DTX or PBS during the whole observation period starting from day -1. All animals received in vitro-generated 2d2.tdRFP Th17 intravenously on day 0. DC depletion in CD11c-DTR/GFP→C57BL/6 reduces the encephalitogenicity of adoptively transferred 2d2.tdRFP Th17. Mann-Whitney-U-test was performed on mean clinical scores (n=11PBS/13DTX); * p < 0.05, ** p < 0.01; see also Appendix Table S1. Pooled data from two independent experiments.


( B ) Mononuclear were isolated on day 30 of shown in ( A ) from CNS of PBS - DTX - treated CD11c - DTR / GFP → C57BL / 6 that had been transferred with 2d2 . tdRFP Th17 . Flow cytometry was performed on surface stained samples . were pre - gated on lymphocyte ( FSC SSC gate ) PI - negative ; pooled data of three ; representative of two independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mononuclear cells were isolated on day 30 of animals shown in (A) from the CNS of PBS- and DTX-treated CD11c-DTR/GFP→C57BL/6 that had been transferred with 2d2.tdRFP Th17. Flow cytometry was performed on surface stained cell samples. Cells were pre-gated on lymphocyte cells (FSC SSC gate) and PI-negative; pooled data of three animals; representative of two independent experiments.


( C ) Mononuclear were isolated from CNS of PBS - DTX - treated 2d2 . tdRFPTh17 - transferred CD11c - DTR / GFP → C57BL / 6mice 2 - 3 d after onset of disease . Upper panels : were stimulated with plate - bound anti - CD3 / anti - CD28 , stained for CD4 cytokines IFN - y , IL - 17 ( pre - gated in addition to FSC / SSC on CD45 + CD4 + ) . Lower panels : expression of transcription factor FoxP3 ; pooled data from three , representative for two independent experiments . ( D ) , ( E ) of cytokine expression data as shown in ( C ) for pooled data from 2 independent experiments ; Mann - Whitney - U - * 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Mononuclear cells were isolated from the CNS of PBS- and DTX-treated 2d2.tdRFPTh17-transferred CD11c-DTR/GFP→C57BL/6mice 2-3 d after onset of the disease. Upper panels: cells were stimulated with plate-bound anti-CD3/anti-CD28, stained for CD4 and cytokines IFN-y, IL-17 (pre-gated in addition to FSC/SSC on CD45+CD4+). Lower panels: expression of transcription factor FoxP3; pooled data from three animals, representative for two independent experiments.(D), (E) Quantification of cytokine expression data as shown in (C) for pooled data from 2 independent experiments; Mann-Whitney-U-Test * p < 0.05.


( F ) 2d2Th17 were transferred into lymphopenic Rag2 - / - c - gamma - / - . Mononuclear were isolated from spleen brain at peak of disease analyzed for IL - 17 GM - CSF production after stimulation with anti - CD3 / anti - CD28 . ( G ) GM - CSF production of IL - 17 producers versus IL - 17 non - producers of CNS - isolated 2d2Th17 ( see also ( F ) ) . Each dot / square represents a single ; Kruskal - Wallis - ; * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 2d2Th17 were transferred into lymphopenic Rag2-/-c-gamma-/-. Mononuclear cells were isolated from the spleen and brain at the peak of the disease and analyzed for IL-17 and GM-CSF production after stimulation with anti-CD3/anti-CD28.(G) GM-CSF production of IL-17 producers versus IL-17 non-producers of CNS-isolated 2d2Th17 cells (see also (F)). Each dot/square represents a single animal; Kruskal-Wallis-test; * p < 0.05, ** p < 0.01, **** p < 0.0001.


( B ) Microarray signal intensities of strongest regulated genes from ( A ) are shown in detailed statistical for different T subgroups . Values are depicted as signal mean ± from three independent experiments . Statistical significance was determined using one - way ANOVA with post - hoc Tukey for multiple comparisons . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Microarray signal intensities of strongest regulated genes from (A) are shown in detailed statistical analysis for the different T cell subgroups. Values are depicted as signal intensity mean ± SEM from three independent experiments. Statistical significance was determined using one-way ANOVA with post-hoc Tukey Test for multiple comparisons. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


TPLSM of EAE lesions in brainstem of adoptive transfer EAE at onset of disease . EAE was induced by transfer of in vitro differentiated 2d2Th17 ( tdRFP - red , IL - 17 - EGFP - green ) into CD11c - DTR / GFPmice ( CD11c - GFP - green ) ; imaging 300 × 300 µm . ( A ) Intravascular IL - 17hi2d2Th17 ( arrow , double positive - > green : IL - 17EGFP red : 2d2 . tdRFP ) rolling towards a CD11c - GFP + ( arrowhead ) . dotted lines mark a venous vessel . ( B ) Time - lapse TPLSM ( maximal projections ) of boxed in ( A ) shows that perivascular elongated CD11c - GFP + ( asterisk ) enters into contact with IL - 17 - expressing ( GFP - green 2d2 . tdRFP - red double - positive ; arrow arrow head ) Th17 ( scale bar : 20 µm ) . ( C ) XYZ - resolved TPLSM depiction reveals that elongated perivascular CD11c - GFP + makes contact with intravascular 2d2Th17 via a filopodium - like dendrite ( xy - plane , 300 × 300 µm , z - depth 70 µm ) .

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

TPLSM of EAE lesions in the brainstem of adoptive transfer EAE at the onset of the disease. EAE was induced by transfer of in vitro differentiated 2d2Th17 cells (tdRFP-red, IL-17-EGFP-green) into CD11c-DTR/GFPmice (CD11c-GFP - green); imaging area 300 × 300 µm.(A) Intravascular IL-17hi2d2Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) rolling cell towards a CD11c-GFP+ cell (arrowhead). White dotted lines mark a venous vessel.(B) Time-lapse TPLSM (maximal intensity projections) of the boxed area in (A) shows that the perivascular elongated CD11c-GFP+ cell (asterisk) enters into contact with IL-17-expressing (GFP - green and 2d2.tdRFP - red double-positive; arrow and arrow head) Th17 cells (scale bar: 20 µm).(C) XYZ-resolved TPLSM depiction reveals that the elongated perivascular CD11c-GFP+ cell makes contact with the intravascular 2d2Th17 cells via a filopodium-like dendrite (xy-plane, 300 × 300 µm, z-depth 70 µm).


( D ) Strong interaction of a perivascular IL - 17hi2d2Th17 with a perivascular CD11c - GFP + ( insert upper left contact ) . ( E ) Time lapse of insert in ( D ) : Stopping motility of round - shaped IL - 17hi2d2Th17 is followed by entry into CNSparenchyma . ( F ) Magnified image of interaction of upper left ( G ) lower right perivascular T - DC contacts as shown in ( D ) ( E ) .

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

(D) Strong interaction of a perivascular IL-17hi2d2Th17 with a perivascular CD11c-GFP+ cell (insert upper left contact).(E) Time lapse of the insert in (D): Stopping motility of the round-shaped IL-17hi2d2Th17 cell is followed by entry into the CNSparenchyma.(F) Magnified image of interaction of the upper left and (G) lower right perivascular T cell-DC contacts as shown in (D) and (E).


( H ) Time - lapse imaging of parenchymal CD11c - GFP + ( arrowhead ) , targeted migration of an IL - 17hi 2d2 Th17 ( arrow , double positive - > green : IL - 17EGFP red : 2d2 . tdRFP ) towards CD11c - GFP + ( arrowhead ) . Scale bar is 20 µm .

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

(H) Time-lapse imaging of parenchymal CD11c-GFP+ cells (arrowhead), targeted migration of an IL-17hi 2d2 Th17 cell (arrow, double positive -> green: IL-17EGFP and red: 2d2.tdRFP) towards the CD11c-GFP+ cell (arrowhead). Scale bar is 20 µm.


( I ) of motility pattern of IL - 17hi 2d2 . tdRFP vs . 2d2 . tdRFP in CD11c - DTR / GFP brainstem lesions before after ( 2 - 5 h ) intraperitoneal DTX injection . Evaluation of mean track velocity of single tracking ( each dot represents a track ) ; pooled data from at least two independent experiments ( > 3 imaging areas ) . For statistical , Mann - Whitney - U - was performed ( * * * p < 0 . 0001 , n . s . = not significant ) . ( K ) of stopping ( red bar ; instantaneous velocity < 2 µm / min ) of data set as shown in ( I ) .

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

(I) Quantification of the motility pattern of IL-17hi 2d2.tdRFP vs. all 2d2.tdRFP in CD11c-DTR/GFP brainstem lesions before and after (2-5 h) intraperitoneal DTX injection. Evaluation of mean track velocity of the single cell tracking (each dot represents a track); pooled data from at least two independent experiments (>3 imaging areas). For the statistical analysis, the Mann-Whitney-U-test was performed (*** p < 0.0001, n.s. = not significant).(K) Percentage of stopping cells (red bar; instantaneous velocity <2 µm/min) of the data set as shown in (I).


Active EAE was induced by subcutaneous immunization with CFA / MOG35 - 55 . were isolated from CNS of EAE at different stages of disease ( pre - onset [ day 8 - 9 ] , peak [ day 14 - 17 ] , chronic [ day 22 - 27 ] ; data from at least two independent experiments ) . Representative samples are shown , which were analyzed by flow cytometry for expression of markers CD11c , CD11b Ly6C / G ( pre - gated on lympho - monocytic by FSC / SSC ; IAb + expression was confirmed for CD11c + in separate stainings ) . ( A ) In CNS , there was a strong increase of CD11c + proportions over different phases of disease . composition of CD11c + subpopulations changed over time , starting with a preponderance of CD11c + Ly6C / G - conventional DCs before onset of EAE . In peak chronic phase there was a continuous increase in proportion of monocyte - derived CD11c + . ( B ) For CNS , of absolute numbers shows a strong increase in CD11c + during course of EAE . ( C ) In CNS , proportions of subpopulations show an increasing proportion of monocyte - derived CD11c + .

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

Active EAE was induced by subcutaneous immunization with CFA/MOG35-55. Cells were isolated from the CNS of EAE animals at different stages of the disease (pre-onset [day 8-9], peak [day 14-17], chronic [day 22-27]; data from at least two independent experiments). Representative samples are shown, which were analyzed by flow cytometry for the expression of the markers CD11c, CD11b and Ly6C/G (pre-gated on lympho-monocytic cells by FSC/SSC; IAb+ expression was confirmed for CD11c+ cells in separate stainings).(A) In the CNS, there was a strong increase of CD11c+ proportions over the different phases of the disease. The composition of CD11c+ subpopulations changed over time, starting with a preponderance of CD11c+Ly6C/G- conventional DCs before onset of EAE. In the peak and chronic phase there was a continuous increase in the proportion of monocyte-derived CD11c+ cells.(B) For the CNS, the quantification of the absolute numbers shows a strong increase in CD11c+ cells during the course of EAE.(C) In the CNS, the proportions of the subpopulations show an increasing proportion of monocyte-derived CD11c+ cells.


( A ) CD11c + , CD4 + CX3CR1 + CD45intIAb + ( microglia ) were isolated from peak EAE from CNS . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleen CD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . CNS CD11c + show gene expression of inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Il23 ( mean ± ) . ( C ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in CNS samples as described

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

(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleen CD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNS CD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Il23 (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above


( B ) CD11c + , CD4 + CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . Relevantly regulated chemokines in other populations than CNS CD11c + are shown ( mean ± ) . ( D ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in spleen samples .

(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). Relevantly regulated chemokines in other cell populations than CNS CD11c+ cells are shown (mean ± SEM).(D) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the spleen samples.


Figure 5Chemokine expression profiling of CD11c + in spleen CNS reveal a key role of CNSCD11c + for inflammatory chemokines . ( A ) CD11c + , CD4 + CX3CR1 + CD45intIAb + ( microglia ) were isolated from peak EAE from CNS . Samples from 6 - 11 were pooled in each experiment ; at least two experiments were performed for each subset . Depicted are normalized mRNA expression ( to expression in spleenCD11c + ) as determined by qRT - PCR relative to housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 ( Eef1a1 ) Peptidylprolyl isomerase A ( Ppia ) . CNSCD11c + show gene expression of inflammatory chemokines Ccl2 , Ccl5 , Cxcl9 , Cxcl10 but not Ccl20 ( mean ± ) . ( B ) CD11c + , CD4 + CD11c - CD11b + IAb + ( monocytes / macrophages ) were isolated from EAE affected mice as described in ( A ) . Relevantly regulated chemokines in other populations than CNSCD11c + are shown ( mean ± ) . ( C ) Quantitative expression of EAE - relevant factors Il23 Csf2 were investigated in CNS samples as described above ( D ) spleen samples . Statistical significance was determined using Kruskal - Wallis . P - values < 0 . 05 were regarded as statistically significant . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

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

Figure 5Chemokine expression profiling of CD11c+ cells in the spleen and CNS reveal a key role of CNSCD11c+ cells for inflammatory chemokines.(A) CD11c+ cells, CD4+ cells and CX3CR1+CD45intIAb+ (microglia) cells were isolated from peak EAE animals from the CNS. Samples from 6-11 animals were pooled in each experiment; at least two experiments were performed for each subset. Depicted are normalized mRNA expression (to expression in spleenCD11c+ cells) levels as determined by qRT-PCR analysis relative to the housekeeping genes Eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) and Peptidylprolyl isomerase A (Ppia). CNSCD11c+ cells show gene expression of the inflammatory chemokines Ccl2, Ccl5, Cxcl9, Cxcl10 but not Ccl20 (mean ± SEM).(B) CD11c+ cells, CD4+ cells and CD11c-CD11b+IAb+ (monocytes/macrophages) were isolated from EAE affected mice as described in (A). Relevantly regulated chemokines in other cell populations than CNSCD11c+ cells are shown (mean ± SEM).(C) Quantitative expression of EAE-relevant factors Il23 and Csf2 were investigated in the CNS samples as described above and (D) the spleen samples.Statistical significance was determined using Kruskal-Wallis test. P-values < 0.05 were regarded as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


( A ) CD11c - GFP + DC cluster formation near venous vessel with ( B ) protrusions attracting 2d2 . tdRFP Th17 .

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(A) CD11c-GFP+ DC cluster formation near venous vessel with (B) protrusions attracting 2d2.tdRFP Th17 cells.


( C ) Low - velocity motility of 2d2 . tdRFPT in DC cluster compared to outside of cluster . SHG in upper panel on left ( arrow ) . In addition , intercellular matrix ( arrow ) are visualized that are only found in inflamed CNSparenchyma are due to second harmonic generation ( SHG ) at 1110 nm wavelength excitation detection at 535 ± 50nm . Tracking of 2d2 . tdRFPTh17 in right panel ( color - coded tracks , low velocity : red , high velocity : yellow ) . ( D ) Quantitative of T tracking : higher mean velocity of 2d2 . tdRFPTh17 in onset of disease with differential distribution at peak of disease . Cluster associated tracks have significantly lower mean velocities than Th17 outside CD11c - GFP clusters . ( E ) Analogously for track straightness ; Kruskal - Wallis Dunn ' s Multiple Comparison ( * * * p < 0 . 0001 ) were performed ; pooled data from at least three independent experiments per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Low-velocity motility of 2d2.tdRFPT cells in DC cluster compared to outside of the cluster. SHG in upper panel on the left (arrow). In addition, intercellular matrix fibers (arrow) are visualized that are only found in inflamed CNSparenchyma and are due to second harmonic generation (SHG) at 1110 nm wavelength excitation and detection at 535 ± 50nm. Tracking of 2d2.tdRFPTh17 in right panel (color-coded tracks, low velocity: red, high velocity: yellow). (D) Quantitative analysis of T cell tracking: higher mean velocity of 2d2.tdRFPTh17 cells in the onset of the disease with differential distribution at the peak of the disease. Cluster associated tracks have significantly lower mean velocities than Th17 cells outside the CD11c-GFP clusters. (E) Analogously for track straightness; Kruskal-Wallis Test and Dunn's Multiple Comparison Test (*** p < 0.0001) were performed; pooled data from at least three independent experiments per group.


A . unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15 - 18 h post - injection . arrowheads represent polar ( PB ) . PN represents pronucleus .

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

A. The unfertilized oocytes were injected with spermatozoa with a variant in IQCN at 15-18 h post-injection. The white arrowheads represent the polar body (PB). PN represents pronucleus.


B . expression localization of PLCζ in sperm from affected individuals .

B. The expression and localization of PLCζ in sperm from the affected individuals.


C . H & E of spermatozoa from affected individuals . black arrow represents normal spermatozoa . black arrowheads represent tapered - head spermatozoa with increased major axis normal minor axis . arrowheads represent thin spermatozoa with decreased major axis minor axis .

O O O O O O O O O O O O O O O O O O O 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. H&E staining of spermatozoa from affected individuals. The black arrow represents normal spermatozoa. The black arrowheads represent tapered-head spermatozoa with increased major axis length and normal minor axis length. The white arrowheads represent thin spermatozoa with decreased major axis length and minor axis length.


D . ultrastructure of spermatozoa from normal affected . squares represent perinuclear ring . Ac represents acrosome . N represents nucleus .

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

D. The ultrastructure of spermatozoa from normal and affected individual. The white squares represent the perinuclear ring. Ac represents acrosome. N represents nucleus.


E . range of acrosomes in sperm heads from normal affected ( n = 7 ) .

E. The range of acrosomes in sperm heads from normal and affected individual (n=7).


A . Fertility assessment experiments in WT Iqcn - / - mice after mating with WT mice ( n = 18 ) .

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

A. Fertility assessment experiments in WT and Iqcn-/- male mice after mating with WT female mice (n = 18).


B . of testes epididymides in WT Iqcn - / - mice .

B. Size of the testes and epididymides in WT and Iqcn-/- male mice.


C . of testis / in WT Iqcn - / - mice ( n = 3 ) .

C. Quantification ratio of the testis weight/body weight in WT and Iqcn-/- male mice (n = 3).


D . Sperm in caudal epididymis from WT Iqcn - / - mice ( n = 3 ) .

D. Sperm counts in the caudal epididymis from WT and Iqcn-/- male mice (n = 3).


E . H & E of sperm from WT Iqcn - / - mice . Red arrowheads represent sperm with no sickle shape heads .

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

E. H&E staining of sperm from WT and Iqcn-/- male mice. Red arrowheads represent sperm with no sickle shape heads.


F . of sperm without sickle shape heads from WT Iqcn - / - mice ( n = 3 ) .

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

F. Percentage of sperm without sickle shape heads from WT and Iqcn-/- male mice (n = 3).


G . Ultrastructure of spermatozoa from WT Iqcn - / - mice . Ac represents acrosome .

G. Ultrastructure of spermatozoa from WT and Iqcn-/- male mice. Ac represents the acrosome.


H . Immunostaining of lectin ( green ) in spermatozoa from WT Iqcn - / - mice .

H. Immunostaining of lectin (green) in spermatozoa from WT and Iqcn-/- male mice.


I . of abnormal acrosomes in spermatozoa from WT Iqcn - / - mice ( n = 3 ) .

I. Percentage of abnormal acrosomes in spermatozoa from WT and Iqcn-/- male mice (n = 3).


J . of spontaneous induced acrosome reactions in spermatozoa from WT Iqcn - / - mice ( n = 3 ) .

J. Percentage of spontaneous and induced acrosome reactions in spermatozoa from WT and Iqcn-/- male mice (n = 3).


A . Representative two pronucleus ( 2PN ) zygotes , two - , blastocysts after ICSI in WT Iqcn - / - mice .

A. Representative two pronucleus (2PN) zygotes, two-cell embryos, and blastocysts after ICSI in WT and Iqcn-/- male mice.


B . of 2PN zygotes , two - blastocysts after ICSI in WT Iqcn - / - mice ( n = 3 ) .

B. Percentage of 2PN zygotes, two-cell embryos and blastocysts after ICSI in WT and Iqcn-/- male mice (n = 3).


C . Profile of Ca2 + responses induced by oocyte injection with spermatozoa from WT Iqcn - / - mice ( n = 5 ) . Spermatozoa from WT mice induced 8 - 9 Ca2 + spikes over a 3 - h period ( blue ) , while spermatozoa from Iqcn - / - mice failed to induce spikes ( red ) .

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

C. Profile of Ca2+ responses induced by oocyte injection with spermatozoa from WT and Iqcn-/- male mice (n = 5). Spermatozoa from WT male mice induced 8-9 Ca2+ spikes over a 3-h period (blue line), while spermatozoa from Iqcn-/- male mice failed to induce spikes (red line).


D . Immunostaining of oocytes injected with spermatozoa from WT Iqcn - / - mice . expression of PLCζ ( red ) α - tubulin ( green ) was detected in oocytes . M represents nucleus F represents nucleus .

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

D. Immunostaining of oocytes injected with spermatozoa from WT and Iqcn-/- male mice. The expression of PLCζ (red) and α-tubulin (green) was detected in oocytes. M represents male nucleus and F represents the female nucleus.


E . Expression localization of PLCζ in sperm from WT Iqcn - / - mice .

E. Expression and localization of PLCζ in sperm from WT and Iqcn-/- male mice.


F . Expression localization of PLCζ during acrosomal biogenesis from WT Iqcn - / - mice . PLCζ was localized in acroplaxome in WT spermatids . PLCζ was only localized in tip of head in cap acrosome phases ( arrowheads ) was lost during maturation phase in Iqcn - / - mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Expression and localization of PLCζ during acrosomal biogenesis from WT and Iqcn-/- male mice. PLCζ was localized in the acroplaxome in WT spermatids. PLCζ was only localized in the tip of the head in the cap and acrosome phases (white arrowheads) and was lost during the maturation phase in Iqcn-/- mice.


A . Expression localization of IQCN during spermiogenesis in testes of WT mice . IQCN ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 . Sb represents late spermatids . Scale bars , 20 μm .

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

A. Expression and localization of IQCN during spermiogenesis in the testes of WT male mice. IQCN (red) was co-localized with α-tubulin (green) from steps 8-14. Sb represents late spermatids. Scale bars, 20 μm.


B . Ultrastructure of testes from WT Iqcn - / - mice from steps 8 - 16 . MA represents manchette . PR represents perinuclear ring . CD represents droplets . Scale bars , 2 μm ( steps 8 - 12 ; 15 - 16 ) 5 μm ( steps 13 - 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 O 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. Ultrastructure of the testes from WT and Iqcn-/- male mice from steps 8-16. MA represents manchette. PR represents perinuclear ring. CD represents cytoplasmic droplets. Scale bars, 2 μm (steps 8-12; 15-16) and 5 μm (steps 13-14).


C . Immunostaining of α - tubulin ( green ) in spermatids at steps 13 - 14 . MA represents manchette . PR represents perinuclear ring .

O O O 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. Immunostaining of α-tubulin (green) in spermatids at steps 13-14. MA represents manchette. PR represents perinuclear ring.


D . of manchette structure in spermatids at steps 13 - 14 from WT Iqcn - / - mice ( n = 10 ) .

D. Length of the manchette structure in spermatids at steps 13-14 from WT and Iqcn-/- male mice (n = 10).


A . Expression localization of calmodulin during spermiogenesis in testes of WT Iqcn - / - mice . Calmodulin ( red ) was co - localized with α - tubulin ( green ) from steps 8 - 14 in WT mice , but failed to co - localize in steps 13 - 14 in Iqcn - / - mice .

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

A. Expression and localization of calmodulin during spermiogenesis in the testes of WT and Iqcn-/- male mice. Calmodulin (red) was co-localized with α-tubulin (green) from steps 8-14 in WT mice, but failed to co-localize in steps 13-14 in Iqcn-/- male mice.


B . Interaction between IQCN calmodulin ( CaM ) following co - IP by IQCN antibody in testes from WT Iqcn - / - mice ( n = 3 ) . IQCN interacts with calmodulin in WT mice , interaction of which is completely lost in Iqcn - / - mice . GAPDH was used as internal control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Interaction between IQCN and calmodulin (CaM) following co-IP by IQCN antibody in testes from WT and Iqcn-/- male mice (n = 3). IQCN interacts with calmodulin in WT mice, the interaction of which is completely lost in Iqcn-/- male mice. GAPDH was used as the internal control.


C . Immunostaining of calmodulin ( red ) α - tubulin ( green ) in HeLa . normal ( WT ) mutant ( c . 910C > T c . 2453 _ 2454del ) plasmids of IQCN were transfected into HeLa . HeLa were treated with an antagonist of calmodulin W - 7 . Multipolar spindles were observed in HeLa transfected with mutant plasmids W - 7 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 O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O

C. Immunostaining of calmodulin (red) and α-tubulin (green) in HeLa cells. The normal (WT) and mutant (c.910C>T and c.2453_2454del) plasmids of IQCN were transfected into HeLa cells. HeLa cells were treated with an antagonist of calmodulin W-7. Multipolar spindles were observed in HeLa cells transfected with mutant plasmids and W-7 treatment.


D . of multipolar spindles in WT mutant IQCN transfected HeLa . ( n = 3 biological replicates )

D. Percentage of multipolar spindles in WT and mutant IQCN transfected HeLa cells. (n = 3 biological replicates)


C ) , of pups gathered in nest location is significantly reduced in Shank2 - / - mice . Mann - Whitney , * * * P < 0 . 001 . Data information : data are presented as mean ± .

O O O O O O O O O O O O O O O O O O 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), the percentage of pups gathered in the nest location is significantly reduced in Shank2-/- mice. Mann-Whitney test, ***P<0.001. Data information: All data are presented as mean ± SEM.


( H ) Shank2 + / + Shank2 - / - mice displayed a significant preference for novel object versus familiar one in session , two - way mixed ANOVA , effect of object : * * * P < 0 . 001 , effect of genotype : P = 0 . 167 , object x genotype interaction : P = 0 . 667 , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : graphs are presented as mean ± , NS : not significant .

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(H) Shank2+/+ and Shank2-/- mice displayed a significant preference for the novel object versus the familiar one in the test session, two-way mixed ANOVA, effect of object: ***P<0.001, effect of genotype: P=0.167, object x genotype interaction: P=0.667, Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All graphs are presented as mean ± SEM, NS: not significant.


( J ) Left panel : Electron of posterior pituitary from Shank2 + / + Shank2 - / - mice in lower ( left panel ) higher magnification . Scale bar ( 500 nm ) . Right diagram : No significant difference was detected in number of vesicles in posterior pituitary between Shank2 + / + Shank2 - / - mice , Mann - Whitney , P = 0 . 507 , Shank2 + / + n = 3 , Shank2 - / - n = 3 . ( K - M ) Additionally , Shank2 - / - mice display no significant difference in : ( K ) Estradiol - , Mann - Whitney , P = 0 . 475 , Shank2 + / + n = 6 , Shank2 - / - n = 7 , ( L ) Progesterone , unpaired , two - Student ' s t - , P = 0 . 570 , Shank2 + / + n = 5 , Shank2 - / - n = 5 or ( M ) Prolactin - plasma concentrations , unpaired , two - Student ' s t - , P = 0 . 980 ; Shank2 + / + n = 3 , Shank2 - / - n = 3 . Data information : data are presented as mean ± , NS : not significant . E , endothelium ; CL , capillary lumen ; M , mitochondria ; NG , neurosecretory granule ; AE , axonal endings , HYP , Hypothalamus ; PIT , Pituitary .

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

(J) Left panel: Electron micrographs of the posterior pituitary gland from Shank2+/+ and Shank2-/- mice in lower (left panel) and higher magnification. Scale bar (500 nm). Right diagram: No significant difference was detected in the number of vesicles in the posterior pituitary gland between Shank2+/+ and Shank2-/- mice, Mann-Whitney test, P=0.507, Shank2+/+ n = 3, Shank2-/- n = 3. (K-M) Additionally, Shank2-/- mice display no significant difference in: (K) Estradiol-, Mann-Whitney test, P=0.475, Shank2+/+ n = 6, Shank2-/- n = 7, (L) Progesterone, unpaired, two-tailed Student's t-test, P=0.570, Shank2+/+ n = 5, Shank2-/- n = 5 or (M) Prolactin- plasma concentrations, unpaired, two-tailed Student's t-test, P=0.980; Shank2+/+ n = 3, Shank2-/- n = 3. Data information: All data are presented as mean ± SEM, NS: not significant. E, endothelium; CL, capillary lumen; M, mitochondria; NG, neurosecretory granule; AE, axonal endings, HYP, Hypothalamus; PIT, Pituitary gland.


( A - G ) qRT - PCR of mRNA expression of hormonal genes hormonal receptors normalized to house - keeping gene HMBS of 9 - 10 week old naive postnatal day 1 Shank2 + / + Shank2 - / - mice : ( A ) Oxytocin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 335 . ( B ) Vasopressin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 945 . ( C ) Prolactin gene expression did not differ between Shank2 + / + Shank2 - / - mice , one - way ANOVA , P = 0 . 202 . Additionally , no significant difference was evident in ( D ) oxytocin receptor , one - way ANOVA , P = 0 . 675 , ( E ) prolactin receptor , one - way ANOVA , P = 0 . 912 , ( F ) vasopressin receptor , one - way ANOVA , P = 0 . 418 ( G ) estrogen receptor alpha expression , one - way ANOVA , P = 0 . 890 in comparison to Shank2 + / + mice . data are presented as mean ± , NS : not significant . Pituitary , ( n = 3 pituitary pooled per value , n = 3 hypothalamus ( Oxytocin , Vasopressin , Oxytocin receptor , Vasopressin receptor , estrogen alpha ) n = 2 - 3 hypothalamus ( Prolactin receptor ) ) . HYP , hypothalamus ; PIT , pituitary .

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

(A-G) qRT-PCR analysis of mRNA expression levels of hormonal genes and hormonal receptors normalized to house-keeping gene HMBS of 9-10 week old naive and postnatal day 1 Shank2+/+ and Shank2-/- mice: (A) Oxytocin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.335. (B) Vasopressin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.945. (C) Prolactin gene expression did not differ between Shank2+/+ and Shank2-/- mice, one-way ANOVA, P=0.202. Additionally, no significant difference was evident in (D) oxytocin receptor, one-way ANOVA, P=0.675, (E) prolactin receptor, one-way ANOVA, P=0.912, (F) vasopressin receptor, one-way ANOVA, P=0.418 and (G) estrogen receptor alpha expression, one-way ANOVA, P=0.890 in comparison to Shank2+/+ mice. All data are presented as mean ± SEM, NS: not significant. Pituitary gland, (n=3 pituitary glands pooled per value, n=3 hypothalamus (Oxytocin, Vasopressin, Oxytocin receptor, Vasopressin receptor, estrogen alpha) n=2-3 hypothalamus (Prolactin receptor)). HYP, hypothalamus; PIT, pituitary gland.


( E - H ) Repeated pup exposure does not induce major components of social attachment behavior in Shank2 - / - : ( E ) pup grooming , unpaired , two - Student ' s t - , * * * P < 0 . 001 ( F ) crouching , Mann - Whitney , * * * P < 0 . 001 ( G ) nest building , Mann - Whitney , * * * P < 0 . 001 , ( H ) maternal interaction , unpaired , two - Student ' s t - , * * * P < 0 . 001 ( trial 5 ) , Shank2 + / + n = 9 , Shank2 - / - n = 9 . Data information : data are presented as mean ± , NS : 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 O O O O O

(E-H) Repeated pup exposure does not induce major components of social attachment behavior in Shank2-/- females: (E) pup grooming, unpaired, two-tailed Student's t-test, ***P<0.001 (F) crouching, Mann-Whitney test, ***P<0.001 and (G) nest building, Mann-Whitney test, ***P<0.001, (H) maternal interaction, unpaired, two-tailed Student's t-test, ***P<0.001 (trial 5), Shank2+/+ n = 9, Shank2-/- n = 9. Data information: All data are presented as mean ± SEM, NS: not significant.


( L ) of synaptic in crude synaptosomes fraction ( P2 ) from hypothalamus of Shank2 + / + Shank2 - / - mice . Shank2 + / + n = 5 ( black bars ) , Shank2 - / - n = 5 ( blue bars ) . Unpaired , two - Student ' s t - .

(L) Levels of synaptic proteins in the crude synaptosomes fraction (P2) from the hypothalamus of Shank2+/+ and Shank2-/- mice. Shank2+/+ n = 5 (black bars), Shank2-/- n = 5 (blue bars). Unpaired, two-tailed Student's t-test.


( D - G ) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2 - / - mice : ( D ) grooming , two - way ANOVA , effect of genotype : * P = 0 . 018 , effect of treatment : * * P = 0 . 002 , genotype x treatment interaction : * * * P = 0 . 001 . ( E ) Crouching , two - way ANOVA , effect of genotype : P = 0 . 091 , effect of treatment : P = 0 . 869 , genotype x treatment interaction : P = 0 . 663 , ( F ) nest building , two - way ANOVA , effect of genotype : P = 0 . 415 , effect of treatment : P = 0 . 341 genotype x treatment interaction : P = 0 . 093 , ( G ) Maternal interaction , two - way ANOVA , effect of genotype : * P = 0 . 034 , effect of treatment : P = 0 . 149 , genotype x treatment interaction : * P = 0 . 045 . Shank2 + / + vehicle injected n = 6 , Shank2 - / - vehicle injected n = 6 , Shank2 + / + CNO injected n = 6 , Shank2 - / - CNO injected n = 7 . Data information : data are presented as mean ± , NS : not significant .

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

(D-G) DREADD activation by CNO injection selectively rescues major components of social attachment behavior in Shank2-/- mice: (D) grooming, two-way ANOVA, effect of genotype: *P=0.018, effect of treatment: **P=0.002, genotype x treatment interaction: ***P=0.001. (E) Crouching, two-way ANOVA, effect of genotype: P=0.091, effect of treatment: P=0.869, genotype x treatment interaction: P=0.663, (F) nest building, two-way ANOVA, effect of genotype: P=0.415, effect of treatment: P=0.341 genotype x treatment interaction: P=0.093, (G) Maternal interaction, two-way ANOVA, effect of genotype: *P=0.034, effect of treatment: P=0.149, genotype x treatment interaction: *P=0.045. Shank2+/+ vehicle injected n = 6, Shank2-/- vehicle injected n = 6, Shank2+/+ CNO injected n = 6, Shank2-/- CNO injected n = 7. Data information: All data are presented as mean ± SEM, NS: not significant.


D H & E ( hematoxylin - eosin ) ( left ) oil red O ( right ) of livers . Scale bar , 50μm .

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

D H&E (hematoxylin-eosin) staining (left) and oil red O staining (right) of livers. Scale bar, 50μm.


E Serum cholesterol concentrations of each group . Liver cholesterol concentrations ( F ) of each group were examined .

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

E Serum cholesterol concentrations of each group. Liver cholesterol concentrations (F) of each group were examined.


H I Cholesterol in p53 + / + p53 - / - HepG2 ( H ) or HCT116 ( I ) were examined . expression was shown by western blotting ( bottom panel ) .

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

H and I Cholesterol levels in p53+/+ and p53-/- HepG2 cells (H) or HCT116 cells (I) were examined. Protein expression was shown by western blotting (bottom panel).


C mRNA of SQLE in different tissues of p53 + / + or p53 - / - mice were determined by qRT - PCR western blot respectively .

O O O O 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 mRNA and protein levels of SQLE in different tissues of p53+/+ or p53-/- mice were determined by qRT-PCR and western blot respectively.


F G p53 + / + p53 - / - HepG2 were cultured for 48 hours in medium containing bovine serum ( Serum ) or lipoprotein - depleted FBS ( LPDS ) . expression was shown by western blotting ( F ) . mRNA of SQLE were examined by qRT - PCR ( G ) . P : premature SREBP2 ; M : mature SREBP2 .

O 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

F and G p53+/+ and p53-/- HepG2 cells were cultured for 48 hours in medium containing fetal bovine serum (Serum) or lipoprotein-depleted FBS (LPDS). Protein expression was shown by western blotting (F). mRNA levels of SQLE were examined by qRT-PCR (G). P: premature SREBP2; M: mature SREBP2.


expression mRNA of p53 + / + p53 - / - HepG2 treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated .

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

Protein expression and mRNA levels of p53+/+ and p53-/- HepG2 cells treated with control siRNA or three separated sets of SREBP2 siRNAs for 72 hours as indicated.


F Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into p53 - / - HCT116 together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative of luciferase are shown . Data represent three independent experiments . expression is shown G Luciferase reporter constructs containing RE1 , RE2 , RE1mut or RE2mut were transfected into HEK293T together with control ( PRK5 - flag vector ) or p53 expression vector ( PRK5 - flag p53 ) for 48 hours . Renilla vector pRL - CMV was used as a transfection internal control . Relative of luciferase are shown . expression is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into p53-/- HCT116 cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown G Luciferase reporter constructs containing RE1, RE2, RE1mut or RE2mut were transfected into HEK293T cells together with control (PRK5-flag vector) or p53 expression vector (PRK5-flag p53) for 48 hours. Renilla vector pRL-CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.


A - C Squalene ( A ) , lanosterol ( B ) cholesterol ( C ) abundance in p53 + / + p53 - / - HepG2 transfected with control or SQLE siRNA for 48 hours were determined via ultra - high pressure liquid chromatography coupled to mass spectrometry ( UHPLC - MS ) . were analyzed by western blotting with specific antibodies ( A bottom panel ) .

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

A-C Squalene (A), lanosterol (B) and cholesterol (C) abundance in p53+/+ and p53-/- HepG2 cells transfected with control or SQLE siRNA for 48 hours were determined via ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS). Protein levels were analyzed by western blotting with specific antibodies (A bottom panel).


A expression of SQLE in human liver tumors ( C ) non - cancerous adjacent tissue ( N ) were analyzed by western blotting .

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

A Protein expression of SQLE in human liver tumors (C) and non-cancerous adjacent tissue (N) were analyzed by western blotting.


B SQLE gene expression in HCC adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma ( normal [ n = 50 ] versus tumors [ n = 371 ] ) . C SQLE gene expression in COAD adjacent normal tissue was analyzed in TCGA Colon Cancer ( normal [ n = 41 ] versus tumors [ n = 286 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SQLE gene expression in HCC and adjacent normal tissue was analyzed in TCGA Liver Hepatocellular Carcinoma (normal [n=50] versus tumors [n=371]). C SQLE gene expression in COAD and adjacent normal tissue was analyzed in TCGA Colon Cancer (normal [n=41] versus tumors [n=286]).


J - M p53 + / + HCT116 ( J K ) p53 + / + HepG2 ( L M ) were transfected with SQLE siRNA or control siRNA in presence or absence of exogenous SQLE cDNA for 48 hours . Cholesterol ( upper panel ) expression ( bottom panel ) were determined ( J L ) . proliferation is shown ( K M ) .

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

J - M p53+/+ HCT116 cells (J and K) and p53+/+ HepG2 cells (L and M) were transfected with SQLE siRNA or control siRNA in the presence or absence of exogenous SQLE cDNA for 48 hours. Cholesterol concentration (upper panel) and protein expression (bottom panel) were determined (J and L). Cell proliferation is shown (K and M).


C Growth of p53 + / + p53 - / - HepG2 transfected with without exogenous SQLE ( HA - SQLE ) in LPDS medium . expression is assayed after transfection 24 hours .

O O O O O O O O O O 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 Growth of p53+/+ and p53-/- HepG2 cells transfected with and without exogenous SQLE (HA-SQLE) in LPDS medium. Protein expression is assayed after transfection 24 hours.


G H Cholesterol , expression ( G ) growth ( H ) of SK - HEP - 1 stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol .

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 B-CONTROLLED_VAR O

G and H Cholesterol levels, protein expression (G) and cell growth (H) of SK-HEP-1 cells stably expressing control shRNA or p53 shRNA in LPDS medium in presence or absence of 5μg cholesterol.


L of SA - β - gal - positive of p53 + / + p53 - / - HepG2 expressing SQLE siRNA or control siRNA ( upper panel ) . expression ( middle panel ) representative ( lower panel ) are shown . Sacle bar , 100 μm .

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

L Percentage of SA-β-gal-positive cells of p53+/+ and p53-/- HepG2 cells expressing SQLE siRNA or control siRNA (upper panel). Protein expression (middle panel) and representative images (lower panel) are shown . Sacle bar, 100 μm.


p53 - / - C57BL / 6N mice ( n = 7 ) were injected with a single dose of diethylnitrosamine ( DEN ) ( 25mg / kg ) at two then fed with HFD diet at 4 . Oral administration of terbinafine were given at 13 old as indicated . Shown are liver incidence ( L ) numbers ( M ) .

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

p53-/- C57BL/6N male mice (n=7) were injected with a single dose of diethylnitrosamine (DEN) (25mg/kg) at two weeks and then fed with HFD diet at 4 weeks. Oral administration of terbinafine were given at 13 weeks old as indicated. Shown are liver tumor incidence (L) and tumor numbers (M).


( N ) H & E ( left ) , Ki - 67 ( middle ) Oil Red O ( right ) of livers of mice .

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

(N) H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice.


( O ) Total liver cholesterol concentrations of mice were examined .

(O) Total liver cholesterol concentrations of mice were examined.


representative liver photos ( C , left ) , liver numbers ( C , right ) , representative ( D , left ) ( D , right ) of Ki - 67 are shown . Red arrows indicate tumors in liver ( C ) .

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

representative liver photos (C, left), liver tumor numbers (C, right), representative images (D, left) and quantification (D, right) of Ki-67 staining are shown. Red arrows indicate the tumors in the liver (C).


E H & E ( left ) , Ki - 67 ( middle ) Oil Red O ( right ) of livers of

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

E H&E (left), Ki-67 (middle) and Oil Red O (right) staining of livers of mice


A : Serial dilutions of stationary phase cultures of indicated strains were spotted on YEPD incubated at indicated temperatures .

A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.


Wild type , mps1 - 3 mps1 - 1 mutant were synchronised in G1 with α - factor at permissive ( 25 ° C ) then released at restrictive ( 34 ° C , t = 0 ) . were collected at indicated time points for FACS of contents ( C )

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

Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents (C)


Wild type , mps1 - 3 mps1 - 1 mutant were synchronised in G1 with α - factor at permissive ( 25 ° C ) then released at restrictive ( 34 ° C , t = 0 ) . were collected at indicated time points for immunofluorescence using anti - tubulin antibodies in order to score metaphase anaphase spindles ( D ) . Budding division were scored on FACS samples .

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

Wild type, mps1-3 and mps1-1 mutant cells were synchronised in G1 with α-factor at permissive temperature (25°C) and then released at restrictive temperature (34°C, t=0). Cells were collected at the indicated time points for immunofluorescence using anti-tubulin antibodies in order to score metaphase and anaphase spindles (D). Budding and nuclear division were scored on the FACS samples.


F : Wild type mps1 mutant bearing TetO / TetR - GFP system to mark centromere of chromosome V [ 43 ] expressing SPBs marker Spc97 - mCherry were synchronised as in ( C - D ) arrested in anaphase through - sensitive cdc15 - 2 allele . At 120 150 minutes , were fixed for scoring chromosome V segregation ( n ≥ 172 ) . Arrowheads indicate sister chromatids of chromosome V . Error bars : SD . N = 3 . Representative of for each genotype are shown on right . Scale bar : 5 μm .

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

F: Wild type and mps1 mutant cells bearing the TetO/TetR-GFP system to mark the centromere of chromosome V [43] and expressing the SPBs marker Spc97-mCherry were synchronised as in (C-D) and arrested in anaphase through the temperature-sensitive cdc15-2 allele. At 120 and 150 minutes, cells were fixed for scoring chromosome V segregation (n≥172). Arrowheads indicate the sister chromatids of chromosome V. Error bars: SD. N=3. Representative images of cells for each genotype are shown on the right. Scale bar: 5 μm.


G : Wild type mps1 - 3 mutant carrying TetO / TetR - GFP markers for CEN5 labelling expressing mCherry - Tub1 were grown at 25 ° C then shifted to 34 ° C for 1 hour before filming . were filmed at 34 ° C every 2 minutes by time lapse microscopy . DIC : differential interference contrast . Scale bar : 5 μm .

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

G: Wild type and mps1-3 mutant cells carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for 1 hour before filming. Cells were filmed at 34°C every 2 minutes by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.


A : Wild type mps1 mutant were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in presence of nocodazole ( t = 0 ) . were collected at indicated time points for FACS of contents .

A: Wild type and mps1 mutant cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.


B : were treated as in ( A ) after 2 hours from release α - factor was re - added to prevent from undergoing a second cycle . samples were collected at indicated time points for western blot of indicated . Pgk1 was used as loading control . Cyc : cycling .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Cells were treated as in (A) and after 2 hours from the release α-factor was re-added to prevent cells from undergoing a second cell cycle. Cell samples were collected at the indicated time points for western blot analysis of the indicated proteins. Pgk1 was used as loading control. Cyc: cycling cells.


C : Top : in vitro kinase assays with recombinant Mps1 Mps1 - 3 purified from E . coli incubated at 34 ° C for indicated times in presence of radioactive ATP a recombinant Spc105 N - terminal fragment ( aa 1 - 320 ) as substrate . Bottom left : kinase activity was quantified on autoradiographs by Image J normalized to of full on Coomassie Blue - stained gel ( right ) . Bottom right : 1μg of recombinant Mps1 kinases used for kinase assays was loaded on SDS page stained with Coomassie Blue for normalisation of kinase assays .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Top: in vitro kinase assays with recombinant Mps1 and Mps1-3 purified from E. coli and incubated at 34°C for the indicated times in the presence of radioactive ATP and a recombinant Spc105 N-terminal fragment (aa 1-320) as substrate. Bottom left: kinase activity was quantified on the autoradiographs by Image J and normalized to the levels of the full length protein on the Coomassie Blue-stained gel (right). Bottom right: 1μg of the recombinant Mps1 kinases used for kinase assays was loaded on SDS page and stained with Coomassie Blue for normalisation of kinase assays.


D : Wild type mps1 - 3 were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in presence of nocodazole . were collected after 90 minutes fixed with formaldehyde for ChIP - seq . ChIP reads were normalised against reads from corresponding input samples , relative enrichment is plotted for chromosome III around centromere ( see centromeric regions of 16 yeast chromosomes in Fig . S2 ) . Y ‐ axis shows enrichment values ( linear scale , range is 0 - 10 ) . Values below 1 . 5 are shown in grey , values above 1 . 5 ( i . e . sequences enriched in ChIP samples ) are red coloured .

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

D: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence of nocodazole. Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S2). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.


E : with indicated genotypes were synchronised in G1 with α - factor at 25 ° C then released in fresh medium at 34 ° C ( t = 0 ) . were collected at indicated time points for FACS of contents .

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

E: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.


A : Serial dilutions of stationary phase cultures of indicated strains were spotted on YEPD incubated at 25 ° C 34 ° C .

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

A: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at 25°C and 34°C.


C : Serial dilutions of stationary phase cultures of indicated strains were spotted on YEPD incubated at indicated temperatures .

C: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.


D : with indicated genotypes were synchronised in G1 with α - factor at 25 ° C then released in fresh medium at 34 ° C in presence of nocodazole ( t = 0 ) . were collected at indicated time points for FACS of contents .

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

D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C in presence of nocodazole (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.


E : were treated as in ( D ) collected at indicated time points for western blot of Mad1 - 3HA . Cyc : cycling .

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

E: Cells were treated as in (D) and collected at the indicated time points for western blot analysis of Mad1-3HA. Cyc: cycling cells.


F : with indicated genotypes were synchronised at 25 ° C then released in fresh medium at 34 ° C ( t = 0 ) . were collected at indicated time points for FACS of contents .

O O O O O O O O O O O O O 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: Cells with the indicated genotypes were synchronised at 25°C and then released in fresh medium at 34°C (t=0). Cells were collected at the indicated time points for FACS analysis of DNA contents.


G - H : with indicated genotypes carrying TetO / TetR - GFP markers for CEN5 labelling expressing mCherry - Tub1 were grown at 25 ° C then shifted to 34 ° C for one hour before filming . were filmed at 34 ° C every 2 or 4 minutes by time lapse microscopy . Chromosome V segregation errors are reported in table ( H ) . Representative are shown as examples in montages ( G ) . Representative montages for wild type mps1 - 3 are shown in Fig . 1F . DIC : differential interference contrast . Scale bar : 5 μm .

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

G-H: Cells with the indicated genotypes carrying the TetO/TetR-GFP markers for CEN5 labelling and expressing mCherry-Tub1 were grown at 25°C and then shifted to 34°C for one hour before filming. Cells were filmed at 34°C every 2 or 4 minutes by time lapse fluorescence microscopy. Chromosome V segregation errors are reported in the table (H). Representative cells are shown as examples in the montages (G). Representative montages for wild type and mps1-3 cells are shown in Fig. 1F. DIC: differential interference contrast. Scale bar: 5 μm.


I : Wild type , mps1 - 3 , mps1 - 3 spc105 - 18 , mps1 - 3 GLC7 - 24 were synchronised in G1 with α - factor at 25 ° C then released at 32 ° C in presence of nocodazole ( note that presence of 3HA tags at C - terminus of Mps1 - 3 slightly decreases maximal of suppression ) . were collected after 90 minutes fixed with formaldehyde for ChIP - seq . ChIP reads were normalised against reads from corresponding input samples , relative enrichment is plotted for chromosome III around centromere ( see centromeric regions of 16 yeast chromosomes in Fig . S5 ) . Y ‐ axis shows enrichment values ( linear scale , range is 0 - 10 ) . Values below 1 . 5 are shown in grey , values above 1 . 5 ( i . e . sequences enriched in ChIP samples ) are red coloured .

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

I: Wild type, mps1-3, mps1-3 spc105-18, and mps1-3 GLC7-24 cells were synchronised in G1 with α-factor at 25°C and then released at 32°C in the presence of nocodazole (note that the presence of 3HA tags at the C-terminus of Mps1-3 slightly decreases the maximal temperature of suppression). Cells were collected after 90 minutes and fixed with formaldehyde for ChIP-seq analysis. ChIP sequence reads were normalised against sequence reads from corresponding input samples, and relative enrichment is plotted for chromosome III around the centromere (see the centromeric regions of all 16 yeast chromosomes in Fig. S5). Y‐axis shows enrichment values (linear scale, range is 0-10). Values below 1.5 are shown in grey, and values above 1.5 (i.e. sequences enriched in ChIP samples) are red coloured.


: Wild type mps1 - 3 were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in presence ( A ) of nocodazole ( t = 0 ) . were collected at indicated time points for western blot of indicated . Equal amounts of extracts were loaded on two different gels , for western blot of Spc105 - 3PK Clb2 / Pgk1 respectively . Clb2 was used as mitotic marker Pgk1 as loading control . Cyc : cycling .

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

: Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (A) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.


Wild type mps1 - 3 were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in absence ( B ) of nocodazole ( t = 0 ) . were collected at indicated time points for western blot of indicated . Equal amounts of extracts were loaded on two different gels , for western blot of Spc105 - 3PK Clb2 / Pgk1 respectively . Clb2 was used as mitotic marker Pgk1 as loading control . Cyc : cycling .

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

Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (B) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins. Equal amounts of protein extracts were loaded on two different gels, for western blot of Spc105-3PK and Clb2/Pgk1 respectively. Clb2 was used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.


with indicated genotypes expressing Bub1 - GFP kinetochore marker Mtw1 - Tomato were grown in SD glu 2 % then shifted to 34 ° C for 1 hour before filming ; they were then filmed every 4 minutes by time lapse microscopy at 34 ° C . Note that BUB1 - GFP is synthetic lethal with mps1 - 3 . Thus , we used mps1 - 3 GALs - MPS1 BUB1 - GFP that were grown in - His RG medium at 30 ° C ; glucose was added to culture for 30 minutes to shut off GALs - MPS1 , followed by shifting to SD medium at 34 ° C for 1 before imaging in same medium . Montages show representative ( C ) . Arrowheads indicate Bub1 - GFP signals at kinetochores . DIC : differential interference contrast . Scale bar : 5 μm .

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Cells with the indicated genotypes expressing Bub1-GFP and the kinetochore marker Mtw1-Tomato were grown in SD glu 2% and then shifted to 34°C for 1 hour before filming; they were then filmed every 4 minutes by time lapse fluorescence microscopy at 34°C. Note that BUB1-GFP is synthetic lethal with mps1-3. Thus, we used mps1-3 GALs-MPS1 BUB1-GFP cells that were grown in -His RG medium at 30°C; glucose was added to the culture for 30 minutes to shut off GALs-MPS1, followed by shifting cells to SD medium at 34°C for 1 before imaging in the same medium. Montages show representative cells (C). Arrowheads indicate Bub1-GFP signals at kinetochores. DIC: differential interference contrast. Scale bar: 5 μm.


Wild type mps1 - 3 were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in presence ( E - F ) of nocodazole ( t = 0 ) . were collected at indicated time points for western blot of indicated for FACS of contents Equal amounts of extracts were loaded on two different gels , for western blot of Bub1 - 3HA / Cdc5 Clb2 / Pgk1 respectively . A asterisk indicates a phosphorylated isoform of Bub1 that in wild type correlates with lack of chromosome biorientation . Cdc5 Clb2 were used as mitotic marker Pgk1 as loading control . Cyc : cycling .

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Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the presence (E-F) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.


Wild type mps1 - 3 were synchronised in G1 with α - factor at 25 ° C then released at 34 ° C in absence ( G - H ) of nocodazole ( t = 0 ) . were collected at indicated time points for western blot of indicated for FACS of contents ( F , H ) . Equal amounts of extracts were loaded on two different gels , for western blot of Bub1 - 3HA / Cdc5 Clb2 / Pgk1 respectively . A asterisk indicates a phosphorylated isoform of Bub1 that in wild type correlates with lack of chromosome biorientation . Cdc5 Clb2 were used as mitotic marker Pgk1 as loading control . Cyc : cycling .

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Wild type and mps1-3 cells were synchronised in G1 with α-factor at 25°C and then released at 34°C in the absence (G-H) of nocodazole (t=0). Cells were collected at the indicated time points for western blot analysis of the indicated proteins and for FACS analysis of DNA contents (F, H). Equal amounts of protein extracts were loaded on two different gels, for western blot of Bub1-3HA/Cdc5 and Clb2/Pgk1 respectively. A white asterisk indicates a phosphorylated isoform of Bub1 that in wild type cells correlates with lack of chromosome biorientation. Cdc5 and Clb2 were used as mitotic marker and Pgk1 as loading control. Cyc: cycling cells.


A : SPC105 - GBD expressing Bub1 - GFP were grown at 30 ° C filmed every 4 minutes at 30 ° C by time lapse microscopy . DIC : differential interference contrast . Scale bar : 5 μm .

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A: SPC105-GBD cells expressing Bub1-GFP were grown at 30°C and filmed every 4 minutes at 30°C by time lapse fluorescence microscopy. DIC: differential interference contrast. Scale bar: 5 μm.


B : Serial dilutions of stationary phase cultures of indicated strains were spotted on YEPD incubated at indicated temperatures .

B: Serial dilutions of stationary phase cultures of the indicated strains were spotted on YEPD and incubated at the indicated temperatures.


C : with indicated genotypes were grown at 25 ° C then shifted at 34 ° C ( t = 0 ) . Every hour after shift same number of was plated on YEPD for each incubated at 25 ° C to determine number of colony - forming units . Percentages of viable have been calculated for each relative to t = 0 . Error bars : SD . N = 3 .

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C: cells with the indicated genotypes were grown at 25°C and then shifted at 34°C (t=0). Every hour after the temperature shift the same number of cells was plated on YEPD for each strain and incubated at 25°C to determine the number of colony-forming units. Percentages of viable cells have been calculated for each strain relative to t=0. Error bars: SD. N=3.


D : with indicated genotypes were synchronised in G1 with α - factor at 25 ° C then released in fresh medium at 34 ° C . were collected at indicated time points for FACS of contents .

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D: Cells with the indicated genotypes were synchronised in G1 with α-factor at 25°C and then released in fresh medium at 34°C. Cells were collected at the indicated time points for FACS analysis of DNA contents.


A . gain of mice with specified genotypes under normal ( ND ) or high - fat diet ( HFD ) .

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A. Body weight gain of male mice with the specified genotypes under normal (ND) or high-fat diet (HFD).


E . Representative pictures of H & E of SubWAT EpiWAT of indicated mice fed HFD .

E. Representative pictures of H&E staining of SubWAT and EpiWAT of indicated male mice fed HFD.


lipids content ( F ) per gram of feces .

lipids content (F) per gram of feces.


D . Western blot of phosphorylated PKD2 ( S876 ) phosphorylated PKD ( S916 / S876 ) in small intestine of Pkd2wt / wt Pkd2ki / ki mice . n = 3 .

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D. Western blot analysis and quantification of phosphorylated PKD2 (S876) and phosphorylated PKD (S916/S876) in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice. n=3.


A . Western blot of phosphorylated PKD ( S916 / S876 ) in small intestine of Pkd2flox / flox Pkd2gutΔ / Δ mice . n = 3 . B . Western blot of phosphorylated PKD ( S744 - 748 ) in small intestine of Pkd2flox / flox Pkd2gutΔ / Δ mice . n = 3 . C

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A. Western blot and quantification of phosphorylated PKD (S916/S876) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. B. Western blot and quantification of phosphorylated PKD (S744-748) in small intestine of Pkd2flox/flox and Pkd2gutΔ/Δ male mice. n=3. C


G . Free - fatty H . Triglycerides content in serum of mice after 17 in HFD . n = 10 .

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G. Free-fatty acids and H. Triglycerides content in serum of male mice after 17 weeks in HFD. n=10.


A . Western blot ( WB ) of specified in small intestine of Pkd2wt / wt Pkd2ki / ki mice after one week of HFD . of bands for each normalized to loading control relative to Pkd2wt / wt . n = 3 .

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A. Western blot (WB) analysis of specified proteins in small intestine of Pkd2wt/wt and Pkd2ki/ki male mice after one week of HFD. Quantification of the bands for each protein normalized to loading control and relative to Pkd2wt/wt. n=3.


H . WB with specified antibodies after an in - vitro kinase .

H. WB with specified antibodies after an in-vitro kinase assay.


C . Western blot of phosphorylated PKD2 in jejunum of C57BL / 6 mice in HFD treated with CRT0066101 or control ( water ) for 13 . n = 3 .

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C. Western blot analysis of phosphorylated PKD2 in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=3.


F . WB of Apolipoprotein A4 ( APOA4 ) in jejunum of C57BL / 6 mice in HFD treated with CRT0066101 or control ( water ) for 13 . of bands normalized to GAPDH relative to control ( water ) n = 6 .

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F. WB of Apolipoprotein A4 (APOA4) in jejunum of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. Quantification of bands normalized to GAPDH and relative to control (water) n=6.


G . of C57BL / 6 mice in HFD treated with CRT0066101 or control ( water ) for 13 . n = 6 for control n = 5 for CRT0066101 .

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G. Body weight of male C57BL/6 mice in HFD and treated with CRT0066101 or control (water) for 13 weeks. n=6 for control and n=5 for CRT0066101.


A . gain of C57BL / 6 mice that , after 7 in HFD , started receiving treatment with CRT0066101 or water . HFD continued along with treatment . n = 8 .

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A. Body weight gain of male C57BL/6 mice that, after 7 weeks in HFD, started receiving treatment with CRT0066101 or water. HFD continued along with treatment. n=8.


F . Western blot of PKD phosphorylation in proximal jejunum of morbidly obese . Samples are loaded according to fasting triglyceride ( lower to higher ) . n = 7 .

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F. Western blot analysis of PKD phosphorylation in proximal jejunum of morbidly obese female patients. Samples are loaded according to fasting triglyceride levels (lower to higher). n=7.


A ) Heat map showing changes in expression for various enzymes regulators of central carbon metabolism in endogenous , CAF1 - knockdown - induced ( through p60 or p150 KD ) 2 - - like . Fold - changes relative to ESCs were calculated on bulk - seq data

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A) Heat map showing changes in RNA expression levels for various enzymes and regulators of central carbon metabolism in endogenous, CAF1-knockdown-induced (through p60 or p150 KD) 2-cell-like cells. Fold-changes relative to ESCs were calculated based on bulk RNA-seq data


A ) Heat map showing changes in expression for various enzymes regulators of central carbon metabolism in endogenous , CAF1 - knockdown - induced ( through p60 or p150 KD ) 2 - - like . Fold - changes relative to ESCs were calculated on bulk - seq

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C ) Oxygen consumption rate of ES ( blue ) 2 - - like ( green ) . medium was formulated to recapitulate standard ES culture conditions contained glucose , L - glutamine pyruvate . Basal , maximal ( FCCP - induced ) non - mitochondrial ( Rotenone Antimycin - A mediated ) respiratory rates are indicated . A representative graph of 3 independent biological replicates performed on Seahorse extracellular flux analyser is shown . Due to low number of 2 - - like available , compared to ESCs , one technical replicate of former was analysed per biological replicate , while three or more technical replicates were performed for latter . Accordingly , mean ± S . D . of technical replicates is shown for ESCs .

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C) Oxygen consumption rate of ES (blue line) and 2-cell-like cells (green line). Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Basal, maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates are indicated. A representative graph of 3 independent biological replicates performed on the Seahorse extracellular flux analyser is shown. Due to the low number of 2-cell-like cells available, compared to ESCs, one technical replicate of the former was analysed per biological replicate, while three or more technical replicates were performed for the latter. Accordingly, mean ± S.D. of technical replicates is shown for ESCs.


D ) Basal oxygen consumption rate of ES ( blue ) , Zscan4 + ( red ) 2 - - like ( green ) across 3 independent biological replicates performed on Seahorse extracellular flux analyser . medium was formulated to recapitulate standard ES culture conditions contained glucose , L - glutamine pyruvate . Boxes indicate range between first third quantile , band specifies median whiskers extend no further than 1 . 5 - times interquartile range . dots indicate measurements obtained in each of technical replicates .

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D) Basal oxygen consumption rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser. Assay medium was formulated to recapitulate standard ES cell culture conditions and contained glucose, L-glutamine and pyruvate. Boxes indicate the range between the first and third quantile, the band specifies the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each of the individual technical replicates.


E ) Oxygen consumption rate of ES ( blue ) , Zscan4 + ( red ) 2 - - like ( green ) in glucose - free media upon acute injection of sodium pyruvate or sodium L - lactate . Note that L - glutamine - but not glucose or pyruvate - was initially present in medium . Maximal ( FCCP - induced ) non - mitochondrial ( Rotenone Antimycin - A mediated ) respiratory rates following pyruvate or lactate treatment are also are indicated . A graph including data from 3 independent biological replicates is presented , mean ± S . D . of technical replicates is shown .

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E) Oxygen consumption rate of ES (blue line), Zscan4+ (red line) and 2-cell-like cells (green line) in glucose-free media and upon acute injection of sodium pyruvate or sodium L-lactate. Note that L-glutamine - but not glucose or pyruvate - was initially present in the assay medium. Maximal (FCCP-induced) and non-mitochondrial (Rotenone and Antimycin-A mediated) respiratory rates following pyruvate or lactate treatment are also are indicated. A graph including data from 3 independent biological replicates is presented, and the mean ± S.D. of technical replicates is shown.


F ) Representative electron microscopy of mitochondria from ES ( n = 49 sections ) 2 - - like ( n = 57 sections ) generated across 2 independent technical biological replicates . Scale bar , 1 μm . Representative from 2 independent biological technical replicates are shown . 162 99 mitochondria were analysed for 2 - - like ES , respectively .

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F) Representative electron microscopy micrographs of mitochondria from ES (n=49 sections) and 2-cell-like cells (n=57 sections) generated across 2 independent technical and biological replicates. Scale bar, 1 μm. Representative images from 2 independent biological and technical replicates are shown. 162 and 99 mitochondria were analysed for 2-cell-like and ES cells, respectively.


G ) Representative single section of CellROX - DeepRed in ES 2 - - like ( green arrow ) obtained using live microscopy . Scale bar , 10 μm . n , indicates number of independent replicates . Representative from 3 independent biological replicates are shown .

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G) Representative single section of CellROX-DeepRed fluorescence in ES and 2-cell-like cells (green arrow) obtained using live cell microscopy. Scale bar, 10 μm. n, indicates the number of independent replicates. Representative images from 3 independent biological replicates are shown.


H ) FACS - assisted of CellROX - DeepRed in ES 2 - - like . Measurements were obtained from 2 independent biological replicates . * * * P < 0 . 005 ; Mann - Whitney U .

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H) FACS-assisted quantification of CellROX-DeepRed fluorescence intensity in ES and 2-cell-like cells. Measurements were obtained from 2 independent biological replicates. *** P < 0.005; Mann-Whitney U test.


A ) ATP content in ES ( blue ) , Zscan4 + ( red ) 2 - - like ( green ) across 4 independent biological replicates .

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A) ATP content in ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 4 independent biological replicates.


B ) Extracellular acidification rate of ES ( blue ) , Zscan4 + ( red ) 2 - - like ( green ) across 3 independent biological replicates performed on Seahorse extracellular flux analyser .

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B) Extracellular acidification rate of ES (blue), Zscan4+ (red) and 2-cell-like cells (green) across 3 independent biological replicates performed on the Seahorse extracellular flux analyser.


C ) Glucose uptake rates in Zscan4 + ( red ) 2 - - like ( green ) were measured using a luciferase - across 4 independent biological replicates are represented relative to those of control ES ( blue ) .

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C) Glucose uptake rates in Zscan4+ (red) and 2-cell-like cells (green) were measured using a luciferase-based assay across 4 independent biological replicates and are represented relative to those of control ES cells (blue).


F ) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES , Zscan4 + 2 - - like . Measurements were quantified relative to glucose uptake rate of ESCs transfected with a negative control siRNA . Shown are mean ± s . d . of indicated number of independent cultures , performed across 2 or more independent biological replicates each .

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F) Glucose uptake rates upon knockdown of Gnpnat1 or a G6pdx were measured in ES, Zscan4+ and 2-cell-like cells. Measurements were quantified relative to the glucose uptake rate of ESCs transfected with a negative control siRNA. Shown are the mean ± s.d. of the indicated number of independent cell cultures, performed across 2 or more independent biological replicates each.


G ) Glucose - 6 - phosphate dehydrogenase activity was measured in ES ( blue ) , Zscan4 + ( red ) 2 - - like ( green ) lysates using a fluorometric . Measurements were obtained from 3 independent biological replicates , performed in 3 technical replicates each . n . s . - not significant ; one - way ANOVA . Data information : In panels A - C G , boxes indicate range between first third quantile , band depicts median whiskers extend no further than 1 . 5 - times interquartile range . dots indicate measurements obtained in each technical replicate .

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G) Glucose-6-phosphate dehydrogenase activity was measured in ES (blue), Zscan4+ (red) and 2-cell-like cell (green) lysates using a fluorometric assay. Measurements were obtained from 3 independent biological replicates, performed in 3 technical replicates each. n.s. - not significant; one-way ANOVA. Data information: In panels A-C and G, boxes indicate the range between the first and third quantile, the band depicts the median and the whiskers extend no further than 1.5-times the interquartile range. Individual dots indicate the measurements obtained in each individual technical replicate.


B ) showing effect of 20 metabolites tested on 2 - - like .

B) Heatmap showing the effect of the 20 metabolites tested on 2-cell-like cell levels.