( C ) . Time course analysis of Xist RNA expression by RT - qPCR during EB differentiation of Rif1 + / + ( Rif1 + / + + OHT ) and Rif1 - / - ( Rif1F / F + OHT ) cells at the indicated timepoints . Rif1 + / + ( solid line ) and Rif1 - / - ( dashed line ) , female ( black ) and male ( grey ) . Data are presented as mean ± standard deviation from three ( female lines ) or two ( male lines ) independent experiments . Statistical significance was determined using 2 - way ANOVA comparing female Rif1 + / + to female Rif1 - / - cell lines ( * * * * p ≤ 0 . 0001 ) . Xist RT - primers Xist ex7 F and R were used . Values are normalised to a geometric mean consisting of the expression of Gapdh , Ubiquitin and β - Actin .

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

(C). Time course analysis of Xist RNA expression by RT-qPCR during EB differentiation of Rif1+/+ (Rif1+/+ +OHT) and Rif1-/- (Rif1F/F +OHT) cells at the indicated timepoints. Rif1+/+ (solid line) and Rif1-/- (dashed line), female (black) and male (grey). Data are presented as mean ± standard deviation from three (female lines) or two (male lines) independent experiments. Statistical significance was determined using 2-way ANOVA comparing female Rif1+/+ to female Rif1-/- cell lines (****p ≤ 0.0001). Xist RT-primers Xist ex7 F and R were used. Values are normalised to a geometric mean consisting of the expression of Gapdh, Ubiquitin and β-Actin.


RIF1 association with the Xist promoter assessed by ChIP - qPCR in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female cell lines , in ESCs ( A ) P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 and 2 are two intergenic regions that serve as negative controls . Peak and cRAD represent two previously identified regions of RIF1 association ( positive control ) . See Appendix Fig . S2C for primer positions within Xist . Mean ± standard deviation from 3 independent experiments ( A ) and 2 independent experiments ( B ) . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association in Rif1 + / + cells on Xist P2 and P1 versus 5 ' . * p ≤ 0 . 05 , * * * * p ≤ 0 . 0001 and ns = not significant .

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

RIF1 association with the Xist promoter assessed by ChIP-qPCR in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female cell lines, in ESCs (A) P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 and 2 are two intergenic regions that serve as negative controls. Peak and cRAD represent two previously identified regions of RIF1 association (positive control). See Appendix Fig. S2C for primer positions within Xist. Mean ±standard deviation from 3 independent experiments (A) and 2 independent experiments (B). p calculated by Student's two-tailed, paired t test comparing RIF1 association in Rif1+/+ cells on Xist P2 and P1 versus 5'. *p ≤ 0.05, ****p ≤ 0.0001 and ns=not significant.


( D ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) and a wild type female mESC line ( grey ) , also harbouring one castaneus and one 129 X chromosome . Allele - specific ChIP - qPCR primers were used , cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2 . Enrichments are presented relative to input DNA . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 , * * * p ≤ 0 . 001 . Below , schematic of the Xist / Tsix alleles in the Fa2L undifferentiated cells . ( E ) . Association of RIF1 with Xist P2 in the Fa2L cells ( black ) upon differentiation . The analysis was performed as in ( D ) . Mean ± standard deviation from 3 independent experiments . p calculated by Student ' s two - tailed , paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2 . * p ≤ 0 . 05 . Below , schematic of the Xist / Tsix alleles in the Fa2L differentiated cells . (

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

(D). Association of RIF1 with Xist P2 in the Fa2L cells (black) and a wild type female mESC line (grey), also harbouring one castaneus and one 129 X chromosome. Allele-specific ChIP-qPCR primers were used, cast indicates association with the castaneus Xist P2 and 129 indicates association with the 129 Xist P2. Enrichments are presented relative to input DNA. Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2, ***p ≤ 0.001. Below, schematic of the Xist/Tsix alleles in the Fa2L undifferentiated cells. (E). Association of RIF1 with Xist P2 in the Fa2L cells (black) upon differentiation. The analysis was performed as in (D). Mean ±standard deviation from 3 independent experiments. p calculated by Student's two-tailed, paired t test comparing RIF1 association with the castaneus and with the 129 X chromosome Xist P2. *p ≤ 0.05. Below, schematic of the Xist/Tsix alleles in the Fa2L differentiated cells. (


( F ) . Quantification by ChIP - qPCR of RIF1 association with the indicated regions in the Fa2L cell line , following treatment with DMSO only ( black ) or flavopiridol ( grey ) . Primers as in ( E ) . ( G ) . Same as in ( F ) but for a wild type female mESC line . All enrichments are presented relative to input DNA . Mean ± standard deviation from three ( F ) and two ( G ) independent experiments are presented . Statistical significance was determined using Student ' s two - tailed , paired t test ( * p ≤ 0 . 05 , * * p ≤ 0 . 01 and ns = not significant ) .

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

(F). Quantification by ChIP-qPCR of RIF1 association with the indicated regions in the Fa2L cell line, following treatment with DMSO only (black) or flavopiridol (grey). Primers as in (E). (G). Same as in (F) but for a wild type female mESC line. All enrichments are presented relative to input DNA. Mean ±standard deviation from three (F) and two (G) independent experiments are presented. Statistical significance was determined using Student's two-tailed, paired t test (*p ≤ 0.05, **p ≤ 0.01 and ns=not significant).


( A ) . Time course analysis of Xist expression by RT - qPCR during EB differentiation of female mESCs following knock down of Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Data are presented as mean ± standard deviation from three independent experiments . Statistical significance was determined using 2 - way ANOVA . Xist primers Xist ex3 F and Xist ex4 R were used . Normalisation was performed using a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . ( * p ≤ 0 . 05 ) . ( B ) . RT - qPCR analysis of Xist expression levels during differentiation of the Fa2L cells , following expression of shRNA against Luciferase ( Control , black ) and Kap1 ( Kap1 KD , grey ) , at the indicated timepoints . Mean ± standard deviation from a minimum of three independent experiments is presented . 2 - way ANOVA was used to determine statistical significance . ns = not significant . (

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

(A). Time course analysis of Xist expression by RT-qPCR during EB differentiation of female mESCs following knock down of Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Data are presented as mean ±standard deviation from three independent experiments. Statistical significance was determined using 2-way ANOVA. Xist primers Xist ex3 F and Xist ex4 R were used. Normalisation was performed using a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. (*p ≤ 0.05). (B). RT-qPCR analysis of Xist expression levels during differentiation of the Fa2L cells, following expression of shRNA against Luciferase (Control, black) and Kap1 (Kap1 KD, grey), at the indicated timepoints. Mean ±standard deviation from a minimum of three independent experiments is presented. 2-way ANOVA was used to determine statistical significance. ns=not significant. (


( C ) . Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase ( Control , black ) and KAP1 ( Kap1 KD , grey ) , during differentiation . Mean ± standard deviation from four independent experiments are shown . Statistical significance was determined using 2 - way ANOVA . ( * * p ≤ 0 . 01 ) . Values have first been normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha .

O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Tsix RNA levels in female mESCs infected with shRNA directed against Luciferase (Control, black) and KAP1 (Kap1 KD, grey), during differentiation. Mean ±standard deviation from four independent experiments are shown. Statistical significance was determined using 2-way ANOVA. (**p ≤ 0.01). Values have first been normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha.


( D ) . RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase ( control ) or against Kap1 ( Kap1 KD ) . Left : Cells with no ( 0 , red ) , one ( 1 , grey ) and two or more ( 2 , black ) Tsix foci were counted in two independent experiments , shown averaged . Statistical significance was determined by χ2 . A minimum of 110 cells were counted per each time point for each line ( * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 ) . Right : examples of cells with one ( top ) or two ( central and bottom ) Tsix FISH signals . Scale bars : 5μm .

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

(D). RNA FISH analysis of Tsix expression during differentiation of female mESCs expressing an shRNA directed against Luciferase (control) or against Kap1 (Kap1 KD). Left: Cells with no (0, red), one (1, grey) and two or more (2, black) Tsix foci were counted in two independent experiments, shown averaged. Statistical significance was determined by χ2. A minimum of 110 cells were counted per each time point for each line (**p ≤ 0.01, ****p ≤ 0.0001). Right: examples of cells with one (top) or two (central and bottom) Tsix FISH signals. Scale bars: 5μm.


( A ) . ChIP - qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs ( Rif1 + / + , same as used in Fig . 3B but without OHT ) and during early differentiation . ZFP629 is a well - characterised KAP1 associated region ( positive control ) . Xite A and C indicate two regions within the Tsix enhancer Xite , Tsix region 1 indicates Tsix major promoter , Tsix region 2 indicates the Dxpas34 region , Tsix region 3 indicates a region slightly downstream of the Dxpas34 region . P1 and P2 indicate the two Xist promoters , 5 ' indicates a region 2 kb upstream of Xist TSS . Inter1 is an intergenic region . See Appendix Fig . S2C for the positions of the primers within Xist and Tsix . The data are presented as mean ± standard deviation from three ( 2d EB and 1d EB ) and two ( ESCs ) independent experiments . Statistical significance was calculated by Student ' s two - tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB ( * p ≤ 0 . 05 and ns = not significant ) . In the inset , Tsix RNA levels were quantified by RT - qPCR during the differentiation of wild type female ESCs shown in Fig . 4F . The average of two experiments is shown . Tsix values are normalised to a geometric mean consisting of the expression of Rplp0 , Ubiquitin and Sdha . Error bars indicate standard deviations .

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

(A). ChIP-qPCR analysis of KAP1 association with the indicated sites in wild type female mESCs (Rif1+/+, same as used in Fig. 3B but without OHT) and during early differentiation. ZFP629 is a well-characterised KAP1 associated region (positive control). Xite A and C indicate two regions within the Tsix enhancer Xite, Tsix region 1 indicates Tsix major promoter, Tsix region 2 indicates the Dxpas34 region, Tsix region 3 indicates a region slightly downstream of the Dxpas34 region. P1 and P2 indicate the two Xist promoters, 5' indicates a region 2 kb upstream of Xist TSS. Inter1 is an intergenic region. See Appendix Fig. S2C for the positions of the primers within Xist and Tsix. The data are presented as mean ±standard deviation from three (2d EB and 1d EB) and two (ESCs) independent experiments. Statistical significance was calculated by Student's two-tailed unpaired t test comparing RIF1 association to Xist P2 and P1 in 2d EB versus 1d EB (*p ≤ 0.05 and ns=not significant). In the inset, Tsix RNA levels were quantified by RT-qPCR during the differentiation of wild type female ESCs shown in Fig. 4F. The average of two experiments is shown. Tsix values are normalised to a geometric mean consisting of the expression of Rplp0, Ubiquitin and Sdha. Error bars indicate standard deviations.


( C ) . KAP1 association with Xist promoter in two independent Rif1 + / + ( Rif1 + / + + OHT , black ) and two Rif1 - / - ( Rif1F / F + OHT , grey ) female mESC cell lines . Ezr is an additional region known to be associated with KAP1 in mESCs . Enrichments are presented relative to input DNA . Mean ± standard deviation from a minimum of three independent experiments per cell line are displayed . Statistical significance was determined using Student ' s two - tailed , unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1 + / + versus Rif1 - / - cells ( * * p ≤ 0 . 01 ) .

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

(C). KAP1 association with Xist promoter in two independent Rif1+/+ (Rif1+/+ +OHT, black) and two Rif1-/- (Rif1F/F +OHT, grey) female mESC cell lines. Ezr is an additional region known to be associated with KAP1 in mESCs. Enrichments are presented relative to input DNA. Mean ±standard deviation from a minimum of three independent experiments per cell line are displayed. Statistical significance was determined using Student's two-tailed, unpaired t test comparing the KAP1 association with Xist P2 and P1 in Rif1+/+ versus Rif1-/- cells (**p ≤ 0.01).


( C ) . HEXIM1 association with the Xist promoter ( P1 and P2 ) in two independent Rif1 + / + and two Rif1 - / - mESC lines , analysed by ChIP - qPCR . Tkbp1 and Myf5 are two control regions . As in the case of KAP1 association , deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2 . Average ± standard deviation of two independent experiments . p values were calculated by two - tailed , unpaired , equal variance t - test . ( * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 ) .

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

(C). HEXIM1 association with the Xist promoter (P1 and P2) in two independent Rif1+/+ and two Rif1-/- mESC lines, analysed by ChIP-qPCR. Tkbp1 and Myf5 are two control regions. As in the case of KAP1 association, deletion of Rif1 induces accumulation of HEXIM1 on Xist P1 and P2. Average ±standard deviation of two independent experiments. p values were calculated by two-tailed, unpaired, equal variance t-test. (**p ≤ 0.01, ***p ≤ 0.001).


( E ) . Upon infection of Fa2L cells with shRNA against Kap1 or control , against Luciferase , HEXIM1 association with P2 was analysed , on both alleles , by ChIP - qPCR . As in the case of KAP1 , HEXIM1 shows preferential association with Xist P2 on the Cast allele ( future Xa , Control ) . The association is lost upon knock down of Kap1 ( Kap1 KD ) . Myf5 serves as a negative and Neat1 as a positive control regions .

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

(E). Upon infection of Fa2L cells with shRNA against Kap1 or control, against Luciferase, HEXIM1 association with P2 was analysed, on both alleles, by ChIP-qPCR. As in the case of KAP1, HEXIM1 shows preferential association with Xist P2 on the Cast allele (future Xa, Control). The association is lost upon knock down of Kap1 (Kap1 KD). Myf5 serves as a negative and Neat1 as a positive control regions.


Pulldowns of pointed end site 1 - 4 mutants using Strep - Hook3 ( D ) or Strep - BICD2 ( E ) . Binding is shown relative to the binding of the wildtype pointed end construct ( dashed lines ) . * shows p < 0 . 05 . For BICD2 , p = 0 . 06 for Site 1 mutant , and p = 0 . 1 for Site 4 mutant . Data presented as mean ± SEM , n = 3 . Statistical significance calculated using ANOVA with Tukey ' s test for multiple comparisons .

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

Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.


A slice through a filtered tomogram showing SR vesicles in contact with a putative T - tubule . Individual RyR1 molecules are indicated by the red circles . The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump . Areas circled in yellow indicate accumulation of density inside the SR . Scale bar : 20 nm . A slice through the structure of RyR1 in contact with putative TT - membrane . A middle slice through the in situ structure of RyR1 at 12 . 6 - Å resolution . The observed additional transmembrane density is indicated by the red arrows . Scale bars in B , C : 10 nm .

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 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 B-MEASURED_VAR 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

A slice through a filtered tomogram showing SR vesicles in contact with a putative T-tubule. Individual RyR1 molecules are indicated by the red circles. The dense protein coat on the surface of the SR vesicles correspond to the SERCA pump. Areas circled in yellow indicate accumulation of density inside the SR. Scale bar: 20 nm. A slice through the structure of RyR1 in contact with putative TT-membrane. A middle slice through the in situ structure of RyR1 at 12.6-Å resolution. The observed additional transmembrane density is indicated by the red arrows. Scale bars in B, C: 10 nm.


Slice through the structures of apoRyR1 with the putative TT - membrane ( left ) and the standalone apoRyR1 ( right , with the flipped handendness and reduced resolution for comparison ) 22 Å away from the middle slice ( Y = 92 in the volume coordinates ) . Scale bar : 10 nm .

O O O O O B-MEASURED_VAR 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

Slice through the structures of apoRyR1 with the putative TT-membrane (left) and the standalone apoRyR1 (right, with the flipped handendness and reduced resolution for comparison) 22 Å away from the middle slice (Y = 92 in the volume coordinates). Scale bar: 10 nm.


The location of the CaM / S100A1 binding site is marked with red circles in slices through maps of the in situ structure ( left ) and the single particle structure ( EMDB : 8393 ) in the same orientation as the in situ structure and filtered to 15 Å ( right ) . Scale bar : 10 nm .

O O O O B-MEASURED_VAR 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

The location of the CaM/S100A1 binding site is marked with red circles in slices through maps of the in situ structure (left) and the single particle structure (EMDB: 8393) in the same orientation as the in situ structure and filtered to 15 Å (right). Scale bar: 10 nm.


A slice through the average structure of the apo - state at a position 22 Å away from the middle slice shown in Fig . 1C . The intra - SR extension is boxed on the density in red .

O O O O O O O O O O O O O O O O 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 slice through the average structure of the apo-state at a position 22 Å away from the middle slice shown in Fig. 1C. The intra-SR extension is boxed on the density in red.


Representative SR vesicles with different distributions of inner - SR density : segregated , evenly distributed , empty , and empty with filaments . RyR1 particles are circled in red , areas circled in yellow indicate accumulation of density inside the SR .

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

Representative SR vesicles with different distributions of inner-SR density: segregated, evenly distributed, empty, and empty with filaments. RyR1 particles are circled in red, areas circled in yellow indicate accumulation of density inside the SR.


The fractions of vesicles that fall into each density category for each RyR1 sample ( EDTA : n = 64 ; 1 mM Ca2 + : n = 32 ; 5 mM Ca2 + : n = 21 , technical replicates ) . Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density , orange to segregated density , and grey to empty vesicles .

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

The fractions of vesicles that fall into each density category for each RyR1 sample (EDTA: n=64; 1 mM Ca2+: n = 32; 5 mM Ca2+: n=21, technical replicates). Blue corresponds to the fraction of SR lumen vesicles showing evenly distributed density, orange to segregated density, and grey to empty vesicles.


Structures of RyR1 determined in presence of increasing Ca2 + concentration ( 1 mM and 5 mM ) , all with intra - SR extensions . Scale bars : 10 nm in ( A ) and ( E ) , 20 nm in ( C ) .

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Structures of RyR1 determined in presence of increasing Ca2+ concentration (1 mM and 5 mM), all with intra-SR extensions. Scale bars: 10 nm in (A) and (E), 20 nm in (C).


B is domain representation on the grayscale image . Please note the location of two transmembrane densities : yellow - shown in Figure 1C , magenta - shown in Figure 3B .

O O O O O O O O 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 is domain representation on the grayscale image. Please note the location of two transmembrane densities: yellow - shown in Figure 1C, magenta - shown in Figure 3B.


Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15 . 4 , 18 , 18 , and 27 Å , respectively , with the percentages of the total particles in each class shown beneath . Red arrows show domain movements compared to class 1 .

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

Slices through the results of classification of the particles in the global average into 4 classes at resolutions 15.4, 18, 18, and 27 Å, respectively, with the percentages of the total particles in each class shown beneath. Red arrows show domain movements compared to class 1.


The first five modes of correlated movements . Direction of motion is indicated by the arrows . Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode . Scale bars : 10 nm .

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

The first five modes of correlated movements. Direction of motion is indicated by the arrows. Informativity of the respective modes measured as variance of the corresponding mode divided by variance of the first mode. Scale bars: 10 nm.


Structure of ryRyR1 at a resolution of 17 . 5 Å . Two views are through the middle slice ( top ) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below ( bottom panels ) . The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo - structure . This movement is highlighted by red arrows in the right panels . Scale bar : 10 nm .

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

Structure of ryRyR1 at a resolution of 17.5 Å. Two views are through the middle slice (top) of the reconstruction and at the level of sarcoplasmic extentions 22 Å below (bottom panels). The movement of the outer domains is associated with a visible change in membrane curvature as compared to the apo-structure. This movement is highlighted by red arrows in the right panels. Scale bar: 10 nm.


( a ) Representative 2D μ - CT scans of RasV12 ; ctrl at day 6 , RasV12 ; scrib - / - larvae at day 6 and day 10 . Muscle ( green ) , fat body ( blue ) and eye - antennal discs / tumor ( red ) are outlined . Scale bar : 1mm . Anterior ( A ) , Right ( R ) .

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(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).


( a ) Quantification of tumor growth of RasV12 ; ctrl day 6 ( n = 15 ) RasV12 ; scrib - / - tumors , day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 4 ) , day 9 ( n = 5 ) and day 10 ( n = 5 ) .

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

(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).


( b ) Quantifications of muscle volume , of larvae carrying tumors of RasV12 ; ctrl ( n = 15 ) , and RasV12 ; scrib - / - at day 6 ( n = 15 ) , day 7 ( n = 5 ) , day 8 ( n = 5 ) , day 9 ( n = 5 ) and dat 10 ( n = 5 ) .

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

(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).


( c ) Quantification of width of Dorsal Oblique 3 ( DO3 ) muscle in larvae carrying tumors of RasV12 ; ctrl at day 6 ( n = 29 ) and RasV12 ; scrib - / - at days 6 ( n = 29 ) and day 10 ( n = 31 ) .

O O O O O O O O O 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

(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).


( d ) Quantification of adipose tissue volume in larvae carrying tumors of RasV12 ; ctrl at day 6 and RasV12 ; scrib - / - at days 6 and day 10 .

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

(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.


( e ) Representative confocal images of adipose tissue in RasV12 ; ctrl and RasV12 ; scrib - / - tumors bearing animals at indicated ages . Lipid droplets are highlighted with Lipid Tox staining . Scale bar :

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

(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm


( f ) Quantification of dry weight of RasV12 ; ctrl ( n = 9 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) and day 10 ( n = 18 ) tumor bearing larvae excluding the tumor weight .

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) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.


( g ) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12 ; ctrl ( n = 14 ) , RasV12 ; scrib - / - day 6 ( n = 15 ) , day 8 ( n = 16 ) and day 10 ( n = 14 ) , each colored line represents a single larva .

O O 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

(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.


( h ) Coomassie feeding assay to asses food intake of RasV12 ; scrib - / - over time , three repeated measurements of an average food intake of 20 larvae .

(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.


( a - e ) Cartoon ( top ) illustrates the genotypes of the larvae , eye - antennal disc ( EAD , circle : tumor cells in green and microenvironment cell in black ) , systemic cells are illustrated as a square ( wild type cells in light brown and atg13 mutant cells in light orange ) at indicated ages . Cartoon ( left ) illustrates the structure of cephalic complex attached to mouth hook . Representative images of Larva ( image of larva using the backlight of microscope ) , cephalic complex ( green highlights the GFP - labelled tumor clones ) , muscle ( Phalloidin in green stains actin and Hoechst in blue stains nucleus ) and adipose tissue ( Lipid Tox in red stains for lipid droplets , Hoechst stains nucleus ) from top to bottom . ( a ) RasV12 ; ctrl tumor bearing animal at day 6 . ( b ) RasV12 ; scrib - / - tumor bearing animal at day 6 . ( c ) RasV12 ; scrib - / - tumor bearing animal at day 8 . ( d ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - at day 8 . ( e ) RasV12 , scrib - / - , atg13 - / - / / atg13 - / - animal complemented with transgenic atg13 , rescuing the tumor growth , at day 8 .

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

(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.


( f ) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 ( shown in yellow dashed line in 2a ) , of animals bearing tumors at day 6 RasV12 ; ctrl ( n = 45 ) , RasV12 ; scrib - / - ( n = 30 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 30 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 29 ) and at day 10 RasV12 ; scrib - / - ( n = 40 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 25 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 28 ) .

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

(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).


( g ) Quantification of tumor volumes day 6 RasV12 ; ctrl ( n = 7 ) , RasV12 ; scrib - / - ( n = 11 ) and day 8 RasV12 ; scrib - / - ( n = 13 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 9 ) and ey3 . 5 - atg13 ; RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 9 ) .

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

(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).


( h ) Quantification of Ventral Longitudinal muscle 4 ( VL4 ) ( shown in yellow dashed line in 2a ) of third segment of larvae carrying at day 6 RasV12 ; ctrl ( n = 15 ) , RasV12 ; scrib - / - ( n = 14 ) , and at day8 RasV12 ; scrib - / - ( n = 11 ) , RasV12 , scrib - / - , atg13 - / - / / atg13 - / - ( n = 12 ) and ey3 . 5 - atg13 ; RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - ( n = 14 ) .

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

(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).


( i ) Quantification of dry weight excluding tumor of RasV12 ; scrib - - , atg13 - / - / / atg13 - / - at day 6 ( n = 8 ) and day 10 ( n = 7 ) .

(i) Quantification of dry weight excluding tumor of RasV12; scrib- -, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).


( j ) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - at day 6 ( n = 15 ) and at day 8 ( n = 15 ) , each colored line represents a single larva .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.


( a ) Changes in groups ( carbohydrates , amino acids and fatty acids ) of storage metabolites measured in circulating haemolymph with progressing wasting , shown as log2 changes measured by LC - MS and calculated per larvae relative to RasV12 ; ctrl at day 6 .

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

(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.


( b ) Volcano plot showing autophagy - dependent changes to amounts of circulating metabolites at day 6 . X - axis shows the log2 of fold change of RasV12 ; atg13 - / - scrib - / - / / atg13 - / - vs . RasV12 ; scrib - / - , y - axis shows - log10 p - value , calculated by t - test . Metabolite names are shown for metabolites with log2 ( FC ) > ± 1 and / or - log10 ( P ) < 2 . Green points indicate log2 ( FC ) > ± 1 , blue indicate - log10 ( P ) < 2 and red indicates for above both thresholds .

O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.


( c ) Autophagy - driven wasting releases metabolites into circulation of 113 reliably detected metabolites , those showing significant differences in any of the three comparisons are shown . Color indicates the log2 ( fold change ) difference and the numbers show the p - value of the comparison . The statistical test to define significance was FDR - adjusted t - test p - value < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05


( a ) The amount of glycogen in the whole larvae measured by biochemical assay ( n = 4 ) , normalized to RasV12 ; ctrl at day 6 , and per number of larvae .

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

(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.


( b - e ) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12 ; ctrl , RasV12 ; scrib - / - and RasV12 ; scrib - / - , atg13 - / - / / atg13 - / - showing glycogen levels ( white ) at day 6 and day 8 .

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

(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.


( g ) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor . A similar , but independent experiment is reported in Holland P . , et al . , 2021 BMC Biol .

(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.


Immunohistochemistry of NSMCE2 on mouse testis . The inset illustrates the accumulation of NSMCE2 on the XY body . Scale bar , 50 μm .

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Immunohistochemistry of NSMCE2 on mouse testis. The inset illustrates the accumulation of NSMCE2 on the XY body. Scale bar, 50 μm.


Immunofluorescence ( IF ) of NSMCE2 and SCP3 on isolated mouse spermatocytes . Threads of NSMCE2 can be detected on regions of weak SCP3 signal , indicative of asynapsis ( arrowheads ) . Scale bar , 2 . 5 μm .

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

Immunofluorescence (IF) of NSMCE2 and SCP3 on isolated mouse spermatocytes. Threads of NSMCE2 can be detected on regions of weak SCP3 signal, indicative of asynapsis (arrowheads). Scale bar, 2.5 μm.


Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes .

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Colocalization of NSMCE2 and BRCA1 at the XY body of mouse spermatocytes.


Representative image of NSMCE2 and BRCA1 foci in control and MMS ‐ treated MEFs . Scale bar , 2 . 5 μm .

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

Representative image of NSMCE2 and BRCA1 foci in control and MMS‐treated MEFs. Scale bar, 2.5 μm.


Quantification of NSMCE2 and BRCA1 foci in wild ‐ type MEFs exposed to IR ( 10 Gy ; 1 ‐ h recovery ) , MMS ( 1 mM for 1 h ; 24 ‐ h recovery ) , and PARP inhibitor ( 1 μM for 1 h ; 24 ‐ h recovery ) . Data indicate mean values and are representative of three independent experiments . Error bars indicate SD .

O O B-MEASURED_VAR 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

Quantification of NSMCE2 and BRCA1 foci in wild‐type MEFs exposed to IR (10 Gy; 1‐h recovery), MMS (1 mM for 1 h; 24‐h recovery), and PARP inhibitor (1 μM for 1 h; 24‐h recovery). Data indicate mean values and are representative of three independent experiments. Error bars indicate SD.


Representative image of IR ‐ ( 10 Gy ; 1 ‐ h recovery ) and MMS ‐ ( 1 mM for 1 ‐ h ; 24 ‐ h recovery ) induced NSMCE2 and γH2AX foci in MEFs . Scale bar , 2 . 5 μm .

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Representative image of IR‐ (10 Gy; 1‐h recovery) and MMS‐ (1 mM for 1‐h; 24‐h recovery) induced NSMCE2 and γH2AX foci in MEFs. Scale bar, 2.5 μm.


Brightfield microscopy of 2 . 5 dpc embryos obtained from crosses of Nsmce2GT / + mice . The right panel is of the same 2 embryos shown on the left panel , 36 h after being cultured in vitro . The arrow indicates embryos that showed asymmetric cell sizes when isolated , which degenerated upon culture . These embryos were identified as Nsmce2GT / GT through genotyping .

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Brightfield microscopy of 2.5 dpc embryos obtained from crosses of Nsmce2GT/+ mice. The right panel is of the same 2 embryos shown on the left panel, 36 h after being cultured in vitro. The arrow indicates embryos that showed asymmetric cell sizes when isolated, which degenerated upon culture. These embryos were identified as Nsmce2GT/GT through genotyping.


Examples of the three genotypes found at Mendelian frequencies ( see Appendix Table S1 ) on 2 . 5 dpc embryos .

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Examples of the three genotypes found at Mendelian frequencies (see Appendix Table S1) on 2.5 dpc embryos.


Examples of the type of nuclei found on Nsmce2GT / GT and Nsmce2 + ( + / + or + / GT ) 2 . 5 dpc embryos , 12 h after in vitro culture . Mutant embryos were identified due to the absence of NSMCE2 signal ( green ) . DAPI was used to stain DNA ( blue ) . White arrows indicate examples of cells with condensed chromosomes or irregular shapes , which are hardly seen in wild type embryos .

O O O O O O O O B-CONTROLLED_VAR 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 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

Examples of the type of nuclei found on Nsmce2GT/GT and Nsmce2+ (+/+ or +/GT) 2.5 dpc embryos, 12 h after in vitro culture. Mutant embryos were identified due to the absence of NSMCE2 signal (green). DAPI was used to stain DNA (blue). White arrows indicate examples of cells with condensed chromosomes or irregular shapes, which are hardly seen in wild type embryos.


Immunoprecipitation of SMC5 ( and IgG , as a control ) from a 48 ‐ h culture of B lymphocytes of wt and Nsmce2SD / SD animals . NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB . The image of the Ponceau staining is shown as a loading control .

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Immunoprecipitation of SMC5 (and IgG, as a control) from a 48‐h culture of B lymphocytes of wt and Nsmce2SD/SD animals. NSMCE2 and SMC6 levels on the immunoprecipitates were analyzed by WB. The image of the Ponceau staining is shown as a loading control.


IF of NSMCE2 foci in MMS ‐ treated Nsmce2 + / + and Nsmce2SD / SD MEFs . Scale bar , 2 . 5 μm .

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IF of NSMCE2 foci in MMS‐treated Nsmce2+/+ and Nsmce2SD/SD MEFs. Scale bar, 2.5 μm.


Representative picture of 6 ‐ month ‐ old Nsmce2 + / + and Nsmce2SD / SD littermates .

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Representative picture of 6‐month‐old Nsmce2+/+ and Nsmce2SD/SD littermates.


Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2SD / SD mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test .

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Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2SD/SD mice. The P‐value was calculated with the Mantel-Cox long‐rank test.


A Kaplan - Meyer survival curves of Nsmce2 + / + and Nsmce2 + / GT mice . The P ‐ value was calculated with the Mantel - Cox long ‐ rank test . * * * P 0 . 001 .

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A Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2+/GT mice. The P‐value was calculated with the Mantel-Cox long‐rank test. ***P 0.001.


B Tumor incidence found on Nsmce2 + / + and Nsmce2 + / GT mice at the time of death ( i . e . moribund mice ) .

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B Tumor incidence found on Nsmce2+/+ and Nsmce2+/GT mice at the time of death (i.e. moribund mice).


C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2 + / + and Nsmce2 + / GT MEFs . β ‐ actin was used as a loading control . Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample .

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C NSMCE2 levels evaluated by Western blot in 2 independent pairs of Nsmce2+/+ and Nsmce2+/GT MEFs. β‐actin was used as a loading control. Numbers shown bellow indicate the relative amounts of NSMCE2 in each sample.


D Sister chromatid exchange ( SCE ) events found per metaphase on Nsmce2 + / + and Nsmce2 + / GT MEFs . * * * P 0 . 001 .

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D Sister chromatid exchange (SCE) events found per metaphase on Nsmce2+/+ and Nsmce2+/GT MEFs. ***P 0.001.


E , F Relative percentages of micronuclei ( E ) and polynucleated cells ( F ) on cultures of Nsmce2 + / + and Nsmce2 + / GT MEFs . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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E, F Relative percentages of micronuclei (E) and polynucleated cells (F) on cultures of Nsmce2+/+ and Nsmce2+/GT MEFs. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


NSMCE2 levels assessed by WB in UQ . CreERT2 transgenic Nsmce2 + / + and Nsmce2lox / lox littermate MEFs , 24 h after exposure to 4 ‐ OHT . Tubulin levels are shown as a loading control .

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NSMCE2 levels assessed by WB in UQ.CreERT2 transgenic Nsmce2+/+ and Nsmce2lox/lox littermate MEFs, 24 h after exposure to 4‐OHT. Tubulin levels are shown as a loading control.


Clonogenic assay on immortalized UQ . CreERT2 / Nsmce2lox / lox MEFs . The picture shows the colonies detected after 10 days of growth in the presence or absence of 4 ‐ OHT , upon staining with crystal violet .

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Clonogenic assay on immortalized UQ.CreERT2/Nsmce2lox/lox MEFs. The picture shows the colonies detected after 10 days of growth in the presence or absence of 4‐OHT, upon staining with crystal violet.


NSMCE2 and BRCA1 foci in MMS ‐ treated ( 1 mM for 1 h ; 24 ‐ h recovery ) UQ . CreERT2 / Nsmce2lox / lox MEFs , 48 h after being treated ( or not ) with 4 ‐ OHT . Scale bar , 5 μm .

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NSMCE2 and BRCA1 foci in MMS‐treated (1 mM for 1 h; 24‐h recovery) UQ.CreERT2/Nsmce2lox/lox MEFs, 48 h after being treated (or not) with 4‐OHT. Scale bar, 5 μm.


SCE events per metaphase on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h . * * * P 0 . 001 .

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SCE events per metaphase on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h. ***P 0.001.


Example of SCE events ( arrows ) found on Nsmce2 ‐ deleted MEFs .

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Example of SCE events (arrows) found on Nsmce2‐deleted MEFs.


SCE events at telomeres on UQ . CreERT2 / Nsmce2lox / lox MEFs grown in the presence of 4 ‐ OHT for 72 h , as detected by CO ‐ FISH ( see ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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SCE events at telomeres on UQ.CreERT2/Nsmce2lox/lox MEFs grown in the presence of 4‐OHT for 72 h, as detected by CO‐FISH (see ). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


Telomere lengths of UQ . CreERT2 Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h .

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Telomere lengths of UQ.CreERT2 Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h.


Example of the intensity of telomere signals ( yellow ) used for ( H ) .

Example of the intensity of telomere signals (yellow) used for (H).


Percentage of cells with micronuclei found on cultures of UQ . CreERT2 / Nsmce2lox / lox MEFs treated or not with 4 ‐ OHT for 72 h . Data are representative of three independent experiments . Error bars indicate SD . * * * P 0 . 001 .

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Percentage of cells with micronuclei found on cultures of UQ.CreERT2/Nsmce2lox/lox MEFs treated or not with 4‐OHT for 72 h. Data are representative of three independent experiments. Error bars indicate SD. ***P 0.001.


Intrauterine dwarfism observed upon NSMCE2 deletion on 17 . 5 dpc embryos . Pregnant females were treated at 14 . 5 dpc for 3 days with intraperitoneal injections of 4 ‐ OHT . The image represents the difference in size observed between Nsmce2 + / + and Nsmce2lox / lox embryos carrying the UQ . CreERT2 transgene ( Nsmce2 + / + and Nsmce2lox / lox , from now on ) .

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Intrauterine dwarfism observed upon NSMCE2 deletion on 17.5 dpc embryos. Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal injections of 4‐OHT. The image represents the difference in size observed between Nsmce2+/+ and Nsmce2lox/lox embryos carrying the UQ.CreERT2 transgene (Nsmce2+/+ and Nsmce2lox/lox, from now on).


Representative picture of Nsmce2 + / + and Nsmce2lox / lox animals fed for 40 weeks with a 4 ‐ OHT ‐ containing diet ( see also Movies EV1 and EV2 ) .

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Representative picture of Nsmce2+/+ and Nsmce2lox/lox animals fed for 40 weeks with a 4‐OHT‐containing diet (see also Movies EV1 and EV2).


Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox / lox mice .

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Images illustrating the presence of altered pigmentation problems that arise on Nsmce2lox/lox mice.


Percentage of fat against total body mass on Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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Percentage of fat against total body mass on Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


White bloodcell counts ( WBC ) from Nsmce2 + / + and Nsmce2lox / lox animals . * * * P 0 . 001 .

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White bloodcell counts (WBC) from Nsmce2+/+ and Nsmce2lox/lox animals. ***P 0.001.


Percentage of γH2AX ‐ positive micronuclei found on the intestinal epithelia of Nsmce2 + / + and Nsmce2lox / lox animals ( see Appendix Fig S11 ) . Data are representative of three independent experiments . Error bars indicate SD . * P 0 . 05 .

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Percentage of γH2AX‐positive micronuclei found on the intestinal epithelia of Nsmce2+/+ and Nsmce2lox/lox animals (see Appendix Fig S11). Data are representative of three independent experiments. Error bars indicate SD. *P 0.05.


A WB illustrating the depletion of NSMCE2 in B cells isolated from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with lipopolysaccharide ( LPS ) . Tubulin was used as a loading control .

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A WB illustrating the depletion of NSMCE2 in B cells isolated from 2‐month‐old CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with lipopolysaccharide (LPS). Tubulin was used as a loading control.


B Representative FACS analysis from the B ‐ cell cultures mentioned in ( A ) . DNA content was measured with propidium iodide ( PI ) . Note the presence of cells with > 4n DNA content in NSMCE2 ‐ deleted cells .

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B Representative FACS analysis from the B‐cell cultures mentioned in (A). DNA content was measured with propidium iodide (PI). Note the presence of cells with > 4n DNA content in NSMCE2‐deleted cells.


C , D Frequency of origin firing ( C ) and fork rate ( D ) were analyzed by DNA fiber analyses ( see ) from fibers prepared from B ‐ cell cultures explained in ( A ) . No significant differences were observed between Nsmce2 + / + and Nsmce2lox / loxB cells . Data are representative of two independent experiments . Error bars indicate SD . Between 200 and 300 tracks were measured to estimate fork rate and 300 - 500 for the calculation of origin firing frequencies .

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C, D Frequency of origin firing (C) and fork rate (D) were analyzed by DNA fiber analyses (see ) from fibers prepared from B‐cell cultures explained in (A). No significant differences were observed between Nsmce2+/+ and Nsmce2lox/loxB cells. Data are representative of two independent experiments. Error bars indicate SD. Between 200 and 300 tracks were measured to estimate fork rate and 300-500 for the calculation of origin firing frequencies.


E Representative images of the DNA fibers used for the analysis explained in ( C , D ) . CldU ( red ) and IdU ( green ) channels are shown .

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E Representative images of the DNA fibers used for the analysis explained in (C, D). CldU (red) and IdU (green) channels are shown.


Representative pictures of the spleens from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes .

Representative pictures of the spleens from 2‐month‐old CD19+/Cre animals of the indicated genotypes.


Numbers of B and T cells from 2 ‐ month ‐ old CD19 + / Cre animals of the indicated genotypes evaluated by flow cytometry ( B220 : B ‐ cell marker ; CD3 : T ‐ cell marker ) .

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Numbers of B and T cells from 2‐month‐old CD19+/Cre animals of the indicated genotypes evaluated by flow cytometry (B220: B‐cell marker; CD3: T‐cell marker).


SCE events per metaphase on B cells obtained from CD19 + / Cre animals of the indicated genotypes , 72 h after being stimulated in vitro with LPS . * * P 0 . 01 , * * * P 0 . 001 .

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SCE events per metaphase on B cells obtained from CD19+/Cre animals of the indicated genotypes, 72 h after being stimulated in vitro with LPS. **P 0.01, ***P 0.001.


Analysis of forward ( FSC ) and side ( SSC ) scatter parameters of B cells obtained from CD19 + / Cre animals of the indicated genotypes , 48 h after being stimulated in vitro with LPS . The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2 .

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Analysis of forward (FSC) and side (SSC) scatter parameters of B cells obtained from CD19+/Cre animals of the indicated genotypes, 48 h after being stimulated in vitro with LPS. The emergence of bigger cells is noticeable on cells lacking both BLM and NSMCE2.


Representative images of B ‐ cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS . Insets provide a magnified view of an individual cell ( arrowhead ) for better comparison of the observed differences in cell size . Scale bar , 100 μm .

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Representative images of B‐cell cultures of the indicated genotypes 48 h after being stimulated in vitro with LPS. Insets provide a magnified view of an individual cell (arrowhead) for better comparison of the observed differences in cell size. Scale bar, 100 μm.


Examples of the types of nuclei found on Blm and Nsmce2 double ‐ mutant B cells ( compared with wild ‐ type B cells ) , in cells obtained from ( E ) . DAPI was used to stain DNA . Whereas wt nuclei are regular in size , double ‐ mutant cells invariably presented enlarged , multilobulated , and irregular nuclei . Scale bar , 2 . 5 μm .

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Examples of the types of nuclei found on Blm and Nsmce2 double‐mutant B cells (compared with wild‐type B cells), in cells obtained from (E). DAPI was used to stain DNA. Whereas wt nuclei are regular in size, double‐mutant cells invariably presented enlarged, multilobulated, and irregular nuclei. Scale bar, 2.5 μm.


d . Real - time reverse transcription quantitative PCR ( RT - qPCR ) showing Fbxw7 , Pten and Trp53 expression in mouse uteri at 4 weeks and 8 weeks postnatal age ( NB Trp53R172H uteri were not analysed at the 4 week timepoint ) . Box extends from 25th to 75th percentiles , with line at median , whiskers indicate minimum and maximum values , and dots individual values ( n = 5 samples per group with exception of Ptendel and Ptendel , Fbxw7mut genotypes at 8 weeks which contain n = 7 samples each ) . Statistical comparison between WT controls and other genotypes was performed by unadjusted , unpaired Mann Whitney test ; * indicates P < 0 . 05 , * * indicates P < 0 .

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d. Real-time reverse transcription quantitative PCR (RT-qPCR) showing Fbxw7, Pten and Trp53 expression in mouse uteri at 4 weeks and 8 weeks postnatal age (NB Trp53R172H uteri were not analysed at the 4 week timepoint). Box extends from 25th to 75th percentiles, with line at median, whiskers indicate minimum and maximum values, and dots individual values (n=5 samples per group with exception of Ptendel and Ptendel, Fbxw7mut genotypes at 8 weeks which contain n=7 samples each). Statistical comparison between WT controls and other genotypes was performed by unadjusted, unpaired Mann Whitney test; * indicates P<0.05, ** indicates P<0.01


e . Western blot of mouse uteri at 8 weeks postnatal age ( NB Trp53R172H uteri were not analysed by Western blotting ) . Values indicate densitometric units ( mean ± SD ) normalised to control group of WT samples in case of Fbxw7 protein and both WT and Fbxw7mut samples in case of Pten and p53 proteins . Image is representative of duplicate technical replicates . Statistical comparison between controls and other groups was performed by unadjusted , unpaired Mann Whitney test ; and are shown next to densitometry results where statistically significant .

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e. Western blot of mouse uteri at 8 weeks postnatal age (NB Trp53R172H uteri were not analysed by Western blotting). Values indicate densitometric units (mean ± SD) normalised to control group of WT samples in case of Fbxw7 protein and both WT and Fbxw7mut samples in case of Pten and p53 proteins. Image is representative of duplicate technical replicates. Statistical comparison between controls and other groups was performed by unadjusted, unpaired Mann Whitney test; and are shown next to densitometry results where statistically significant.


f . Immunohistochemical ( IHC ) analysis of mouse uteri at 8 weeks postnatal age . Images are representative of minimum of 3 biological replicates for each genotype and protein . Scale bar indicates 100μm .

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f. Immunohistochemical (IHC) analysis of mouse uteri at 8 weeks postnatal age. Images are representative of minimum of 3 biological replicates for each genotype and protein. Scale bar indicates 100μm.


a . Kaplan - Meier curves showing survival of genetically engineered mouse models ( GEMM ) of indicated genotypes and group sizes . Statistical comparison between groups was performed by unadjusted log - rank test .

a. Kaplan-Meier curves showing survival of genetically engineered mouse models (GEMM) of indicated genotypes and group sizes. Statistical comparison between groups was performed by unadjusted log-rank test.


c . Low and high power images of GEMM uteri at 8 weeks age and at study endpoint after H & E staining or IHC for cytokeratin 8 ( CK8 ) ( epithelia ) . Scale bars in low and high magnification panels indicate 1000μm and 100μm respectively . Images are representative of a minimum of 5 mice per genotype per timepoint , with exception of wild - type and Fbxw7mut mice at 8 weeks age where 3 mice were analysed .

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c. Low and high power images of GEMM uteri at 8 weeks age and at study endpoint after H&E staining or IHC for cytokeratin 8 (CK8) (epithelia). Scale bars in low and high magnification panels indicate 1000μm and 100μm respectively. Images are representative of a minimum of 5 mice per genotype per timepoint, with exception of wild-type and Fbxw7mut mice at 8 weeks age where 3 mice were analysed.


e . H & E - stained sections of high grade external sarcomas in Trp53del , Trp53del ; Fbxw7mut , Trp53mut and Trp53mut ; Fbxw7mut mice . Images are representative of minimum of 3 mice examined for each genotype . Scale bar indicates 100μm .

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e. H&E-stained sections of high grade external sarcomas in Trp53del, Trp53del; Fbxw7mut, Trp53mut and Trp53mut; Fbxw7mut mice. Images are representative of minimum of 3 mice examined for each genotype. Scale bar indicates 100μm.


a . ( Left panel ) Schematic illustrating gene expression profiling of mouse uterine samples of indicated genotypes and group sizes at 8 weeks age , with groups compared by enrichment analysis . ( Right panel ) Dot plot indicating enrichment of MSigDB C6 oncogenic gene sets in Ptendel ; Fbxw7mut uteri vs Ptendel uteri . Arrows indicate enrichment of LEF1 signatures .

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a. (Left panel) Schematic illustrating gene expression profiling of mouse uterine samples of indicated genotypes and group sizes at 8 weeks age, with groups compared by enrichment analysis. (Right panel) Dot plot indicating enrichment of MSigDB C6 oncogenic gene sets in Ptendel; Fbxw7mut uteri vs Ptendel uteri. Arrows indicate enrichment of LEF1 signatures.


b . Dot plot showing enrichment analysis of 1 , 064 putative FBXW7 substrates against GO biological processes .

b. Dot plot showing enrichment analysis of 1,064 putative FBXW7 substrates against GO biological processes.


a . HEK293T cells were transfected with constructs encoding FLAG - V5 - tagged FBXW7α and Myc - tagged LEF1 . FLAG - V5 - tagged FBXW7 α was immunoprecipitated ( IP ) from cell extracts with anti - FLAG antibody before immunoblotting for tagged proteins as indicated ( upper panels ) . Lower panels show inputs . b . HEK293T cells were transfected with constructs encoding FLAG - V5 - tagged FBXW7α and Myc - tagged TCF7L2 . IP was performed as in ( a ) before immunoblotting for tagged proteins as indicated . Data information : All images shown are representative of a minimum of two independent

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a. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged LEF1. FLAG-V5-tagged FBXW7 α was immunoprecipitated (IP) from cell extracts with anti-FLAG antibody before immunoblotting for tagged proteins as indicated (upper panels). Lower panels show inputs. b. HEK293T cells were transfected with constructs encoding FLAG-V5-tagged FBXW7α and Myc-tagged TCF7L2. IP was performed as in (a) before immunoblotting for tagged proteins as indicated. Data information: All images shown are representative of a minimum of two independent experiments


g . HEK293T cells were transfected with Myc - tagged FBXW7α and either FLAG - V5 - tagged full length LEF1 ( FL ) or truncated LEF1 in which the N terminal domain required for β - catenin binding has been deleted . Immunoblotting for indicated proteins was performed following IP by anti - FLAG antibody . Data information : All images shown are representative of a minimum of two independent

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g. HEK293T cells were transfected with Myc-tagged FBXW7α and either FLAG-V5-tagged full length LEF1 (FL) or truncated LEF1 in which the N terminal domain required for β-catenin binding has been deleted. Immunoblotting for indicated proteins was performed following IP by anti-FLAG antibody. Data information: All images shown are representative of a minimum of two independent experiments


a . Dot plot showing log2 - transformed expression of FBXW7 , LEF1 and other wnt pathway genes in endometrial epithelial cell subsets defined by single cell RNAseq ( scRNAseq )

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a. Dot plot showing log2-transformed expression of FBXW7, LEF1 and other wnt pathway genes in endometrial epithelial cell subsets defined by single cell RNAseq (scRNAseq)


b . Representative LEF1 immunohistochemistry in GEMM uteri from mice of indicated genotypes at 8 weeks age . For each of the four columns ( i . e genotypes ) , upper and lower images are taken from littermate females housed in the same cage . Images are representative of 3 - 4 mice as shown in ( c ) . Scale bar indicates 100μm .

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b. Representative LEF1 immunohistochemistry in GEMM uteri from mice of indicated genotypes at 8 weeks age. For each of the four columns (i.e genotypes), upper and lower images are taken from littermate females housed in the same cage. Images are representative of 3-4 mice as shown in (c). Scale bar indicates 100μm.


c . Percentage of mice with epithelial LEF1 expression in endometria at 8 weeks age according to genotype . P value was derived from binomial logistic regression with epithelial LEF1 expression as the dependent variable .

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c. Percentage of mice with epithelial LEF1 expression in endometria at 8 weeks age according to genotype. P value was derived from binomial logistic regression with epithelial LEF1 expression as the dependent variable.


d . Immunohistochemistry for LEF1 and β - catenin from endometrial carcinomas in Ptendel ± Fbxw7mut mice . High power images show representative staining in LEF1 negative and positive glands . Scale bar bar indicates 1000μm .

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d. Immunohistochemistry for LEF1 and β-catenin from endometrial carcinomas in Ptendel ± Fbxw7mut mice. High power images show representative staining in LEF1 negative and positive glands. Scale bar bar indicates 1000μm.


( A - C ) AFM and TEM analysis of unloaded ( u - NPs ) and cholesterol loaded NPs ( NPs - Chol ) . AFM " height " images ( left column ) , 3D reconstruction ( middle column ) and TEM micrograph ( right column ) of u - NPs ( A ) , NPs - Chol1 ( B ) and NPs - Chol3 ( C ) .

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(A-C) AFM and TEM analysis of unloaded (u-NPs) and cholesterol loaded NPs (NPs-Chol). AFM "height" images (left column), 3D reconstruction (middle column) and TEM micrograph (right column) of u-NPs (A), NPs-Chol1 (B) and NPs-Chol3 (C).


( D ) Release profile in water of cholesterol ( continuous line , - ) and NBD - Chol ( dotted line , - - - ) from NPs - Chol1 and NPs - NBD - Chol1 , respectively .

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(D) Release profile in water of cholesterol (continuous line, -) and NBD-Chol (dotted line, - - -) from NPs-Chol1 and NPs-NBD-Chol1, respectively.


( E ) In vitro release of NBD - Chol from NPs at different time - intervals in NS cells . Data in the graph represent mean ( µg ) ± SEM of total NBD - Chol ( embedded into and released from NPs ; red columns ) and NBD - Chol released after NPs degradation ( purple columns ) present in the homogenates of NS cells treated with NPs - NBD -

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(E) In vitro release of NBD-Chol from NPs at different time-intervals in NS cells. Data in the graph represent mean (µg) ± SEM of total NBD-Chol (embedded into and released from NPs; red columns) and NBD-Chol released after NPs degradation (purple columns) present in the homogenates of NS cells treated with NPs-NBD-Chol1


( A - B ) Representative confocal images of liver ( A ) and brain ( B ) slices from R6 / 2 mice ip injected with C - NPs ( left ) or with g7 - NPs ( right ) and sacrificed after 4 hrs . ( C ) Quantification of g7 - NPs localized in the liver , striatum , and cortex of WT ( n = 3 ) and R6 / 2 mice ( n = 3 ) . Data represent mean ( µg ) ± SEM .

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(A-B) Representative confocal images of liver (A) and brain (B) slices from R6/2 mice ip injected with C-NPs (left) or with g7-NPs (right) and sacrificed after 4 hrs. (C) Quantification of g7-NPs localized in the liver, striatum, and cortex of WT (n=3) and R6/2 mice (n=3). Data represent mean (µg) ± SEM.


( D - E ) g7 - NPs in brain slices from R6 / 2 mice administered with a single ip injection and sacrificed after 24 hrs ( D , left ) or 2 weeks ( D , right ) and after multiple ip injections within 1 week ( E ) .

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(D-E) g7-NPs in brain slices from R6/2 mice administered with a single ip injection and sacrificed after 24 hrs (D, left) or 2 weeks (D, right) and after multiple ip injections within 1 week (E).


( F - I ) Representative confocal images of immunostaining against IBA1 ( F ) , GFAP ( G ) , calbindin ( H ) , and DARPP - 32 ( I ) on coronal sections of brains isolated from R6 / 2 mice ip injected with g7 - NPs and sacrificed at the indicated time points . White arrowheads indicate intracellular g7 - NPs .

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(F-I) Representative confocal images of immunostaining against IBA1 (F), GFAP (G), calbindin (H), and DARPP-32 (I) on coronal sections of brains isolated from R6/2 mice ip injected with g7-NPs and sacrificed at the indicated time points. White arrowheads indicate intracellular g7-NPs.


( A ) Representative confocal image ( crop ) of brain slices from R6 / 2 mice ip injected with rhodamine labeled g7 - NPs - NBD - Chol and sacrificed after 12 hrs , showing co - localization of NBD - Chol ( green ) and Rhodamine ( NPs , red ) . Scale bar : 5m .

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(A) Representative confocal image (crop) of brain slices from R6/2 mice ip injected with rhodamine labeled g7-NPs-NBD-Chol and sacrificed after 12 hrs, showing co-localization of NBD-Chol (green) and Rhodamine (NPs, red). Scale bar: 5m.


( B - C ) Representative confocal image ( low magnification ) of brain slices from R6 / 2 mice ip injected with g7 - NPs - NBD - Chol and sacrificed after 24 hrs ( B ) or 2 weeks ( C ) and relative co - localization of NBD - Chol and g7 - NPs . Scale bar : 10 m ( D ) g7 - NPs quantification in brain slices at the same time points in ( B - C ) . DAPI was used to counterstain nuclei . Data in ( D ) are expressed as number of g7 - NPs ( evaluated based their size ) for 100 m2 sem . Statistics : * * P < 0 , 01 ( 48 hrs vs 7 days ; 7 days vs 14 days ) , * * * P < 0 , 001 ( 24 hrs vs 7 days ; 7 days vs 14 days ) determined by one - way ANOVA followed by Newmann - Keuls multiple comparison test .

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(B-C) Representative confocal image (low magnification) of brain slices from R6/2 mice ip injected with g7-NPs-NBD-Chol and sacrificed after 24 hrs (B) or 2 weeks (C) and relative co-localization of NBD-Chol and g7-NPs. Scale bar: 10 m (D) g7-NPs quantification in brain slices at the same time points in (B-C). DAPI was used to counterstain nuclei. Data in (D) are expressed as number of g7-NPs (evaluated based their size) for 100 m2 sem. Statistics: **P<0,01 (48 hrs vs 7 days; 7 days vs 14 days), ***P<0,001 (24 hrs vs 7 days; 7 days vs 14 days) determined by one-way ANOVA followed by Newmann-Keuls multiple comparison test.


( A , D ) Spontaneous IPSCs ( A ) and spontaneous EPSCs ( D ) were recorded from striatal MSNs ( WTs = 52 ; R6 / 2 - untreated = 27 ; R6 / 2 - Chol = 29 ) at a holding potential of + 10 mV and - 70mV respectively . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled . ( B , E ) Amplitude - frequency histogram and average frequency ( inset ) of IPSCs ( B ) and EPSCs ( E ) from R6 / 2 - Chol , R6 / 2 - untreated and WT MSNs . ( C , F ) Cumulative inter - event histogram showing the release probability of IPSCs ( C ) and EPSCs ( F ) in all groups .

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(A,D) Spontaneous IPSCs (A) and spontaneous EPSCs (D) were recorded from striatal MSNs (WTs=52; R6/2-untreated=27; R6/2-Chol=29) at a holding potential of +10 mV and -70mV respectively. As no differences were found between R6/2 mice treated with saline (R6/2) or with empty g7-NPs (R6/2-emp), data were pooled. (B, E) Amplitude-frequency histogram and average frequency (inset) of IPSCs (B) and EPSCs (E) from R6/2-Chol, R6/2-untreated and WT MSNs. (C,F) Cumulative inter-event histogram showing the release probability of IPSCs (C) and EPSCs (F) in all groups.


( B ) Fall latency from an accelerating rotarod for 5 - 11 - week - old WT and R6 / 2 mice during cholesterol supplementation ( WT = 17 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 13 ) .

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(B) Fall latency from an accelerating rotarod for 5-11-week-old WT and R6/2 mice during cholesterol supplementation (WT=17; R6/2-untreated=21; R6/2-Chol=13).


( C ) Rearing activity in open field at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(C) Rearing activity in open field at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( D ) global activity in the open field test at 10 weeks of age ( WT = 14 ; R6 / 2 - untreated = 21 ; R6 / 2 - Chol = 15 ) .

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(D) global activity in the open field test at 10 weeks of age (WT=14; R6/2-untreated=21; R6/2-Chol=15).


( E ) Index of discrimination ( % ) in WT , R6 / 2 - untreated and R6 / 2 - Chol mice during disease progression , at 8 weeks of age ( WT = 24 ; R6 / 2 - untreated = 36 ; R6 / 2 - Chol = 21 ) , at 10 weeks of age ( WT = 25 ; R6 / 2 - untreated = 35 ; R6 / 2 - Chol = 20 ) and at 12 weeks of age ( WT = 24 ; R6 / 2 - untreated = 30 ; R6 / 2 - Chol = 19 ) ; the index above zero indicates a preference for the novel object ; the index below zero indicates a preference for the familiar object . As no differences were found between R6 / 2 mice treated with saline ( R6 / 2 ) or treated with empty g7 - NPs ( R6 / 2 - emp ) , data were pooled .

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(E) Index of discrimination (%) in WT, R6/2-untreated and R6/2-Chol mice during disease progression, at 8 weeks of age (WT=24; R6/2-untreated=36; R6/2-Chol=21), at 10 weeks of age (WT=25; R6/2-untreated=35; R6/2-Chol=20) and at 12 weeks of age (WT=24; R6/2-untreated=30; R6/2-Chol=19); the index above zero indicates a preference for the novel object; the index below zero indicates a preference for the familiar object. As no differences were found between R6/2 mice treated with saline (R6/2) or treated with empty g7-NPs (R6/2-emp), data were pooled.


( A - B ) protein levels ( A ) and relative densitometry quantification ( B ) of several synaptic proteins in triton - insoluble ( synaptic enriched ) fractions purified from total brains . Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6 / 2 mice by cholesterol supplementation .

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(A-B) protein levels (A) and relative densitometry quantification (B) of several synaptic proteins in triton-insoluble (synaptic enriched) fractions purified from total brains. Levels of PSD95 and NMDA receptor subunits GluN1 and GluN2B are rescued in R6/2 mice by cholesterol supplementation.


( C - D - E ) mRNA levels for Bdnf ( C ) , Snap25 ( D ) in cortex and hippocampus ; Complexin II ( E ) in cortex , hippocampus and striatum from a subset of WT ( n = 4 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - chol animals ( n = 3 ) . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D-E) mRNA levels for Bdnf (C), Snap25 (D) in cortex and hippocampus; Complexin II (E) in cortex, hippocampus and striatum from a subset of WT (n=4), R6/2-untreated (n=7) and R6/2-chol animals (n=3). As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( F - G ) Representative images of Nissl Staining ( F ) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT ( n = 7 ) , R6 / 2 ( n = 7 ) , R6 / 2 - emp ( n = 6 ) , R6 / 2 - Chol ( n = 8 )

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(F-G) Representative images of Nissl Staining (F) and ventricle volume revealed by Neurolucida Analysis at 12 weeks of age in WT (n= 7), R6/2 (n=7), R6/2-emp (n=6), R6/2-Chol (n=8)


( A - B ) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT ( n = 6 ) , R6 / 2 - untreated ( n = 7 ) and R6 / 2 - Chol ( n = 5 ) mice . Data represent mean ± SEM .

(A-B) Lathosterol and 24OHC measured by mass spectrometry in the brain of WT (n=6), R6/2-untreated (n=7) and R6/2-Chol (n=5) mice. Data represent mean ± SEM.


( C - D ) mRNA levels of hmgcr and fdft1 in liver and lung of WT ( n = 7 ) , R6 / 2 - untreated ( n = 8 ) and R6 / 2 - Chol ( n = 4 ) mice . As no differences were found between R6 / 2 mice treated with saline or treated with empty g7 - NPs , data were pooled .

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(C-D) mRNA levels of hmgcr and fdft1 in liver and lung of WT (n=7), R6/2-untreated (n=8) and R6/2-Chol (n=4) mice. As no differences were found between R6/2 mice treated with saline or treated with empty g7-NPs, data were pooled.


( E - F ) mRNA levels of inflammatory genes in liver and lung of the of WT ( n = 7 ) , R6 / 2 ( n = 4 ) , R6 / 2 - emp ( n = 4 ) and R6 / 2 - Chol ( n = 5 ) mice .

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(E-F) mRNA levels of inflammatory genes in liver and lung of the of WT (n=7), R6/2 (n=4), R6/2-emp (n=4) and R6/2-Chol (n=5) mice.


C , Quantification of telomeric allelic imbalances ( TAI ) in soft tissue and bone sarcoma .

C, Quantification of telomeric allelic imbalances (TAI) in soft tissue and bone sarcoma.


E - G , Ex vivo treatment of HRDhigh ( NMFH - 1 , OH931 , PM197 , USZ - 21 _ MFS2 and USZ - 21 _ UPS1 ) sarcoma models compared with HRDlow ( USZ - 20 _ REA1 , USZ - 21 _ LG1 , USZ - 22 _ EMC2 , USZ - 20 _ SFT1 and USZ - 21 _ CIC1 ) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin , doxorubicin and trabectedin .

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E-G, Ex vivo treatment of HRDhigh (NMFH-1, OH931, PM197, USZ-21_MFS2 and USZ-21_UPS1) sarcoma models compared with HRDlow (USZ-20_REA1, USZ-21_LG1, USZ-22_EMC2, USZ-20_SFT1 and USZ-21_CIC1) sarcoma models for 4 days with six doses of the chemotherapy agents oxaliplatin, doxorubicin and trabectedin.


M - O , HRDhigh sarcoma cell models and UWB1 . 289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin . Note that no sensitivity to olaparib in monotherapy was observed at 3

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M-O, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses olaparib alone and in combination with 1 nM trabectedin. Note that no sensitivity to olaparib in monotherapy was observed at 3 days


Q - S , HRDhigh sarcoma cell models and UWB1 . 289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin .

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Q-S, HRDhigh sarcoma cell models and UWB1.289 treated for 3 days with five doses adavosertib alone and in combination with 100 nM doxorubicin.


A , Immunofluorescence showing nuclear expression of the DNA damage marker γH2A . X ( magenta ) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1 . 289 cells . B , Immunofluorescence showing RAD51 nuclear foci ( magenta ) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model ( USZ - 21 _ LG1 ) . C , D , Quantification of γH2A . X ( C ) or RAD51 ( D ) nuclear intensity per cell compared to untreated control cells . E ,

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A, Immunofluorescence showing nuclear expression of the DNA damage marker γH2A.X (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination in HRDhigh and HRDlow sarcoma cell models as well as ovarian carcinoma UWB1.289 cells. B, Immunofluorescence showing RAD51 nuclear foci (magenta) upon 6 h treatment with 10 nM trabectedin and 100 nM olaparib in combination only in the HRDlow sarcoma cell model (USZ-21_LG1). C, D, Quantification of γH2A.X (C) or RAD51 (D) nuclear intensity per cell compared to untreated control cells. E,


E , Immunohistochemistry ( IHC ) in human tissue sections showing RAD51 ( brown ) expression in HRDlow but not HRDhigh sarcoma patients . Small - nucleated lymphocytes and / or proliferating cells appeared RAD51 - positive in HRDhigh models .

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E, Immunohistochemistry (IHC) in human tissue sections showing RAD51 (brown) expression in HRDlow but not HRDhigh sarcoma patients. Small-nucleated lymphocytes and/or proliferating cells appeared RAD51-positive in HRDhigh models.


F , Immunofluorescence in human tissue sections showing RAD51 ( magenta ) nuclear foci in an HRDlow but not an HRDhigh sarcoma patient .

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F, Immunofluorescence in human tissue sections showing RAD51 (magenta) nuclear foci in an HRDlow but not an HRDhigh sarcoma patient.


H , Magnetic resonance imaging ( MRI ) of LMS patient since primary diagnosis . Open arrowheads point at metastatic lesions .

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H, Magnetic resonance imaging (MRI) of LMS patient since primary diagnosis. Open arrowheads point at metastatic lesions.


J , RAD51 IHC in the metastatic patient ' s tissue . Compare RAD51 nuclear expression in normal tissue ( arrowhead points at gastric gland ) but not in tumorous gastric tissue .

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J, RAD51 IHC in the metastatic patient's tissue. Compare RAD51 nuclear expression in normal tissue (arrowhead points at gastric gland) but not in tumorous gastric tissue.


( A ) Genome - wide ubiquitin binding profiles identify numerous regions of H2B - ubiquitylation in WT cells and distinct sites of non - H2B ubiquitylation ( ' ubiquitin - hotspot ' , ub - hotspot , ub - HS ) which persist in h2b - K123R and rad6 ∆ strains , and increase in cdc48 mutants ( cdc48 - 6 ) . A 90 kb stretch of chromosome XIII ( ChrXIII ) is depicted . Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody ( see also Appendix Fig . S1A ) and enriched DNA was analyzed on NimbleGen arrays ( Chip - chip ) . DNA from non - specific IgG - ChIP experiments served as background control . Significantly enriched regions are marked by bars above the respective ChIP - chip tracks and are summarized in Dataset EV1 . Data represent means from two independent replicates , except for ubiquitin ( FK2 ) in rad6 ∆ and IgG - ChIP in WT ( n = 1 ) . All experiments , including those using cdc48 - 6 and other temperature - sensitive ( ts ) alleles , were performed at 30 ° C ( semipermissive temperature for ts - alleles ) unless stated otherwise .

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(A) Genome-wide ubiquitin binding profiles identify numerous regions of H2B-ubiquitylation in WT cells and distinct sites of non-H2B ubiquitylation ('ubiquitin-hotspot', ub-hotspot, ub-HS) which persist in h2b-K123R and rad6∆ strains, and increase in cdc48 mutants (cdc48-6). A 90 kb stretch of chromosome XIII (ChrXIII) is depicted. Chromatin immunoprecipitation was performed using the FK2 ubiquitin antibody (see also Appendix Fig. S1A) and enriched DNA was analyzed on NimbleGen arrays (Chip-chip). DNA from non-specific IgG-ChIP experiments served as background control. Significantly enriched regions are marked by bars above the respective ChIP-chip tracks and are summarized in Dataset EV1. Data represent means from two independent replicates, except for ubiquitin (FK2) in rad6∆ and IgG-ChIP in WT (n = 1). All experiments, including those using cdc48-6 and other temperature-sensitive (ts) alleles, were performed at 30°C (semipermissive temperature for ts-alleles) unless stated otherwise.