( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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

(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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - / - 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 : All data are presented as mean ± SEM , 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 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 ) staining ( left ) and oil red O staining ( 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 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 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 ) .

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

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

O O O O O 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

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.


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 .

O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) 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 ) .

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

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 Protein expression of SQLE in human liver tumors ( C ) and 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 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 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 ] ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 + / + and p53 - / - HepG2 cells transfected with and without exogenous SQLE ( HA - SQLE ) in LPDS medium . Protein 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 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 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 .

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-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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) .

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

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 ) and Oil Red O ( right ) staining of livers of mice .

(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ) and Oil Red O ( right ) staining 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 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 the indicated strains were spotted on YEPD and incubated at the indicated temperatures .

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 the indicated temperatures.


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 )

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 : 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 : 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 .

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

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 the indicated strains were spotted on YEPD and incubated at 25 ° C and 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 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 the indicated strains were spotted on YEPD and incubated at the indicated temperatures .

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


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 .

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

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

O O O O O O O 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 : 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 .

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

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 : 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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.


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 .

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

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 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 .

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

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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O 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: 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 the indicated strains were spotted on YEPD and incubated at the indicated temperatures .

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

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: 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 : 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 .

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

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

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


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

O O O O O O O O O O O O O O O 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. 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 acids and H . Triglycerides content in serum of male mice after 17 weeks in HFD . n = 10 .

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

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


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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. 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 assay .

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


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 .

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

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 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 .

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

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 . 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 .

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 B-CONTROLLED_VAR O

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 . 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 .

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

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 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 .

O O O O O O O O O O O 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. 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 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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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

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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 .

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) and 2 - cell - like cells ( 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 ) and 2 - cell - like cells ( green ) across 3 independent biological replicates performed on the 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 ) 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 ) .

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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 + 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 .

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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 ) 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 .

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

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


Percentage of 2 - cell - like cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . 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 .

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Percentage of 2-cell-like cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. 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.


Percentage of Zscan4 + cells in cultures treated with increasing concentrations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across 3 biological replicates . 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 .

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Percentage of Zscan4+ cells in cultures treated with increasing concentrations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across 3 biological replicates. 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.


Percentage of Zscan4 + cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . 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 .

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Percentage of Zscan4+ cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. 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.


Percentage of 2 - cell - like cells in cultures treated with varying combinations of sodium acetate , sodium L - lactate or D - ribose . Shown are the mean ± s . d . of 2 independent cell cultures , performed across biological replicates . 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 .

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Percentage of 2-cell-like cells in cultures treated with varying combinations of sodium acetate, sodium L-lactate or D-ribose. Shown are the mean ± s.d. of 2 independent cell cultures, performed across biological replicates. 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.


A ) Immunofluorescence staining for OCT4 , ZSCAN4 and 2C : : tbGFP in control and acetate treated ESC cultures . Green arrows indicate 2 - cell - like cells , and inlets highlight their DAPI structure . Scale bar , 20 μm .

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A) Immunofluorescence staining for OCT4, ZSCAN4 and 2C::tbGFP in control and acetate treated ESC cultures. Green arrows indicate 2-cell-like cells, and inlets highlight their DAPI structure. Scale bar, 20 μm.


B ) RT - qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h . Shown are the mean ± s . d . of 3 independent cell cultures , performed in 2 technical replicates .

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B) RT-qPCR of the indicated genes in ESC cultures treated with sodium acetate for 24h. Shown are the mean ± s.d. of 3 independent cell cultures, performed in 2 technical replicates.


C ) Percentage of 2 - cell - like cells obtained upon transfection of control or Dux - targeting siRNAs in control conditions or in combination with sodium acetate treatment . Measurements were obtained from 2 independent cell cultures , performed across 3 independent biological replicates . 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 technical replicate .

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C) Percentage of 2-cell-like cells obtained upon transfection of control or Dux-targeting siRNAs in control conditions or in combination with sodium acetate treatment. Measurements were obtained from 2 independent cell cultures, performed across 3 independent biological replicates. 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 technical replicate.


D ) Percentage of 2 - cell - like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment . Shown are the mean ± s . d . of 4 independent cell cultures , performed across 2 independent biological replicates .

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D) Percentage of 2-cell-like cells obtained upon transfection siRNAs targeting the indicated chromatin factors in control conditions or in combination with sodium acetate treatment. Shown are the mean ± s.d. of 4 independent cell cultures, performed across 2 independent biological replicates.


F ) Proportion of Zscan4 + cells at various timepoints during the time - lapse experiment . Shown are the mean ± s . d . of 3 independent experiments .

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F) Proportion of Zscan4+ cells at various timepoints during the time-lapse experiment. Shown are the mean ± s.d. of 3 independent experiments.


Glycolytic flux of wildtype ( WT ) and TM6 strains strongly correlates with fructose - 1 , 6 - bisphosphate ( FBP ) concentration . The glycolytic flux is reported here as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were obtained on the basis of physiological and metabolome data , and via a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . While on high glucose , the WT strain accomplishes a high glucose uptake rate ( and thus glycolytic flux ) , the mutant strain ( TM6 ) only generates a low glucose uptake ( and thus glycolytic flux ) . On maltose , also the mutant strain achieves a high glycolytic flux , since maltose is transported by a separate transporter ( Chang et al , 1989 ) . To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the model - derived glycolytic flux ( A ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics .

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Glycolytic flux of wildtype (WT) and TM6 strains strongly correlates with fructose-1,6-bisphosphate (FBP) concentration. The glycolytic flux is reported here as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were obtained on the basis of physiological and metabolome data, and via a novel method to estimate intracellular fluxes (Niebel et al, 2019). While on high glucose, the WT strain accomplishes a high glucose uptake rate (and thus glycolytic flux), the mutant strain (TM6) only generates a low glucose uptake (and thus glycolytic flux). On maltose, also the mutant strain achieves a high glycolytic flux, since maltose is transported by a separate transporter (Chang et al, 1989). To assess the linear correlation between the FBP concentration and the glycolytic flux across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the model-derived glycolytic flux (A) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


FBP concentration as a function of cellular growth rate shows weaker correlation . For metabolite levels and growth rates , error bars correspond to the standard deviation between three independent experiments ; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space ( cf . Methods ) . The carbon sources were used at a final concentration of 10 gL - 1 and are indicated : Glucose ( GLU ) ; Galactose ( GAL ) ; Maltose ( MAL ) and Pyruvate ( PYR ) . To assess the linear correlation between the FBP concentration and the growth rate ( B ) across the studied conditions , we implemented Pearson ' s correlation analysis assisted by bootstrapping . Specifically , we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions ( combinations of strains and carbon sources ) , biological and technical replicates . We paired each of these FBP measurements with the mean and standard deviation of the growth rate ( B ) in the corresponding metabolic condition . We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition , and implemented ordinary nonparametric bootstrapping with 100 , 000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth - rate distributions to calculate the correlation statistics .

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FBP concentration as a function of cellular growth rate shows weaker correlation. For metabolite levels and growth rates, error bars correspond to the standard deviation between three independent experiments; for glycolytic fluxes to the mean and standard deviations of the sampled flux solution space (cf. Methods). The carbon sources were used at a final concentration of 10 gL-1 and are indicated: Glucose (GLU); Galactose (GAL); Maltose (MAL) and Pyruvate (PYR). To assess the linear correlation between the FBP concentration and the growth rate (B) across the studied conditions, we implemented Pearson's correlation analysis assisted by bootstrapping. Specifically, we used in total 53 FBP concentration measurements corresponding to six different metabolic conditions (combinations of strains and carbon sources), biological and technical replicates. We paired each of these FBP measurements with the mean and standard deviation of the growth rate (B) in the corresponding metabolic condition. We assumed the normal distribution of the flux and growth rate with the given mean and standard deviation in every condition, and implemented ordinary nonparametric bootstrapping with 100,000 iterations by randomly sampling values with replacement from the 53 FBP measurements and flux or growth-rate distributions to calculate the correlation statistics.


CggR intracellular abundance in the wildtype ( WT ) strain on glucose strongly varies with the promoter used . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL - 1 . Error bars represent the standard deviation of at least three replicate experiments .

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CggR intracellular abundance in the wildtype (WT) strain on glucose strongly varies with the promoter used. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose as carbon source at a final concentration of 10 gL-1. Error bars represent the standard deviation of at least three replicate experiments.


The relative abundance of CggR ( normalized to the abundance measured on glucose and the same promoter ) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells , but not with PTEFmut2 and PCMV . The CggR intracellular levels were quantified by proteomics in steady - state cultures grown in minimal media with glucose , galactose , maltose or pyruvate as carbon sources at a final concentration of 10 gL - 1 . WT data includes all three promoters , whereas TM6 only includes the PTEFmut2 and PTEFmut7 data . Error bars represent the standard deviation of at least three replicate experiments .

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The relative abundance of CggR (normalized to the abundance measured on glucose and the same promoter) is almost constant with PTEFmut7 across multiple growth rates in WT and TM6 cells, but not with PTEFmut2 and PCMV. The CggR intracellular levels were quantified by proteomics in steady-state cultures grown in minimal media with glucose, galactose, maltose or pyruvate as carbon sources at a final concentration of 10 gL-1. WT data includes all three promoters, whereas TM6 only includes the PTEFmut2 and PTEFmut7 data. Error bars represent the standard deviation of at least three replicate experiments.


Thermal shift assays were used to determine the melting curves of wild - type CggR and mutants . Error bars correspond to the standard deviation of at least five replicates .

Thermal shift assays were used to determine the melting curves of wild-type CggR and mutants. Error bars correspond to the standard deviation of at least five replicates.


CggR - FBP affinity constants ( KD ' s ) of the wildtype and mutant variants , determined by fitting a simple cooperative binding model to the melting curves data . Error bars indicate the 95 % confidence intervals .

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CggR-FBP affinity constants (KD's) of the wildtype and mutant variants, determined by fitting a simple cooperative binding model to the melting curves data. Error bars indicate the 95% confidence intervals.


Quantification of CggR binding to DNA . The CggR bound to DNA fraction was calculated by dividing the intensity of the protein - DNA complex band by the total DNA . The background - subtracted total intensities of the CggR - DNA complex and the free - DNA bands were assessed with ImageJ . Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype .

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Quantification of CggR binding to DNA. The CggR bound to DNA fraction was calculated by dividing the intensity of the protein-DNA complex band by the total DNA. The background-subtracted total intensities of the CggR-DNA complex and the free-DNA bands were assessed with ImageJ. Error bars correspond to the standard deviation of three replicate experiments for the mutants and six for the wildtype.


The four reporter plasmids were transferred to the wildtype strain containing the CggR ( R250A ) under the control of the PTEFmut7 . The strength of the four promoters was assessed by quantifying YFP ( FL1 ) and mCherry ( FL3 ) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l - 1 glucose . The FL1 and FL3 fluorescence shown is the non - background corrected median of 100 000 cell events . The non - FL control is the signal from a wildtype strain grown under the same conditions . The background fluorescence , assessed by the wildtype harboring the YCplac33 plasmid , was substracted from FL1 and FL3 . The final reporter activity is the ratio of the background - corrected YFP and mCherry values . Error bars represent the standard deviation of three independently determined ratios from three replicate experiments .

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The four reporter plasmids were transferred to the wildtype strain containing the CggR (R250A) under the control of the PTEFmut7. The strength of the four promoters was assessed by quantifying YFP (FL1) and mCherry (FL3) fluorescence in exponentially growing wildtype cells in minimal medium with 10 g l-1 glucose. The FL1 and FL3 fluorescence shown is the non-background corrected median of 100 000 cell events. The non-FL control is the signal from a wildtype strain grown under the same conditions. The background fluorescence, assessed by the wildtype harboring the YCplac33 plasmid, was substracted from FL1 and FL3. The final reporter activity is the ratio of the background-corrected YFP and mCherry values. Error bars represent the standard deviation of three independently determined ratios from three replicate experiments.


Reporter activity of the sensor across ( D ) multiple FBP levels Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments .

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Reporter activity of the sensor across (D) multiple FBP levels Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Reporter activity of the sensor across glycolytic fluxes . The glycolytic flux is reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and fructose - 1 , 6 - bisphosphate ( FBP ) . Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) . Reporter activity is given by the YFP / mCherry ratio , calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry . Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid ( Appendix Table S8 ) . The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR . Error bars represent the standard deviation of at least three replicate experiments .

Reporter activity of the sensor across glycolytic fluxes. The glycolytic flux is reported as the flux between the metabolites fructose 6-phosphate (F6P) and fructose-1,6-bisphosphate (FBP). Glycolytic fluxes were here estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019). Reporter activity is given by the YFP/mCherry ratio, calculated through the quantification of YFP and mCherry fluorescence along culture time using flow cytometry. Both YFP and mCherry fluorescence levels were first corrected for background using the same strains harboring the YCplac33 plasmid (Appendix Table S8). The control is the wildtype and TM6 strains expressing only the reporter plasmid without CggR. Error bars represent the standard deviation of at least three replicate experiments.


Fraction of CggR bound to FBP across FBP concentrations . The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant . The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound / bound state using the Tm at 0 mM FBP as unbound and at 36 mM ( corresponding to maximum FBP concentration used ) as total bound states . The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one - site specific binding model in GraphPad . The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant . Vertical lines delimit the physiological FBP range .

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Fraction of CggR bound to FBP across FBP concentrations. The red arrows indicate the shift in the percentage of CggR bound to FBP achieved in the R250A variant. The percentage of CggR molecules bound to FBP was calculated after normalizing the Tm values for unbound/bound state using the Tm at 0 mM FBP as unbound and at 36 mM (corresponding to maximum FBP concentration used) as total bound states. The curve fitting of the normalized values of CggR fraction bound to FBP was performed using a one-site specific binding model in GraphPad. The solid line corresponds to the wildtype CggR and the dashed line to the R250A variant. Vertical lines delimit the physiological FBP range.


Sub - populations of WT and TM6 cells , grown separately and mixed in different fractions as indicated in percentages , can easily be distinguished by flow cytometry , FL1 YFP channel , FL3 mCherry channel . Histogram of single - cell ratios of FL1 / FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry . Here , a sub - population of minimally 5 % can be distinguished .

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Sub-populations of WT and TM6 cells, grown separately and mixed in different fractions as indicated in percentages, can easily be distinguished by flow cytometry, FL1 YFP channel, FL3 mCherry channel. Histogram of single-cell ratios of FL1/FL3 fluorescence intensities of mixed WT and TM6 populations analyzed by flow cytometry. Here, a sub-population of minimally 5% can be distinguished.


Tukey boxplots showing the YFP / mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux . At least 35 cells were analyzed in each condition . The glycolytic flux is here reported as the flux between the metabolites fructose 6 - phosphate ( F6P ) and FBP . Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes ( Niebel et al , 2019 ) .

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Tukey boxplots showing the YFP/mCherry ratio of individual cells measured by microscopy as a function of glycolytic flux. At least 35 cells were analyzed in each condition. The glycolytic flux is here reported as the flux between the metabolites fructose 6-phosphate (F6P) and FBP. Glycolytic fluxes were estimated on the basis of physiological and metabolome data and a novel method to estimate intracellular fluxes (Niebel et al, 2019).


YFP / mCherry ratio measured by microscopy in co - existing dividing ( high - flux ) versus non - dividing ( low - flux ) isogenic TM6 cells on 10 gL - 1 glucose .

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YFP/mCherry ratio measured by microscopy in co-existing dividing (high-flux) versus non-dividing (low-flux) isogenic TM6 cells on 10 gL-1 glucose.


Brightfield ( BF ) , YFP and mCherry microscopy images for a co - existing dividing ( high - flux ) and a non - dividing ( low - flux ) TM6 cell expressing the flux sensor in 10g L - 1 glucose minimal medium .

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Brightfield (BF), YFP and mCherry microscopy images for a co-existing dividing (high-flux) and a non-dividing (low-flux) TM6 cell expressing the flux sensor in 10g L-1 glucose minimal medium.


The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor - expressing strain ( F ) , which reflects the cell - cycle dynamics of intracellular FBP concentration and glycolytic flux . In a control strain , lacking CggR , the production rates of YFP and mCherry are coupled ( G ) . The uncoupling was calculated for individual cell - cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale ( see more details in Materials and methods ; Appendix Figure S10 ) . Each curve represents the mean across the indicated number of cell cycles in a replicate experiment . The corresponding shaded areas denote the 95 % confidence intervals of the means ( bootstrapping with 5 000 iterations ) . We smoothed the single - cell - cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression , and used these trajectories to derive the YFP and mCherry production rates , accounting for fluorescent - protein maturation in a first - order kinetics model . To align the cell - cycle trajectories and to calculate the phase , we used the array of three cell cycle events E { cytokinesis ( cyt ) , budding , next cyt } as reference points . The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes , with the mean duration presented in parentheses for each replicate experiment . The cells belonged to the TM6 strain and were cultivated on 20 gL - 1 glucose in the microfluidic device .

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The production rates of YFP and mCherry are uncoupled during the cell cycle in the biosensor-expressing strain (F), which reflects the cell-cycle dynamics of intracellular FBP concentration and glycolytic flux. In a control strain, lacking CggR, the production rates of YFP and mCherry are coupled (G). The uncoupling was calculated for individual cell-cycle trajectories as the difference between the YFP and mCherry production rates normalised to have the same scale (see more details in Materials and methods; Appendix Figure S10). Each curve represents the mean across the indicated number of cell cycles in a replicate experiment. The corresponding shaded areas denote the 95% confidence intervals of the means (bootstrapping with 5 000 iterations). We smoothed the single-cell-cycle trajectories of YFP and mCherry signals as well as cell volume via the Gaussian process regression, and used these trajectories to derive the YFP and mCherry production rates, accounting for fluorescent-protein maturation in a first-order kinetics model. To align the cell-cycle trajectories and to calculate the phase, we used the array of three cell cycle events E {cytokinesis (cyt), budding, next cyt} as reference points. The cell cycles used for the analysis had the duration in the interval between 150 and 300 minutes, with the mean duration presented in parentheses for each replicate experiment. The cells belonged to the TM6 strain and were cultivated on 20 gL-1 glucose in the microfluidic device.


( A - F ) Representative PAS - stained sections of the pons ( A ) and hippocampal ( C ) regions of the brain and heart ( E ) of one year old HOIL - 1 [ C458S ] and WT mice are shown . Scale bar = 100 μm . Arrow heads indicate α - amylase - resistant PAS - positive polyglucosan deposits . Graphs showing α - amylase resistant PAS scores of the pons ( B ) and hippocampal ( D ) regions of the brain and the heart ( F ) of HOIL - 1 [ C458S ] ( red ) and WT ( blue ) mice aged 0 . 5 , 1 . 0 and 1 . 5 years . The number of biological replicates analysed at each age is indicated . The word zero highlighted in blue indicates that no α - amylase resistant , PAS - positive material could be detected in the WT mice . The error bars show mean + SEM . Statistical significance between the genotypes was calculated by using two - way ANOVA and Šidák ' s multiple comparison ' s test . * * * * denotes p < 0 . 0001 .

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(A-F) Representative PAS-stained sections of the pons (A) and hippocampal (C) regions of the brain and heart (E) of one year old HOIL-1[C458S] and WT mice are shown. Scale bar = 100 μm. Arrow heads indicate α-amylase-resistant PAS-positive polyglucosan deposits. Graphs showing α-amylase resistant PAS scores of the pons (B) and hippocampal (D) regions of the brain and the heart (F) of HOIL-1[C458S] (red) and WT (blue) mice aged 0.5, 1.0 and 1.5 years. The number of biological replicates analysed at each age is indicated. The word zero highlighted in blue indicates that no α-amylase resistant, PAS-positive material could be detected in the WT mice. The error bars show mean + SEM. Statistical significance between the genotypes was calculated by using two-way ANOVA and Šidák's multiple comparison's test. **** denotes p<0.0001.


( A ) HOIL - 1 activity towards purified bovine liver glycogen was measured using Cy5 - labelled ubiquitin . Control assays lacking ATP , UBE2L3 , HOIL - 1 and glycogen are indicated , as are subsequent treatments with 50 μg / mL human salivary α ‑ amylase and 1 . 5 M hydroxylamine ( NH2OH ) . The gel has been stained using the periodic acid - Schiff ( PAS ) method to visualise glycogen ( purple - magenta ) and Coomassie Blue stain to visualise protein .

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(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.


( B ) Prior to PAS and Coomassie staining , the gel shown in ( A ) was stained with Flamingo fluorescent protein stain ( shown in red ) and glycogen ubiquitylation observed by visualising the fluorescent Cy5 - ubiquitin signal ( shown in green ) .

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(B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green).