The violin plot depicting the smaller genome size of HAC isolates ( n = 820 ) than that of DPC ( n = 407 ) and HPC ( n = 262 ) . Inside the violins are the box and whisker plots , with the dot showing the median , the box showing the quartiles , and the whisker showing the 95 % percentiles . P - values were calculated using unpaired t - test ( * * , P < 0 . 05 ) .

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

The violin plot depicting the smaller genome size of HAC isolates (n=820) than that of DPC (n=407) and HPC (n=262). Inside the violins are the box and whisker plots, with the dot showing the median, the box showing the quartiles, and the whisker showing the 95% percentiles. P-values were calculated using unpaired t-test (**, P<0.05).


The survival curve for zebrafish inoculated with representative S . suis isolates from HAC and HPC . In total twenty - five representative isolates were tested , including fourteen isolates from HAC and ten healthy - pig isolates from HPC , as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control . The cumulative mortalities were calculated using the combined data from the isolates in each group . As expected , all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates ( P < 0 . 05 ) . Survival curve and statistical analysis were created using Kaplan - Meier and SPSS 23 . 0 . P - values were calculated using Log rank test . P < 0 . 05 was considered significant ( * * , P < 0 . 05 ; NS , P > 0 . 05 ) . No significant difference ( NS , P = 0 . 072 ) was found between HAC isolates and SC19 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 survival curve for zebrafish inoculated with representative S. suis isolates from HAC and HPC. In total twenty-five representative isolates were tested, including fourteen isolates from HAC and ten healthy-pig isolates from HPC, as well as the strain SC19 in HAC responsible for the 2005 human outbreak as a positive control. The cumulative mortalities were calculated using the combined data from the isolates in each group. As expected, all 14 isolates from HAC displayed a significantly higher mortality than the HPC isolates (P<0.05). Survival curve and statistical analysis were created using Kaplan-Meier and SPSS 23.0. P-values were calculated using Log rank test. P<0.05 was considered significant (**, P<0.05; NS, P>0.05). No significant difference (NS, P=0.072) was found between HAC isolates and SC19.


( C ) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype ( left ) . Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting ( right ) . Cropped blots are

O O O O O O O O O O O O O O O O O O O O O O O O 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) NSI mice homozygous for the Fah knockout displayed a lethal neonatal phenotype (left). Fah expression was detected in the livers of homozygous NSI mice with Fah knockout and Fah intact NSI mice by Western blotting (right). Cropped blots are shown


( F ) Representative PCR amplification of MYC , TP53R249S , KRASG12D , NRASG12D , CTNNB1S45F , BRAFV600E , AXIN1G652S , IL6 , PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC - PHHs ( Tumor ) and their corresponding overexpression vector plasmids as positive controls ( OCs )

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(F) Representative PCR amplification of MYC, TP53R249S, KRASG12D, NRASG12D, CTNNB1S45F, BRAFV600E, AXIN1G652S, IL6, PIK3CAE542K and CSF1RY969C in the genomic DNA of dissected primary tumors harvested from NSIF mice transplanted with OC-PHHs (Tumor) and their corresponding overexpression vector plasmids as positive controls (OCs)


( I ) Expression of MYC , p53 , and KRASG12D was detected in the iHCC samples ( n = 3 , biological replicates ) by Western blotting . Cropped blots are

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

(I) Expression of MYC, p53, and KRASG12D was detected in the iHCC samples (n=3, biological replicates) by Western blotting. Cropped blots are shown


( B ) Genomic DNA of colonies that were derived from single cells of iHCC1 - 1 and iHCC2 - 1 tumor samples were extracted for PCR amplification of MYC , TP53R249S , and

O O O O O O O O O O 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) Genomic DNA of colonies that were derived from single cells of iHCC1-1 and iHCC2-1 tumor samples were extracted for PCR amplification of MYC, TP53R249S, and KRASG12D


( B ) Volcano plot of RNA - seq data showing genes with differential expression ( 1 , 591 upregulated and 2 , 243 downregulated genes , fold change ≥ 2 and P value ≤ 0 . 05 ) in iHCC cells compared to PHHs . TP53 , KRAS , MYC , MUC1 , and AFP were highly expressed in iHCC cells , whereas hepatocyte signature markers , including FOXA2 , FOXA3 , ATF5 , HNF4A , and ALB were highly expressed in

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of RNA-seq data showing genes with differential expression (1,591 upregulated and 2,243 downregulated genes, fold change≥2 and P value≤0.05) in iHCC cells compared to PHHs. TP53, KRAS, MYC, MUC1, and AFP were highly expressed in iHCC cells, whereas hepatocyte signature markers, including FOXA2, FOXA3, ATF5, HNF4A, and ALB were highly expressed in PHHs


( D ) Heat map analysis of the log2 ( RPKM ) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells . These genes were classified into three categories based on their

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

(D) Heat map analysis of the log2(RPKM) values of selected genes found to be highly expressed or downregulated in PHHs and iHCC cells. These genes were classified into three categories based on their functions


( A ) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs . Within these 20 genes , seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA - HCC ( TCGA - LIHC )

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

(A) Heat map shows the top 20 genes encoding cell surface markers found to be highly expressed in iHCC samples compared to the corresponding PHHs. Within these 20 genes, seven genes highlighted in red were selected because their expression was correlated with poor prognosis of HCC patients based on TCGA-HCC (TCGA-LIHC) analysis


( B ) Representative cytotoxicity CARFAP - T and CARMUC1 - T cells assessed by coincubation with iHCC2 - 1 cells . P = 0 . 0038 ( CARFAP - T vs . mock T ) , and P < 0 . 0478 ( CARMUC1 - T vs . mock - T ) . P values were determined by paired t - test , * * P ≤ 0 . 05 . Cells were seeded at E : T ratios ranging from 4 : 1 to 1 : 4 ; data are presented as the mean ± SD . N - numbers refer to biological replicates ( n = 3 )

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(B) Representative cytotoxicity CARFAP-T and CARMUC1-T cells assessed by coincubation with iHCC2-1 cells. P=0.0038 (CARFAP-T vs. mock T), and P<0.0478 (CARMUC1-T vs. mock-T). P values were determined by paired t-test, **P≤0.05. Cells were seeded at E: T ratios ranging from 4:1 to 1:4; data are presented as the mean ± SD. N-numbers refer to biological replicates (n=3)


( D ) Clonogenic assay of iHCC1 - 1 cells treated with a MEK inhibitor ( trametinib ) and / or a CDK4 / 6 inhibitor ( palbociclib ) ; Representative images were shown based on three biological

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

(D) Clonogenic assay of iHCC1-1 cells treated with a MEK inhibitor (trametinib) and/or a CDK4/6 inhibitor (palbociclib); Representative images were shown based on three biological replicates


( F ) Clonogenic assays ( Top ) of iHCC1 - 1 cells treated with XL413 . The data are the representative of three biological replications . ( Bottom ) Growth curves of iHCC in xenografts ( n = 5 ) treated with PBS or XL413 at 50 mm3 tumor volume . P = 0 . 0486 ( XL413 vs . PBS group ) by two - way ANOVA with Sidak ' s multiple comparison test at Day24 , * P ≤ 0 . 05 . Data are presented as the mean ± SD . N - numbers refer to biological replicates .

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 B-CONTROLLED_VAR 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

(F) Clonogenic assays (Top) of iHCC1-1 cells treated with XL413. The data are the representative of three biological replications. (Bottom) Growth curves of iHCC in xenografts (n=5) treated with PBS or XL413 at 50 mm3 tumor volume. P=0.0486 (XL413 vs. PBS group) by two-way ANOVA with Sidak's multiple comparison test at Day24, *P≤0.05. Data are presented as the mean ± SD. N-numbers refer to biological replicates.


( a ) Silver - stained gel image of the affinity - purified complexes of PSPC1 , SFPQ and NONO . The respective proteins are marked along with AcTEV protease used for the elution of purified proteins .

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(a) Silver-stained gel image of the affinity-purified complexes of PSPC1, SFPQ and NONO. The respective proteins are marked along with AcTEV protease used for the elution of purified proteins.


( f ) Western blotting of affinity purified complexes of PSPC1 , SFPQ and NONO with SWI / SNF antibodies . cBAF , PBAF , ncBAF specific , as well as common subunits , are marked .

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(f) Western blotting of affinity purified complexes of PSPC1, SFPQ and NONO with SWI/SNF antibodies. cBAF, PBAF, ncBAF specific, as well as common subunits, are marked.


( a ) Silver - stained gel image of the affinity - purified complexes of ARID1A and ARID1B . The bait and a few other SWI / SNF subunits are marked as per their molecular weight .

(a) Silver-stained gel image of the affinity-purified complexes of ARID1A and ARID1B. The bait and a few other SWI/SNF subunits are marked as per their molecular weight.


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( c ) SWI / SNF

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

Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (c) SWI/SNF subunits


Heat maps showing the enriched proteins ( dNSAFavg * 1000 ) normalized to the bait identified in MudPIT analysis focusing on various ( e ) PSPs .

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

Heat maps showing the enriched proteins (dNSAFavg*1000) normalized to the bait identified in MudPIT analysis focusing on various (e) PSPs.


( f ) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components . SMARCA4 was used as a positive control for purification .

(f) Western blotting of affinity purified complexes of ARID1A and ARID1B with bonafide paraspeckle components. SMARCA4 was used as a positive control for purification.


( a ) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo - FLAG - tagged ARID1B . Input and eluted samples were resolved on a gel followed by western blotting with various antibodies . Western with SWI / SNF subunits - SMARCA4 and SMARCD1 were used as the control for purification , while beta - actin was used as a loading control .

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

(a) Halo purification was performed from nuclear extracts of scramble and various knockdown HEK293FRT cells expressing Halo-FLAG-tagged ARID1B. Input and eluted samples were resolved on a gel followed by western blotting with various antibodies. Western with SWI/SNF subunits- SMARCA4 and SMARCD1 were used as the control for purification, while beta-actin was used as a loading control.


( e , f ) ( e ) RIP qPCR and ( f ) PCR analysis performed using an antibody against endogenous ARID1B . for monitoring NEAT1 _ 2 levels . RIP with NONO and IgG was used as a positive and negative control respectively . GAPDH levels were also assessed in the assay . Data are expressed as a percent of Input .

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

(e, f) (e) RIP qPCR and (f) PCR analysis performed using an antibody against endogenous ARID1B. for monitoring NEAT1_2 levels. RIP with NONO and IgG was used as a positive and negative control respectively. GAPDH levels were also assessed in the assay. Data are expressed as a percent of Input.


( c ) smFISH analysis was performed to detect NEAT1 signal in the nucleus . The scale bar corresponds to 10 µm . Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control . The number of nuclei ( n ) used for quantification is mentioned .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) smFISH analysis was performed to detect NEAT1 signal in the nucleus. The scale bar corresponds to 10 µm. Graph showing the FISH signal quantification across the knockdowns in comparison to scramble control. The number of nuclei (n) used for quantification is mentioned.


A Determination of a time window for 2a2iL treatment needed to induce a naïve - like pluripotency state in primed hPSCs . One day treatment with 2a followed by the addition of 2a2iL for up to 14 days . Scale bars : 200 µm .

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

A Determination of a time window for 2a2iL treatment needed to induce a naïve-like pluripotency state in primed hPSCs. One day treatment with 2a followed by the addition of 2a2iL for up to 14 days. Scale bars: 200 µm.


C Characteristics of 2a2iL - RH6 cells represented by dome - shaped colonies , ALP activity , and a normal male karyotype ( 46 , XY ) . 2a2iL - RH6 expressed the common pluripotency markers OCT4 , NANOG , SOX2 , and TRA - 1 - 81 , but not SSEA1 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) and Propidium iodide ( PI ) . Scale bars : 200 , 100 , and 50 µm in the inset .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Characteristics of 2a2iL-RH6 cells represented by dome-shaped colonies, ALP activity, and a normal male karyotype (46, XY). 2a2iL-RH6 expressed the common pluripotency markers OCT4, NANOG, SOX2, and TRA-1-81, but not SSEA1. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) and Propidium iodide (PI). Scale bars: 200, 100, and 50 µm in the inset.


A Doubling time in pRH6 versus 2a2iL - RH6 . * * P < 0 . 01 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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

A Doubling time in pRH6 versus 2a2iL-RH6. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


B Cloning efficiency in the presence ( + ) and absence ( − ) of ROCK inhibitor ( ROCKi ) in pRH6 versus 2a2iL - RH6 . ROCKi ( − ) * P < 0 . 05 , ROCKi ( + ) * P < 0 . 01 ( t - test ) , data presented as mean ± SD ( n = 3 ) .

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

B Cloning efficiency in the presence (+) and absence (−) of ROCK inhibitor (ROCKi) in pRH6 versus 2a2iL-RH6. ROCKi (−)*P<0.05, ROCKi (+) *P<0.01 (t-test), data presented as mean ± SD (n=3).


C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL - RH6 . The ratio of firefly luciferase expression to Renilla luciferase expression ( expressed from co - transfected plasmid ) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL - RH6 . OCT4 PE - Luc * * * P < 0 . 001 , OCT4 DE - Luc * P < 0 . 05 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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C Firefly luciferase expression of constructs that contain distal and proximal enhancers of OCT4 after transfection of pRh6 and 2a2iL-RH6. The ratio of firefly luciferase expression to Renilla luciferase expression (expressed from co-transfected plasmid) measured 48 h after transfection showed proximal enhancer activity in pRH6 and distal enhancer activation in 2a2iL-RH6. OCT4 PE-Luc ***P<0.001, OCT4 DE-Luc *P<0.05 (t-test). Data presented as mean ± SD (n=3).


D qRT - PCR analysis of pluripotency - related genes in pRH6 and 2a2iL - RH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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

D qRT-PCR analysis of pluripotency-related genes in pRH6 and 2a2iL-RH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


E Immunofluorescence staining for several naïve specific markers in 2a2iL - RH6 . Scale bar : 100 µm .

E Immunofluorescence staining for several naïve specific markers in 2a2iL-RH6. Scale bar: 100 µm.


F Western blot analysis of naïve - related pluripotency markers in pRH6 and 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

F Western blot analysis of naïve-related pluripotency markers in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence staining against pRH5 and 2a2iL - RH5 ( 46 , XX ) for H3K27me3 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Reactivation of inactive X - chromosome in female 2a2iL - RH5 was confirmed by the lack of H3K27me3 nuclear foci . Scale bar : 50 µm .

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G Immunofluorescence staining against pRH5 and 2a2iL-RH5 (46, XX) for H3K27me3. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Reactivation of inactive X-chromosome in female 2a2iL-RH5 was confirmed by the lack of H3K27me3 nuclear foci. Scale bar: 50 µm.


A Phase contrast images of embryoid bodies ( EBs ) from 2a2iL - RH6 cells that differentiated into neurons . Scale bars : 200 and 100 µm in the inset .

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A Phase contrast images of embryoid bodies (EBs) from 2a2iL-RH6 cells that differentiated into neurons. Scale bars: 200 and 100 µm in the inset.


B qRT - PCR analysis of 20 day - EBs from pRH6 and 2a2iL - RH6 for markers of different embryonic germ layer derivatives . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

B qRT-PCR analysis of 20 day-EBs from pRH6 and 2a2iL-RH6 for markers of different embryonic germ layer derivatives. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Immunofluorescence images of differentiated 2a2iL - RH6 cells that depict expression of markers for neuron - like cells ( TUJ - 1 ) , cardiac - muscle - like cells ( CTNT ) , and hepatocyte - like cells ( SOX17 ) . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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

C Immunofluorescence images of differentiated 2a2iL-RH6 cells that depict expression of markers for neuron-like cells (TUJ-1), cardiac-muscle-like cells (CTNT), and hepatocyte-like cells (SOX17). Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


D 2a2iL - RH6 cells form teratomas that consist of three embryonic germ layer - derived tissues . ( i ) Rosette structures ( ectoderm ) , ( ii ) cartilage ( mesoderm ) , and ( iii ) primitive gut ( endoderm ; scale bar 50 µm ) .

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

D 2a2iL-RH6 cells form teratomas that consist of three embryonic germ layer-derived tissues. (i) Rosette structures (ectoderm), (ii) cartilage (mesoderm), and (iii) primitive gut (endoderm; scale bar 50 µm).


E From left to right : Phase contrast and GFP representation in 2a2iL - RH6 - eGFP ; presence of injected 2a2iL - RH6 - eGFP in the mouse blastocyst and integration into the inner cell mass ( ICM ) after 3 , 24 and 48 h incubation periods . Scale bars : 200 , 100 , and 50 µm in the inset .

E From left to right: Phase contrast and GFP representation in 2a2iL-RH6-eGFP; presence of injected 2a2iL-RH6-eGFP in the mouse blastocyst and integration into the inner cell mass (ICM) after 3, 24 and 48 h incubation periods. Scale bars: 200, 100, and 50 µm in the inset.


F Fluorescence images of tissue - sections from human - mouse chimeras in E10 . 5 showed cells positive for GFP ( top ) and human nuclear antigen ( HNA ) ( bottom ) in different parts of the embryos , including the head ( i ) , trunk ( ii ) , and tail ( iii ) with the higher magnification in the right panels . Scale bars : 200 and 100 µm in the inset .

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F Fluorescence images of tissue-sections from human-mouse chimeras in E10.5 showed cells positive for GFP (top) and human nuclear antigen (HNA) (bottom) in different parts of the embryos, including the head (i), trunk (ii), and tail (iii) with the higher magnification in the right panels. Scale bars: 200 and 100 µm in the inset.


G Genomic PCR analysis for a human specific mitochondrial sequence ( 391 bp ) in human - mouse chimeras .

G Genomic PCR analysis for a human specific mitochondrial sequence (391 bp) in human-mouse chimeras.


H Immunofluorescence staining against NANOG shows lack of expression in E10 . 5 human - mouse chimera tissue sections . Scale bars : 200 and 100 µm in the inset .

H Immunofluorescence staining against NANOG shows lack of expression in E10.5 human-mouse chimera tissue sections. Scale bars: 200 and 100 µm in the inset.


I Immunofluorescence staining against TUJ1 , BRACHYURY ( BRA ) , and SOX17 in E10 . 5 human - mouse chimera tissue sections represent differentiation - derivatives of 2a2iL - RH6 . Scale bars : 200 and 100 µm in the inset .

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I Immunofluorescence staining against TUJ1, BRACHYURY (BRA), and SOX17 in E10.5 human-mouse chimera tissue sections represent differentiation-derivatives of 2a2iL-RH6. Scale bars: 200 and 100 µm in the inset.


B Morphology of ICM - outgrowths by protocols 1 and 2 during the first 4 days after seeding . nRH11 - 14 ( naïve Royan H11 , 12 , 13 , and 14 ) : naïve hESC lines produced from human embryos using 2a2iL condition . Scale bar : 100 µm .

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B Morphology of ICM-outgrowths by protocols 1 and 2 during the first 4 days after seeding. nRH11-14 (naïve Royan H11, 12, 13, and 14): naïve hESC lines produced from human embryos using 2a2iL condition. Scale bar: 100 µm.


C Comparison of the efficiency of naïve - like hESCs derivation from human blastocysts by two protocols .

C Comparison of the efficiency of naïve-like hESCs derivation from human blastocysts by two protocols.


D Representative example of naïve - like hESCs ( nRH13 ) at passage 33 . Dome - shaped colonies were stained for ALP activity and karyotyped . Scale bars : 200 and 100 µm in the inset .

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D Representative example of naïve-like hESCs (nRH13) at passage 33. Dome-shaped colonies were stained for ALP activity and karyotyped. Scale bars: 200 and 100 µm in the inset.


E Expression of the common pluripotency markers OCT4 , SOX2 , and TRA - 1 - 81 , and naïve - related pluripotency markers NANOG , KLF17 , and TFCP2L1 in nRH13 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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E Expression of the common pluripotency markers OCT4, SOX2, and TRA-1-81, and naïve-related pluripotency markers NANOG, KLF17, and TFCP2L1 in nRH13. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


F qRT - PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line , pRH6 . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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F qRT-PCR analysis for pluripotency markers in nRH13 in comparison to a primed hESC line, pRH6. *P<0.05, **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Expression of lineage markers after embryoid body ( EB ) formation and spontaneous differentiation in nRH13 cells . * P < 0 . 05 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

G Expression of lineage markers after embryoid body (EB) formation and spontaneous differentiation in nRH13 cells. *P<0.05, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


A Pairwise Pearson correlation coefficient heatmap of transcriptome profiles ( RNA - seq ) from 2a2iL - , pRH5 , and pRH6 cell lines . Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples .

A Pairwise Pearson correlation coefficient heatmap of transcriptome profiles (RNA-seq) from 2a2iL-, pRH5, and pRH6 cell lines. Each cell of the heatmap shows the pairwise Pearson correlation coefficient heatmap of genes with the largest expression variances in a pair of samples.


C The volcano plot shows a differential expression between 2a2iL - versus primed hPSCs lines . X - axis shows the log2 fold - change , and the y - axis shows the negative log10 scaled adjusted P - value ( Benjamini - Hochberg ) . Red and blue points represent up - regulated genes in 2a2iL - and primed cells , respectively ( absolute log - 2 fold - change > 1 , adjusted P - value < 0 . 05 ) . Gray points indicate non - significant genes between these states .

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C The volcano plot shows a differential expression between 2a2iL- versus primed hPSCs lines. X-axis shows the log2 fold-change, and the y-axis shows the negative log10 scaled adjusted P-value (Benjamini-Hochberg). Red and blue points represent up-regulated genes in 2a2iL- and primed cells, respectively (absolute log-2 fold-change >1, adjusted P-value <0.05). Gray points indicate non-significant genes between these states.


A Position of TGF - β family members in the volcano plot of differentially expressed genes in 2a2iL - and primed hPSCs . X - axis shows log2 fold - change and y - axis shows negative log10 adjusted P - value < 0 . 05 ( Adjusted P - value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate ) . Red and blue points represent up - regulated genes in 2a2iL - and primed hPSCs , respectively . Genes with no significant expression change are shown in gray .

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A Position of TGF-β family members in the volcano plot of differentially expressed genes in 2a2iL- and primed hPSCs. X-axis shows log2 fold-change and y-axis shows negative log10 adjusted P-value <0.05 (Adjusted P-value was obtained using the Benjamini and Hochberg correction to determine the false discovery rate). Red and blue points represent up-regulated genes in 2a2iL- and primed hPSCs, respectively. Genes with no significant expression change are shown in gray.


B qRT - PCR analysis of several TGF - β - related genes in pRH6 and 2a2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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B qRT-PCR analysis of several TGF-β-related genes in pRH6 and 2a2iL-RH6. **P<0.01, ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


C Inhibition of the TGF - β pathway by two small molecules , SB43 and A83 . Left panel : morphology of naïve - like cells three days after treatment . Right panel : morphology of treated cells after passaging . Scale bar : 100 µm .

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C Inhibition of the TGF-β pathway by two small molecules, SB43 and A83. Left panel: morphology of naïve-like cells three days after treatment. Right panel: morphology of treated cells after passaging. Scale bar: 100 µm.


D Heatmap shows expression of putative LRH - 1 target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs , with a range from dark blue for lower expression to dark red for higher expression . E Heatmap shows expression of putative RARγ target genes in this study ' s transcriptome profiles . Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs , with a range from dark blue for lower expression to dark red for higher expression .

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D Heatmap shows expression of putative LRH-1 target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression. E Heatmap shows expression of putative RARγ target genes in this study's transcriptome profiles. Colors indicate log2 fold change of marker genes in the naïve versus primed hPSCs, with a range from dark blue for lower expression to dark red for higher expression.


A qRT - PCR analysis of ligands associated with TGF - β signaling during conversion of primed to naïve - like pluripotency by 2a2iL induction protocol at days 1 , 3 , and 5 . 2iL was selected as a control group . * * * P < 0 . 0001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

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A qRT-PCR analysis of ligands associated with TGF-β signaling during conversion of primed to naïve-like pluripotency by 2a2iL induction protocol at days 1, 3, and 5. 2iL was selected as a control group. ***P<0.0001. One-way ANOVA. Data presented as mean ± SD (n=3).


B Inhibition of TGF - β pathway using SB43 during conversion of primed to naïve pluripotency . Scale bar : 100 µm .

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B Inhibition of TGF-β pathway using SB43 during conversion of primed to naïve pluripotency. Scale bar: 100 µm.


C Morphology of TGFβ2iL - treated RH6 cells along with ALP activity . Scale bar : 200 µm .

C Morphology of TGFβ2iL-treated RH6 cells along with ALP activity. Scale bar: 200 µm.


D qRT - PCR analysis of naïve related genes in pRH6 , 2a2iL - , and TGFβ2iL - RH6 . * * P < 0 . 01 , * * * P < 0 . 001 . One - way ANOVA . Data presented as mean ± SD ( n = 3 ) .

D qRT-PCR analysis of naïve related genes in pRH6, 2a2iL-, and TGFβ2iL-RH6. **P<0.01, ***P<0.001. One-way ANOVA. Data presented as mean ± SD (n=3).


E Bar plot representing cell proliferation rates of 2a2iL - and TGFβ2iL - RH6 by cell counts . * * P < 0 . 01 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

E Bar plot representing cell proliferation rates of 2a2iL- and TGFβ2iL-RH6 by cell counts. **P<0.01 (t-test). Data presented as mean ± SD (n=3).


F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL - RH6 based on the number of ALP positive colonies . * * * P < 0 . 001 ( t - test ) . Data presented as mean ± SD ( n = 3 ) .

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F Bar plot of the cloning efficiency of 2a2iL and TGFβ2iL-RH6 based on the number of ALP positive colonies. ***P<0.001 (t-test). Data presented as mean ± SD (n=3).


G Immunofluorescence images show the expression of naïve pluripotency markers in TGF - β2iL - RH6 . Nuclei were stained with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) . Scale bar : 100 µm .

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G Immunofluorescence images show the expression of naïve pluripotency markers in TGF-β2iL-RH6. Nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI). Scale bar: 100 µm.


A . In the Discovery cohort , Kaplan - Meier estimates of disease - free survival ( DFS ) in years ( y ) in patients with low ( red ) versus high ( blue ) nuclear PRMT5 expression ( upper panel ) and in 2 groups of patients according to their treatment . Patients treated with tamoxifen ( Tam , middle panel ) and with aromatase inhibitors ( AIs , lower panel ) . P - values are calculated with the logrank test . B . The same analyses were performed in the Validation cohort .

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A. In the Discovery cohort, Kaplan-Meier estimates of disease-free survival (DFS) in years (y) in patients with low (red) versus high (blue) nuclear PRMT5 expression (upper panel) and in 2 groups of patients according to their treatment. Patients treated with tamoxifen (Tam, middle panel) and with aromatase inhibitors (AIs, lower panel). P-values are calculated with the logrank test. B. The same analyses were performed in the Validation cohort.


A . GST and GST - PRMT5 fusion proteins were incubated with in vitro - translated ERα , the interaction was then visualized by Western blotting using an anti - ERα antibody . The corresponding Coomassie - stained gel is shown in the right panel .

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A. GST and GST-PRMT5 fusion proteins were incubated with in vitro-translated ERα, the interaction was then visualized by Western blotting using an anti-ERα antibody. The corresponding Coomassie-stained gel is shown in the right panel.


B . ERα / PRMT5 interaction was assessed by co - immunoprecipitation of MCF7 cell extracts treated or not with E2 for the indicated times , using an anti - PRMT5 antibody . The presence of PRMT5 , ERα and β - actin was evaluated by Western blot analysis .

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B. ERα/PRMT5 interaction was assessed by co-immunoprecipitation of MCF7 cell extracts treated or not with E2 for the indicated times, using an anti-PRMT5 antibody. The presence of PRMT5, ERα and β-actin was evaluated by Western blot analysis.


C . The same experiment was performed for MCF7 cells treated or not with tamoxifen ( Tam ) for the indicated times . The scale bar is 10µM .

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C. The same experiment was performed for MCF7 cells treated or not with tamoxifen (Tam) for the indicated times. The scale bar is 10µM.


D . Proximity Ligation Assay ( PLA ) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2 , Tam 1 µM or both for 6 h . After fixation , PLA experiments were performed to evaluate the interactions between ERα / PRMT5 using specific antibodies . The detected dimers are represented by red dots . The nuclei were counterstained with mounting medium containing DAPI ( blue ) ( Obj : X60 ) . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test .

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D. Proximity Ligation Assay (PLA) to detect interactions between ERα and PRMT5 in MCF7 cells treated with E2, Tam 1 µM or both for 6 h. After fixation, PLA experiments were performed to evaluate the interactions between ERα/PRMT5 using specific antibodies. The detected dimers are represented by red dots. The nuclei were counterstained with mounting medium containing DAPI (blue) (Obj:X60). The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.


F . Paraffin - embedded sections from a fresh tumor were incubated with or without G595 , and PRMT5 and SDMA expression were assessed by IHC .

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F. Paraffin-embedded sections from a fresh tumor were incubated with or without G595, and PRMT5 and SDMA expression were assessed by IHC.


C . MCF7 cells were treated with or without E2 , Tam or both for 6 h . ERα methylation was determined by PLA using an anti - ERα and a pan methyl recognizing symmetrical dimethylation ( SDMA ) . The scale bar is 10µM . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test .

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C. MCF7 cells were treated with or without E2, Tam or both for 6 h. ERα methylation was determined by PLA using an anti-ERα and a pan methyl recognizing symmetrical dimethylation (SDMA). The scale bar is 10µM. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test.


D . The same experiment was performed with G595 to verify that ERα methylation was PRMT5 - dependent . Red dots reflect ERα methylation . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . The sale bar is 40µM . Western blotting was performed to assess ERα , PRMT5 expression and the global profile of methylation using a pan SDMA antibody .

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D. The same experiment was performed with G595 to verify that ERα methylation was PRMT5-dependent. Red dots reflect ERα methylation. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. The sale bar is 40µM. Western blotting was performed to assess ERα, PRMT5 expression and the global profile of methylation using a pan SDMA antibody.


E . MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti - ERα antibody . ERα , SDMA and GAPDH expression was evaluated in the input .

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E. MCF7 cells were treated as in C and lysates were immunoprecipitated with a pan SDMA antibody and immunoblotted with an anti-ERα antibody. ERα, SDMA and GAPDH expression was evaluated in the input.


B . MCF7 cells were treated with Tam for 6 h in the presence or absence of G595 , cell extracts were then subjected to ChIP assay using anti - ERα or anti - SMRT antibodies . The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters . The results are expressed relative to the signal obtained from input chromatin . The mean ± SEM of at least three experiments is shown . P - values were determined using a Student t - test .

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B. MCF7 cells were treated with Tam for 6 h in the presence or absence of G595, cell extracts were then subjected to ChIP assay using anti-ERα or anti-SMRT antibodies. The precipitated DNA fragments were used for qPCR analysis using specific primers for the indicated promoters. The results are expressed relative to the signal obtained from input chromatin. The mean ± SEM of at least three experiments is shown. P-values were determined using a Student t-test.


C . MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT - QPCR with specific primers for GREB1 . The values were normalized against 28S mRNA and represent the ± SEM of three experiments .

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C. MCF7 cells were treated with Tam for 6 h and total RNA was prepared and cDNAs were analyzed by RT-QPCR with specific primers for GREB1. The values were normalized against 28S mRNA and represent the ± SEM of three experiments.


B . MCF7 cells were treated with Tam for 6h in the presence or in absence of G595 , then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins . The scale bar is 10µM . Quantification was performed as in Figure 2 . The mean ± SEM of three independent experiments is shown . P - values were determined using a Student t - test . Western blotting was performed to assess ERα , SIRT2 , SAP18 and GAPDH expression and the global profile of SDMA .

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B. MCF7 cells were treated with Tam for 6h in the presence or in absence of G595, then ERα interaction with SIRT2 and SAP18 was determined by PLA using specific antibodies against both proteins. The scale bar is 10µM. Quantification was performed as in Figure 2. The mean ± SEM of three independent experiments is shown. P-values were determined using a Student t-test. Western blotting was performed to assess ERα, SIRT2, SAP18 and GAPDH expression and the global profile of SDMA.


A . ERα methylation SDMA was studied by PLA , and PRMT5 and MEP50 expression were analyzed by IHC on formalin - fixed MCF7 xenografts treated or not with Tam ( Obj : X40 ) . The scale bar is 50 µM . Histogram quantifying the number of dots / cells and the H - scores for nuclear PRMT5 and MEP50 are presented on the right . The mean ± SEM of 7 tumors is shown . P - values were determined using a Student t - test .

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A. ERα methylation SDMA was studied by PLA, and PRMT5 and MEP50 expression were analyzed by IHC on formalin-fixed MCF7 xenografts treated or not with Tam (Obj: X40). The scale bar is 50 µM. Histogram quantifying the number of dots/cells and the H-scores for nuclear PRMT5 and MEP50 are presented on the right. The mean ± SEM of 7 tumors is shown. P-values were determined using a Student t-test.


B . ERα methylation SDMA , PRMT5 and MEP50 expression were studied as in A on a Tam - resistant formalin - fixed PDX model HBCx - 34 Tam R treated or not with Tam . ERα methylation SDMA was analyzed as described above and the H - score for nuclear PRMT5 and MEP50 was evaluated as in A . The scale bar is 50 µM . The mean ± SEM of 3 tumors is shown . P - values were determined using a Student t - test .

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B. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed PDX model HBCx-34 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 3 tumors is shown. P-values were determined using a Student t-test.


C . ERα methylation SDMA , PRMT5 and MEP50 expression were studied as in A on a Tam - resistant formalin - fixed the PDX model HBCx - 22 Tam R treated or not with Tam . ERα methylation SDMA was analyzed as described above and the H - score for nuclear PRMT5 and MEP50 was evaluated as in A . The scale bar is 50 µM . The mean ± SEM of 7 tumors is shown .

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C. ERα methylation SDMA, PRMT5 and MEP50 expression were studied as in A on a Tam-resistant formalin-fixed the PDX model HBCx-22 Tam R treated or not with Tam. ERα methylation SDMA was analyzed as described above and the H-score for nuclear PRMT5 and MEP50 was evaluated as in A. The scale bar is 50 µM. The mean ± SEM of 7 tumors is shown.


A . For each tumor , we analyzed the level of ERα SDMA methylation by PLA . The H - score for nuclear PRMT5 expression and the number of dots / cell are listed under the figures . The scale bar is 20µM .

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A. For each tumor, we analyzed the level of ERα SDMA methylation by PLA. The H-score for nuclear PRMT5 expression and the number of dots/cell are listed under the figures. The scale bar is 20µM.


RFP + ( G1 phase ) , GFP + ( G2 phase ) , and GFP + / RFP + ( S phase ) RPE - FUCCI cells were sorted by flow cytometry , RNA was extracted and analyzed using qRT - PCR ( n = 3 ) . Expression is normalized to GAPDH mRNA . Bars show mean ± SD .

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RFP+ (G1 phase), GFP+ (G2 phase), and GFP+/RFP+ (S phase) RPE-FUCCI cells were sorted by flow cytometry, RNA was extracted and analyzed using qRT-PCR (n=3). Expression is normalized to GAPDH mRNA. Bars show mean ± SD.


RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS . Nocodazole was added after 4 h to arrest cells in mitosis . Alternatively , RO3306 inhibitor was added to prevent mitotic entry . Where indicated sample was collected by mitotic shake - off ( MS ) to obtain pure mitotic population . Samples were analyzed by immunoblotting ( upper panel ) . Staining for pS10 - H3 was used as a marker of mitosis , TFIIH as a loading control . In parallel , cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining . Representative plot is shown , M indicates percentage of mitotic cells ( middle panel ) . Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample ( lower panel ) . Bars show median ± SD .

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RPE cells were released for indicated times from a thymidine block and collected cells were analyzed by immunoblotting and FACS. Nocodazole was added after 4 h to arrest cells in mitosis. Alternatively, RO3306 inhibitor was added to prevent mitotic entry. Where indicated sample was collected by mitotic shake-off (MS) to obtain pure mitotic population. Samples were analyzed by immunoblotting (upper panel). Staining for pS10-H3 was used as a marker of mitosis, TFIIH as a loading control. In parallel, cell cycle progression was followed by flow cytometry using DAPI and mpm2 staining. Representative plot is shown, M indicates percentage of mitotic cells (middle panel). Signal of the rabbit FAM110A antibody was quantified in three independent replicates and normalized to the loading and to the thymidine arrested sample (lower panel). Bars show median ± SD.


RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA , probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ - tubulin , and were analyzed by confocal microscopy . Shown is maximal projection of representative mitotic ( upper panel ) and interphase ( lower panel ) cells . Scale bars indicate 10 μm .

RPE cells transfected with control or FAM110A siRNA were fixed with 4 % PFA, probed with rabbit polyclonal antibody against FAM110A and mouse monoclonal to γ-tubulin, and were analyzed by confocal microscopy. Shown is maximal projection of representative mitotic (upper panel) and interphase (lower panel) cells. Scale bars indicate 10 μm.


Quantification of the mitotic index in three experiments from ( A ) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * p < 0 . 05 , * * p < 0 . 005 ) .

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Quantification of the mitotic index in three experiments from (A) and in RPE cells transfected with siRNAs to FAM110B and CSPP1 that were analyzed in parallel. Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (* p<0.05, ** p<0.005).


RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase . Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown . Scale bars indicate 20 μm , arrowheads point to misaligned chromosomes . More than 30 metaphases were evaluated per condition ( n = 3 ) . Bars indicate median ± SD . Statistical significance was determined by two - tailed t - test , ( * * * p < 0 . 001 , * p < 0 . 05 ) . Quantification of lagging chromosomes in anaphase cells from ( E ) . More than 30 anaphases were evaluated per condition ( n = 3 ) . Representative images of abnormal anaphase are shown , scale bars indicate 20 μm , arrowheads point to incorrectly segregating chromosomes . Statistical significance was determined by two - tailed t - test . Bars indicate median ± SD ( * * * p < 0 . 001 ) .

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RPE cells transfected with control or FAM110A siRNA were grown for 48 h and treated with MG132 30 min prior fixation to trap mitotic cells in metaphase. Impaired chromosomal alignment and multipolar spindles were scored and representative images are shown. Scale bars indicate 20 μm, arrowheads point to misaligned chromosomes. More than 30 metaphases were evaluated per condition (n=3). Bars indicate median ±SD. Statistical significance was determined by two-tailed t-test, (*** p<0.001, * p<0.05).   Quantification of lagging chromosomes in anaphase cells from (E). More than 30 anaphases were evaluated per condition (n=3). Representative images of abnormal anaphase are shown, scale bars indicate 20 μm, arrowheads point to incorrectly segregating chromosomes. Statistical significance was determined by two-tailed t-test. Bars indicate median ±SD (*** p<0.001).


U2OS cells stably expressing H2B - EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals . Time frame closest to the nuclear envelope breakdown ( NEB ) was set as 0 min . Representative examples are shown ( n = 3 ) . Black bottom line indicates the time frames from NEB to the metaphase - to - anaphase transition . Scale bars indicate 10 μm .

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

U2OS cells stably expressing H2B-EGFP were transfected with indicated siRNAs and after 48 h they were filmed in 5 min intervals. Time frame closest to the nuclear envelope breakdown (NEB) was set as 0 min. Representative examples are shown (n=3). Black bottom line indicates the time frames from NEB to the metaphase-to-anaphase transition. Scale bars indicate 10 μm.


Progression through mitosis was categorized in cells from ( A ) ( 100 / condition ) as follows : nuclear envelope breakdown to the metaphase - to - anaphase transition ( MAT ) , anaphase , telophase and interphase . Data from one of three experiments are shown , each bar indicates one cell . Cells that died during imaging are shown in grey .

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

Progression through mitosis was categorized in cells from (A) (100/condition) as follows: nuclear envelope breakdown to the metaphase-to-anaphase transition (MAT), anaphase, telophase and interphase. Data from one of three experiments are shown, each bar indicates one cell. Cells that died during imaging are shown in grey.


Time from the nuclear envelope breakdown to MAT was quantified in cells from ( B ) . Shown is median ± SD ( n = 3 ) . Statistical significance was determined by ANOVA ( * * * * p < 0 . 0001 ) .

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

Time from the nuclear envelope breakdown to MAT was quantified in cells from (B). Shown is median ±SD (n=3). Statistical significance was determined by ANOVA (****p<0.0001).


Cell extracts from asynchronously growing or mitotic RPE - EGFP or RPE - EGFP - FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting . Antibody against pS10H3 was used as a marker of mitosis .

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Cell extracts from asynchronously growing or mitotic RPE-EGFP or RPE-EGFP-FAM110A cells were incubated with GFP Trap and bound proteins were analyzed by immunoblotting. Antibody against pS10H3 was used as a marker of mitosis.


RPE cells arrested in mitosis by nocodazole ( NZ ) were treated with DMSO , PLK1 inhibitor BI2536 ( 100 nM ) , Aurora - A inhibitor MLN8054 ( 0 . 25 μM ) , CDK1 inhibitor RO3306 ( 20 nM ) or CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min , collected and analyzed by immunoblotting with indicated antibodies . Asynchronously growing RPE cells are shown for comparison ( left line ) . Staining for H3 - pS10 served as a marker of mitosis ( n = 3 ) .

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

RPE cells arrested in mitosis by nocodazole (NZ) were treated with DMSO, PLK1 inhibitor BI2536 (100 nM), Aurora-A inhibitor MLN8054 (0.25 μM), CDK1 inhibitor RO3306 (20 nM) or CK1 inhibitor PF-670462 (1 μM) for 60 min, collected and analyzed by immunoblotting with indicated antibodies. Asynchronously growing RPE cells are shown for comparison (left line). Staining for H3-pS10 served as a marker of mitosis (n=3).


U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 ( 100 nM ) and collected by mitotic shake off . Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF - 670462 ( 1 μM ) for 60 min . Whole cell lysates were probed with indicated antibodies . Staining for pS10 - H3 served as a marker of mitosis .

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

U2OS cells were trapped in mitosis by overnight treatment with nocodazole or PLK1 inhibitor BI2536 (100 nM) and collected by mitotic shake off. Part of the nocodazole arrested cells was further treated with CK1 inhibitor PF-670462 (1 μM) for 60 min. Whole cell lysates were probed with indicated antibodies. Staining for pS10-H3 served as a marker of mitosis.


RPE cells or RPE cells stably expressing indicated EGFP - FAM110A variants were transfected with control ( siNC ) , FAM110A or CSNK1D siRNA and grown for 48 h . Alternatively , cells were incubated with PF670462 for the last 12 h . Cells were treated with MG132 for 30 min prior fixation . Impaired chromosomal alignment was scored in > 30 metaphase cells per condition . Error bars indicate median ± SD . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 , * * * p < 0 . 001 ) .

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

RPE cells or RPE cells stably expressing indicated EGFP-FAM110A variants were transfected with control (siNC), FAM110A or CSNK1D siRNA and grown for 48 h. Alternatively, cells were incubated with PF670462 for the last 12 h. Cells were treated with MG132 for 30 min prior fixation. Impaired chromosomal alignment was scored in >30 metaphase cells per condition. Error bars indicate median ±SD. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001, *** p<0.001).


Representative immunoblot analysis of samples from ( D ) . Full arrow indicates migration of the EGFP - FAM110A - WT , empty arrow of endogenous FAM110A .

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Representative immunoblot analysis of samples from (D). Full arrow indicates migration of the EGFP-FAM110A-WT, empty arrow of endogenous FAM110A.


RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation . Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ - Tubulin . Representative images are shown , scale bars indicate 10 μm . Quantification of ( A ) . Plotted is median FAM110A intensity at spindle poles positive for γ - tubulin ± SD . Each dot represents a single spindle pole . Statistical significance was determined by ANOVA ( n = 3 ) ( * * * * p < 0 . 0001 ) .

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

RPE1 were transfected with control siRNA or CSNK1D siRNA or were treated with PF670462 prior fixation. Cells were probed with rabbit polyclonal to FAM110A and mouse monoclonal to γ-Tubulin. Representative images are shown, scale bars indicate 10 μm.   Quantification of (A). Plotted is median FAM110A intensity at spindle poles positive for γ-tubulin ± SD. Each dot represents a single spindle pole. Statistical significance was determined by ANOVA (n=3) (**** p<0.0001).


Cells expressing the wild - type or mutant EGFP - FAM110A were fixed and stained for γ - tubulin and α - tubulin and analyzed by confocal microscopy ( z - stack 0 . 16 µm ) . Representative images of maximal projections of the metaphase cells are shown , scale bars indicate 10 μm . Merged images show signal of DAPI , γ - tubulin and EGFP . Insets show a single Z plane at the spindle pole .

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

Cells expressing the wild-type or mutant EGFP-FAM110A were fixed and stained for γ-tubulin and α-tubulin and analyzed by confocal microscopy (z-stack 0.16 µm). Representative images of maximal projections of the metaphase cells are shown, scale bars indicate 10 μm. Merged images show signal of DAPI, γ-tubulin and EGFP. Insets show a single Z plane at the spindle pole.


( a ) Different VPS34 complexes were immunoprecipitated ( IP ) from MEFs in the presence ( N ) or absence ( − A ) of amino acids using the indicated antibodies and assayed for kinase activity ( left , top panel ) . Inputs were immunoblotted using the antibodies indicated ( left , lower panels ) . Quantification of VPS34 activity is from 3 biological repeats ( right panel ; data shown are mean + s . d . ) . AR , autoradiography .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Different VPS34 complexes were immunoprecipitated (IP) from MEFs in the presence (N) or absence (−A) of amino acids using the indicated antibodies and assayed for kinase activity (left, top panel). Inputs were immunoblotted using the antibodies indicated (left, lower panels). Quantification of VPS34 activity is from 3 biological repeats (right panel; data shown are mean+s.d.). AR, autoradiography.


( b ) Immunoprecipitation of three VPS34 complexes , normalized for VPS34 , was performed using the indicated antibodies under nutrient - rich ( N ) or starvation ( - A ) conditions . VPS34 - binding partners were analysed by western blotting .

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

(b) Immunoprecipitation of three VPS34 complexes, normalized for VPS34, was performed using the indicated antibodies under nutrient-rich (N) or starvation (-A) conditions. VPS34-binding partners were analysed by western blotting.


( c ) ATG14L - containing VPS34 complexes were immunopurified from WT , ULK1 − / − 2KD ( ULK def . ) and FIP200 − / − MEFs , and measured for lipid kinase activity as in a .

O O O O O O O O O O O O O O O O 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) ATG14L-containing VPS34 complexes were immunopurified from WT, ULK1−/− 2KD (ULK def.) and FIP200−/− MEFs, and measured for lipid kinase activity as in a.


( d ) VPS34 was immunopurified from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a .

(d) VPS34 was immunopurified from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( e ) Immunoprecipitation of Beclin - 1 - containing VPS34 complexes from WT , ULK - deficient and FIP200 − / − MEFs . A kinase assay was performed as in a .

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

(e) Immunoprecipitation of Beclin-1-containing VPS34 complexes from WT, ULK-deficient and FIP200−/− MEFs. A kinase assay was performed as in a.


( f ) LC3B puncta and PtdIns ( 3 ) P levels were analysed with anti - LC3B and the biotin - 2XFYVE domain probe . Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown ( scale bars , 10 μm ) . ( g ) Quantification of LC3B puncta from f . Details of quantification are provided in the Methods ; Data shown in g - i are mean + s . d . from a minimum of 6 unique fields of view from a representative experiment ( see statistical source data in Supplementary Table S1 ) . ( h ) Total PtdIns ( 3 ) P was quantified from the experiment in f . Error bars were calculated as in g . ( i ) Quantification of PtdIns ( 3 ) P that co - localizes with LC3B on amino - acid withdrawal from the experiment described in f .

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

(f) LC3B puncta and PtdIns(3)P levels were analysed with anti-LC3B and the biotin-2XFYVE domain probe. Representative immunofluorescence images of LC3B and 2XFYVE domain binding are shown (scale bars, 10 μm). (g) Quantification of LC3B puncta from f. Details of quantification are provided in the Methods; Data shown in g-i are mean+s.d. from a minimum of 6 unique fields of view from a representative experiment (see statistical source data in Supplementary Table S1). (h) Total PtdIns(3)P was quantified from the experiment in f. Error bars were calculated as in g. (i) Quantification of PtdIns(3)P that co-localizes with LC3B on amino-acid withdrawal from the experiment described in f.


( j ) VPS34 activity with or without ULK1 overexpression was assayed . A representative experiment of four repeats is shown .

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

(j) VPS34 activity with or without ULK1 overexpression was assayed. A representative experiment of four repeats is shown.


( a ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 . ATG14L - containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ - 32P [ ATP ] . Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography ( AR , left panels ) . Western blotting was performed ( right panels ) . Results are representative of two unique experiments .

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

(a) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1. ATG14L-containing VPS34 complexes were immunopurified and subjected to an in vitro ULK1 kinase assay in the presence of γ-32P[ATP]. Bound ATG14L complexes and soluble ULK1 were separated and phosphorylation was detected by autoradiography (AR, left panels). Western blotting was performed (right panels). Results are representative of two unique experiments.


( b ) Full - length murine GST - Beclin - 1 and various truncations ( as labelled ) were subjected to an in vitro HA - ULK1 kinase assay . GST - Beclin - 1 6 ( S / T ) A has serine / threonine residues 4 , 7 , 10 , 14 , 29 and 42 mutated to alanine . ULK1 inputs were determined by western blotting , Beclin - 1 inputs by Coomassie ( Coom ) and target phosphorylation by autoradiography .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Full-length murine GST-Beclin-1 and various truncations (as labelled) were subjected to an in vitro HA-ULK1 kinase assay. GST-Beclin-1 6(S/T)A has serine/threonine residues 4, 7, 10, 14, 29 and 42 mutated to alanine. ULK1 inputs were determined by western blotting, Beclin-1 inputs by Coomassie (Coom) and target phosphorylation by autoradiography.


( c ) GST - Beclin - 1 ( 1 - 85 ) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry . Ser 14 , Ser 15 in humans ( outlined ) , is the main phosphorylation site identified ( for mass spectrometry data , see Supplementary Fig . S2b , c ) . Mass spectrometry was performed on a single experiment .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GST-Beclin-1 (1-85) was subjected to an in vitro ULK1 kinase reaction and analysed by mass spectrometry. Ser 14, Ser 15 in humans (outlined), is the main phosphorylation site identified (for mass spectrometry data, see Supplementary Fig. S2b,c). Mass spectrometry was performed on a single experiment.


( d ) Beclin - 1 Ser 14 is the main in vitro ULK1 phosphorylation site . Beclin - 1 WT , and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay . The reaction was developed by autoradiography and stained for Beclin - 1 input levels by Coomassie blue . Results are representative of two unique experiments .

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

(d) Beclin-1 Ser 14 is the main in vitro ULK1 phosphorylation site. Beclin-1 WT, and S4A and S14A mutants were subjected to an in vitro ULK1 kinase assay. The reaction was developed by autoradiography and stained for Beclin-1 input levels by Coomassie blue. Results are representative of two unique experiments.


( e ) HEK293 cells were transfected with the indicated plasmids under nutrient - rich conditions . Beclin - 1 was immunoprecipitated and immunoblotted with pBeclin - 1 ( Ser 14 ) , or anti - Beclin - 1 as a loading control . ULK1 inputs are included below immunoprecipitated samples .

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

(e) HEK293 cells were transfected with the indicated plasmids under nutrient-rich conditions. Beclin-1 was immunoprecipitated and immunoblotted with pBeclin-1(Ser 14), or anti-Beclin-1 as a loading control. ULK1 inputs are included below immunoprecipitated samples.


( f ) Purified GST - Beclin - 1 ( 1 - 85 ) was subjected to in vitro phosphorylation by GST - ULK1 ( top panel ) and GST - ULK2 ( bottom panel ) . Reactions were immunoblotted with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O 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) Purified GST-Beclin-1 (1-85) was subjected to in vitro phosphorylation by GST-ULK1 (top panel) and GST-ULK2 (bottom panel). Reactions were immunoblotted with the indicated antibodies.


( g ) HEK293 cells were transfected with ATG14L , VPS34 and Beclin - 1 and grown under nutrient - rich conditions . ATG14L - containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig . 1j . Inputs were immunoblotted with the indicated antibodies . Representative of four unique experiments .

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

(g) HEK293 cells were transfected with ATG14L, VPS34 and Beclin-1 and grown under nutrient-rich conditions. ATG14L-containing VPS34 complexes were immunoprecipitated and lipid kinase activity was assayed as described in Fig. 1j. Inputs were immunoblotted with the indicated antibodies. Representative of four unique experiments.


( h ) Stable lines containing Beclin - 1 ( WT or S14A ) were used for Beclin - 1 immunoprecipitation . Binding partners were determined by SDS - PAGE analysis and western blot using the indicated antibodies . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

(h) Stable lines containing Beclin-1(WT or S14A) were used for Beclin-1 immunoprecipitation. Binding partners were determined by SDS-PAGE analysis and western blot using the indicated antibodies. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) WT MEFs were cultured with or without amino acids . ATG14L - associated Beclin - 1 was immunoprecipitated and treated with lambda phosphatase treatment ( PPase ) as indicated . Western blotting ( immunoblotting , IB ) was performed with the indicated antibodies . Beclin - 1 Ser 14 phosphorylation was quantified ( shown under top panel ) and normalized to total Beclin - 1 .

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

(a) WT MEFs were cultured with or without amino acids. ATG14L-associated Beclin-1 was immunoprecipitated and treated with lambda phosphatase treatment (PPase) as indicated. Western blotting (immunoblotting, IB) was performed with the indicated antibodies. Beclin-1 Ser 14 phosphorylation was quantified (shown under top panel) and normalized to total Beclin-1.


( b ) WT MEFs were starved for the indicated times . Beclin - 1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated ( top panels ) . Whole - cell lysates were immunoblotted with pULK1 S757 ( mTORC1 - target site ) , pS6K and ULK1 antibodies ( bottom panels ) . Two unique experiments were performed .

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(b) WT MEFs were starved for the indicated times. Beclin-1 was immunopurified by ATG14L immunoprecipitation and immunoblotted as indicated (top panels). Whole-cell lysates were immunoblotted with pULK1 S757 (mTORC1-target site), pS6K and ULK1 antibodies (bottom panels). Two unique experiments were performed.


( c ) WT or FIP200 − / − MEFs were incubated under nutrient - rich , amino - acid - deprived or Torin - 1 ( + T , an mTOR inhibitor ) conditions . Beclin - 1 was purified and immunoblotted as in Fig . 3b .

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

(c) WT or FIP200−/− MEFs were incubated under nutrient-rich, amino-acid-deprived or Torin-1 (+T, an mTOR inhibitor) conditions. Beclin-1 was purified and immunoblotted as in Fig. 3b.


( d ) WT or ULK - deficient MEFs were incubated with or without amino acids . Beclin - 1 was purified and immunoblotted as in a . Two unique experiments were performed . Uncropped images of blots are shown in Supplementary Fig . S4 .

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

(d) WT or ULK-deficient MEFs were incubated with or without amino acids. Beclin-1 was purified and immunoblotted as in a. Two unique experiments were performed. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Beclin - 1 alone ( lanes 1 - 4 ) or Beclin - 1 and ATG14L ( lanes 5 - 8 ) were overexpressed in HEK293 cells . Beclin - 1 was purified either by direct immunoprecipitation ( lanes 1 - 4 ) or by ATG14L immunoprecipitation ( lanes 5 - 8 ) . Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1 . Reactions were immunoblotted with the indicated antibodies . Black line denotes discontinuous lanes from the same gel . Two unique experiments were performed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Beclin-1 alone (lanes 1-4) or Beclin-1 and ATG14L (lanes 5-8) were overexpressed in HEK293 cells. Beclin-1 was purified either by direct immunoprecipitation (lanes 1-4) or by ATG14L immunoprecipitation (lanes 5-8). Immunoprecipitated samples were subjected to an in vitro ULK1 kinase assay with increasing amounts of ULK1. Reactions were immunoblotted with the indicated antibodies. Black line denotes discontinuous lanes from the same gel. Two unique experiments were performed.


( b ) Beclin - 1 alone or bound to ATG14L was purified as described in a . Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points . Western blotting was performed with the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O 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) Beclin-1 alone or bound to ATG14L was purified as described in a. Equal amounts of ULK1 were added to each complex and reactions were quenched at the indicated time points. Western blotting was performed with the indicated antibodies.


( c ) An ATG14L - FLAG - 6His - inducible U2OS cell line was induced for 16 h in the presence of amino acids . Endogenous Beclin - 1 was immunoprecipitated and immunoblotted as in Fig . 3a . ATG14L input levels were detected by immunoblotting . Two unique experiments were performed .

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(c) An ATG14L-FLAG-6His-inducible U2OS cell line was induced for 16 h in the presence of amino acids. Endogenous Beclin-1 was immunoprecipitated and immunoblotted as in Fig. 3a. ATG14L input levels were detected by immunoblotting. Two unique experiments were performed.


( d ) HEK293 cells transfected with either ATG14L or Beclin - 1 , or both , in conjunction with ULK1 , were immunoprecipitated as indicated and blotted with the indicated antibodies .

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(d) HEK293 cells transfected with either ATG14L or Beclin-1, or both, in conjunction with ULK1, were immunoprecipitated as indicated and blotted with the indicated antibodies.


( e ) HEK293 cells were transfected with Beclin - 1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD , which is defective in Beclin - 1 binding , under nutrient - rich conditions . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

(e) HEK293 cells were transfected with Beclin-1 and ULK1 in the presence of ATG14L WT or ATG14LΔCCD, which is defective in Beclin-1 binding, under nutrient-rich conditions. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( f ) HEK293 cells were transfected with ULK1 and Beclin - 1 in conjunction with either ATG14L WT , or one of two mutants ( ΔBATS , ΔN ) that are defective in phagophore localization . Samples were handled as in e . Uncropped images of blots are shown in Supplementary Fig . S4 . Lysates were resolved by SDS - PAGE and blotted with the indicated antibodies .

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(f) HEK293 cells were transfected with ULK1 and Beclin-1 in conjunction with either ATG14L WT, or one of two mutants (ΔBATS,ΔN) that are defective in phagophore localization. Samples were handled as in e. Uncropped images of blots are shown in Supplementary Fig. S4. Lysates were resolved by SDS-PAGE and blotted with the indicated antibodies.


( a ) HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated in the presence of amino acids . Lysates were immunoblotted with the indicated antibodies . A representative experiment of three repeats is shown .

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(a) HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated in the presence of amino acids. Lysates were immunoblotted with the indicated antibodies. A representative experiment of three repeats is shown.


( b ) UVRAG bridges the interaction between Beclin - 1 and ULK1 . HEK293 cells were transfected with Beclin - 1 , with or without UVRAG , in conjunction with ULK1 as indicated . Lysates were immunoprecipitated with anti - HA ( Beclin - 1 ) antibody and blotted with the indicated antibodies . A representative experiment of three repeats is shown . Uncropped images of blots are shown in Supplementary Fig . S4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) UVRAG bridges the interaction between Beclin-1 and ULK1. HEK293 cells were transfected with Beclin-1, with or without UVRAG, in conjunction with ULK1 as indicated. Lysates were immunoprecipitated with anti-HA(Beclin-1) antibody and blotted with the indicated antibodies. A representative experiment of three repeats is shown. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) HEK293 cells were transfected with the indicated plasmids . Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated . Lysates were immunoblotted with the indicated antibodies .

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(a) HEK293 cells were transfected with the indicated plasmids. Cells were grown in the presence of amino acids and treated with NH4Cl to block autophagic turnover where indicated. Lysates were immunoblotted with the indicated antibodies.


( b ) HEK293 cells transfected with Beclin - 1 ATG14L were grown in the presence or absence of amino acids . Lysates were immunoblotted with the indicated antibodies ( left panel ) and quantified by densitometry ( right panel ) . Data represent mean + s . d . of three unique experiments .

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(b) HEK293 cells transfected with Beclin-1 ATG14L were grown in the presence or absence of amino acids. Lysates were immunoblotted with the indicated antibodies (left panel) and quantified by densitometry (right panel). Data represent mean+s.d. of three unique experiments.


( c ) Beclin - 1 - shRNA reconstituted lines ( WT or mutant ) and controls ( scramble shRNA or Beclin - 1 shRNA ) were grown with or without amino acids and assessed for autophagy ( left panel ) and Beclin - 1 levels ( right panel ) .

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(c) Beclin-1-shRNA reconstituted lines (WT or mutant) and controls (scramble shRNA or Beclin-1 shRNA) were grown with or without amino acids and assessed for autophagy (left panel) and Beclin-1 levels (right panel).


( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm .

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(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm.


( d ) Autophagosome ( denoted by arrowheads ) generation on amino - acid withdrawal was analysed by electron microscopy . Cell lines and conditions from c were used and representative images from the indicated amino - acid - starved lines are shown ; scale bars , 0 . 4 μm . ( e ) Quantification of d . Fold induction was determined by arbitrarily making the nutrient - rich condition 1 ( solid bars ) for each line . Error bars represent the s . d . of the mean value over an average of 20 fields of view within a representative experiment . NS , not significant .

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(d) Autophagosome (denoted by arrowheads) generation on amino-acid withdrawal was analysed by electron microscopy. Cell lines and conditions from c were used and representative images from the indicated amino-acid-starved lines are shown; scale bars, 0.4 μm. (e) Quantification of d. Fold induction was determined by arbitrarily making the nutrient-rich condition 1 (solid bars) for each line. Error bars represent the s.d. of the mean value over an average of 20 fields of view within a representative experiment. NS, not significant.


( f ) HA - Beclin - 1 WT or S14D was transiently expressed in FIP200 − / − MEFs grown under nutrient - rich conditions . Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA - Beclin - 1 . Scale bars , 20 μm . ( g ) Quantification of LC3B puncta from confocal in f . In the HA - Beclin - 1 - or HA - Beclin - 1 - S14D - transfected samples , only the HA - positive cells were counted for LC3B puncta . Error bars were processed as in e . Mean value shown ; P values determined by Student ' s t - test using 10 unique fields of view from f . Uncropped images of blots are shown in Supplementary Fig . S4 .

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(f) HA-Beclin-1 WT or S14D was transiently expressed in FIP200−/− MEFs grown under nutrient-rich conditions. Indirect immunofluorescence was performed using antibodies against endogenous LC3B and HA-Beclin-1. Scale bars, 20 μm. (g) Quantification of LC3B puncta from confocal in f. In the HA-Beclin-1- or HA-Beclin-1-S14D-transfected samples, only the HA-positive cells were counted for LC3B puncta. Error bars were processed as in e. Mean value shown; P values determined by Student's t-test using 10 unique fields of view from f. Uncropped images of blots are shown in Supplementary Fig. S4.


( a ) Bec - 1C . elegans were reconstituted with either WT or mutant GFP - BEC - 1 . Stable worm lines with Bec - 1 rescue were obtained and embryos were stained with anti - PGL - 1 antibody . Arrowheads indicate normal PGL - 1 staining in germline cells . ( b ) Quantification of PGL - 1 puncta outside germline cells ( left panel ) . Error bars represents s . d . between 3 unique embryos in a representative experiment . Reconstituted Bec - 1 ( WT and mutant ) levels in Bec − / − stable worms were compared by western blotting . Mean value presented .

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(a) Bec-1C. elegans were reconstituted with either WT or mutant GFP-BEC-1. Stable worm lines with Bec-1 rescue were obtained and embryos were stained with anti- PGL-1 antibody. Arrowheads indicate normal PGL-1 staining in germline cells. (b) Quantification of PGL-1 puncta outside germline cells (left panel). Error bars represents s.d. between 3 unique embryos in a representative experiment. Reconstituted Bec-1 (WT and mutant) levels in Bec−/− stable worms were compared by western blotting. Mean value presented.


( c ) Spectrum of defects in PGL granule degradation in bec - 1 mutant rescue embryos . Mutant embryos exhibited either high levels of diffuse PGL - 1 staining ( middle - left panel ; one - third of the embryos ) , or large punctuate PGL - 1 structures in somatic cells ( bottom - left panel ; two - thirds of the embryos ) . Both diffuse or punctuate PGL - 1 staining in somatic cells have been described in autophagy - deficient embryos .

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(c) Spectrum of defects in PGL granule degradation in bec-1 mutant rescue embryos. Mutant embryos exhibited either high levels of diffuse PGL-1 staining (middle-left panel; one-third of the embryos), or large punctuate PGL-1 structures in somatic cells (bottom-left panel; two-thirds of the embryos). Both diffuse or punctuate PGL-1 staining in somatic cells have been described in autophagy-deficient embryos.


( d ) Embryos from the lines described in a were labelled with anti - LGG - 1 , along with WT and unc - 51 worms . Representative embryos at ∼ 100 cell stage are shown . ( e ) Quantification of LGG - 1 per embryo from labelling in d . Error bars generated as in b . Mean value presented . Uncropped images of blots are shown in Supplementary Fig . S4 . Scale bar , 10 μm .

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(d) Embryos from the lines described in a were labelled with anti-LGG-1, along with WT and unc-51 worms. Representative embryos at ∼ 100 cell stage are shown. (e) Quantification of LGG-1 per embryo from labelling in d. Error bars generated as in b. Mean value presented. Uncropped images of blots are shown in Supplementary Fig. S4. Scale bar, 10 μm.


( A ) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h . Then , WB was used to measure p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein expression . " Nor " = normal medium . Actin served as the loading control . The red text " - Cys " indicates that cystine deficiency most significantly activates AMPK and its substrates .

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(A) 293T cells were cultured in complete medium made of dialyzed serum for 24 h which was then replaced with medium in which one amino acid was eliminated and cultured for 8 h. Then, WB was used to measure p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein expression. "Nor" = normal medium. Actin served as the loading control. The red text "-Cys" indicates that cystine deficiency most significantly activates AMPK and its substrates.


( F ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor protein in 293T cells transfected with shRNAs targeting LKB1 , CaMKK2 , TAK1 or a non - targeting control ( NTC ) that were treated with cystine - deficient medium for 8 h . Actin served as the loading control .

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(F) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor protein in 293T cells transfected with shRNAs targeting LKB1, CaMKK2, TAK1 or a non-targeting control (NTC) that were treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( I ) 293T cells cultured with cystine - deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti - AMPKα , followed by WB analysis with anti - AMPKα and anti - CaMKK2 .

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(I) 293T cells cultured with cystine-deficient medium or complete medium for 8 h were harvested and subjected to immunoprecipitation with anti-AMPKα, followed by WB analysis with anti-AMPKα and anti-CaMKK2.


( E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK ACC , Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine - deficient medium for 8 h . Actin served as the loading control .

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(E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK ACC, Raptor protein expression in 293T cells transfected with shRNAs targeting CARS that were further treated with cystine-deficient medium for 8 h. Actin served as the loading control.


( J ) WB analysis of p - AMPK and total AMPK protein expression in CARS - overexpressing 293T cells and RCC4 cells that were treated with 1 µg / ml STO - 609 or DMSO for 8 h . Actin served as the loading control .

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(J) WB analysis of p-AMPK and total AMPK protein expression in CARS-overexpressing 293T cells and RCC4 cells that were treated with 1 µg/ml STO-609 or DMSO for 8 h. Actin served as the loading control.


( D , E ) WB analysis of p - AMPK , p - ACC , p - Raptor and total AMPK , ACC , Raptor , AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine - deficient medium for 8 h ( D ) or transfected with Flag - CARS for 48 h ( E ) . Actin served as the loading control .

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(D, E) WB analysis of p-AMPK, p-ACC, p-Raptor and total AMPK, ACC, Raptor, AMPKγ2 protein expression in 293T cells transfected with shRNAs targeting AMPKγ2 that were further treated with cystine-deficient medium for 8 h (D) or transfected with Flag-CARS for 48 h (E). Actin served as the loading control.


( F ) 293T cells were transfected with HA - AMPKγ2 alone or with Flag - CARS for 48 h and then cultured with cystine - deficient medium or complete medium for 8 h . Cell lysates were immunoprecipitated with anti - Flag , and a WB analysis was performed .

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(F) 293T cells were transfected with HA-AMPKγ2 alone or with Flag-CARS for 48 h and then cultured with cystine-deficient medium or complete medium for 8 h. Cell lysates were immunoprecipitated with anti-Flag, and a WB analysis was performed.


( B ) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(B) The cell death level was determined in 293T cells with the same treatment conditions as in Figure 4A. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( C , D ) The crystal violet assay ( C ) and apoptosis rate assay ( D ) were performed in 293T cells transfected with shRNAs targeting NTC , CARS , CaMKK2 or AMPKγ2 and further treated with cystine - deficient medium for 24 h with or without 1 mM AICAR . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(C, D) The crystal violet assay (C) and apoptosis rate assay (D) were performed in 293T cells transfected with shRNAs targeting NTC, CARS, CaMKK2 or AMPKγ2 and further treated with cystine-deficient medium for 24 h with or without 1 mM AICAR. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.


( F WB analysis of p - AMPK , total AMPK , CARS , AMPKγ2 , and CaMKK2 protein expression with 786 - O cells transfected with Flag - EV , Flag - CARS , Flag - CaMKK2 or Flag - AMPKγ2 and further treated with cystine - deficient medium for 8 h . Actin served as the loading control . Data are presented as the mean ( ± SD ) of three independent experiments . NS , not significant ; * P < 0 . 05 [ two - tailed Student ' s t - test ] , compared with the indicated groups .

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(F WB analysis of p-AMPK, total AMPK, CARS, AMPKγ2, and CaMKK2 protein expression with 786-O cells transfected with Flag-EV, Flag-CARS, Flag-CaMKK2 or Flag-AMPKγ2 and further treated with cystine-deficient medium for 8 h. Actin served as the loading control. Data are presented as the mean (± SD) of three independent experiments. NS, not significant; *P < 0.05 [two-tailed Student's t-test], compared with the indicated groups.