Figure 2qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues , normalized to GAPDH . Western blot analysis of Dnmt2 protein expression in haematopoietic tissues . Proliferation analysis of primary bone marrow suspension cells . Primary bone marrow suspension cells isolated from the femurs of 8 - day - old mice were grown together with the attached stromal cells . Dnmt2 − / − population doubling levels of three biological replicates , each plated three times , were calculated relative to wild - type culture . Reduced replating abilities of primary Dnmt2 − / − bone marrow cells in a myeloid colony formation assay . For primary ( 1 ° ) colony formation , 1 . 25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation . For secondary ( 2 ° ) colony formation , cells were harvested and 0 . 1 % of primary colonies were replated into semisolid medium . Results are from two independent experiments analyzing 9 wild - type and 13 Dnmt2 − / − mice in total . Reduced secondary CFU - preB colony formation of Dnmt2 − / − cells . Numbers of CFU - preB - cell colonies are indicated per 5 × 104 wild - type or Dnmt2 − / − total bone marrow cells . 10 % of primary colonies were replated into secondary CFU - preB assays . Stable engraftment ( upper panel ) and multi - lineage reconstitution ( lower panel ) of wild - type and Dnmt2 − / − bone marrow transplants . CD11b : macrophages ; Ly6G : granulocytes ; CD45R : B cells ; CD3 : T cells .

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Figure 2qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues, normalized to GAPDH.Western blot analysis of Dnmt2 protein expression in haematopoietic tissues.Proliferation analysis of primary bone marrow suspension cells. Primary bone marrow suspension cells isolated from the femurs of 8-day-old mice were grown together with the attached stromal cells. Dnmt2−/− population doubling levels of three biological replicates, each plated three times, were calculated relative to wild-type culture.Reduced replating abilities of primary Dnmt2−/−bone marrow cells in a myeloid colony formation assay. For primary (1°) colony formation, 1.25 × 104 total bone marrow cells were plated in semisolid medium allowing myeloid differentiation. For secondary (2°) colony formation, cells were harvested and 0.1% of primary colonies were replated into semisolid medium. Results are from two independent experiments analyzing 9 wild-type and 13 Dnmt2−/−mice in total.Reduced secondary CFU-preB colony formation of Dnmt2−/− cells. Numbers of CFU-preB-cell colonies are indicated per 5 × 104 wild-type or Dnmt2−/− total bone marrow cells. 10% of primary colonies were replated into secondary CFU-preB assays.Stable engraftment (upper panel) and multi-lineage reconstitution (lower panel) of wild-type and Dnmt2−/− bone marrow transplants. CD11b: macrophages; Ly6G: granulocytes; CD45R: B cells; CD3: T cells.


Figure 3Flow cytometry analysis of bone marrow primary mesenchymal cells . Quantification of fibroblast colony - forming units ( CFU - F ) . Quantification of alkaline phosphatase colony - forming units ( CFU - ALP ) . Dnmt2 expression after 3 days of mesenchymal cell differentiation . Alizarin Red staining , indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2 − / − mice . Scale bar : 50 μm . Quantification was performed with three biological replicates . qPCR analysis of the osteoblast markers Osterix and Osteocalcin . Oil Red O / hematoxylin staining , showing reduced adipocyte differentiation of primary stromal cells from Dnmt2 − / − mice . Scale bar : 50 μm . Quantification was performed with three biological replicates . qPCR analysis of the adipocyte markers CEBp alpha and Glut4 .

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Figure 3Flow cytometry analysis of bone marrow primary mesenchymal cells.Quantification of fibroblast colony-forming units (CFU-F).Quantification of alkaline phosphatase colony-forming units (CFU-ALP).Dnmt2 expression after 3 days of mesenchymal cell differentiation.Alizarin Red staining, indicating enhanced osteoblast differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.qPCR analysis of the osteoblast markers Osterix and Osteocalcin.Oil Red O/hematoxylin staining, showing reduced adipocyte differentiation of primary stromal cells from Dnmt2−/− mice. Scale bar: 50 μm. Quantification was performed with three biological replicates.qPCR analysis of the adipocyte markers CEBp alpha and Glut4.


Figure 4Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines ( boxes ) within tRNAs from bone marrow ( BM ) , mesenchymal stromal cells ( MSC ) , and in vitro differentiated adipocytes and osteoblasts . Methylation levels are indicated on a color scale from green ( unmethylated ) to red ( completely methylated ) . Representative Northern blot showing tRNA ( Gly ) and tRNA ( Asp ) fragmentation in bone marrow from 8 - day - old and adult mice . tRNA ( Ser ) was included as a Dnmt2 non - substrate tRNA , and 5S rRNA was included as a loading control . tRNA fragment coverage for the Dnmt2 substrates tRNA ( Gly ) and tRNA ( Asp ) , and the non - substrate tRNA ( Ser ) in bone marrow , as determined by deep sequencing . Wild - type coverage is represented in red , and Dnmt2 − / − coverage is indicated in blue . The plots illustrate the specific increase of 5 ′ - halves from tRNA - Gly and 3 ′ - halves from tRNA - Asp fragments in Dnmt2 mutants .

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Figure 4Bisulfite sequencing maps of 454 reads showing the average methylation status of individual cytosines (boxes) within tRNAs from bone marrow (BM), mesenchymal stromal cells (MSC), and in vitro differentiated adipocytes and osteoblasts. Methylation levels are indicated on a color scale from green (unmethylated) to red (completely methylated).Representative Northern blot showing tRNA(Gly) and tRNA(Asp) fragmentation in bone marrow from 8-day-old and adult mice. tRNA(Ser) was included as a Dnmt2 non-substrate tRNA, and 5S rRNA was included as a loading control.tRNA fragment coverage for the Dnmt2 substrates tRNA(Gly) and tRNA(Asp), and the non-substrate tRNA(Ser) in bone marrow, as determined by deep sequencing. Wild-type coverage is represented in red, and Dnmt2−/− coverage is indicated in blue. The plots illustrate the specific increase of 5′-halves from tRNA-Gly and 3′-halves from tRNA-Asp fragments in Dnmt2 mutants.


Figure 5Analysis of protein synthesis by dynamic SILAC . The top 10 % of proteins that are translated slower in Dnmt2 − / − versus wild - type cells are indicated in orange . The top 10 % of proteins that are translated faster in Dnmt2 − / − versus wild - type cells are shown in blue . A change of > twofold is indicated by the black lines . Dimethyl - labeling analysis of 8 - day - old mouse bone marrow tissue . Proteins that were down - regulated ( < twofold change ) in Dnmt2 - deficient cells are indicated in red , and up - regulated proteins ( > twofold change ) are shown in green . Nestin and periostin are marked with arrows . Correlation between protein and mRNA expression for differentially expressed proteins . Proteins that were down - regulated ( < twofold change ) in Dnmt2 - deficient cells are indicated in red , and up - regulated proteins ( > twofold change ) are shown in green . The Pearson correlation coefficient is − 0 . 03 . Representative staining of nestin and periostin in femur sections from 8 - day - old wild - type and Dnmt2 − / − mice . The anti - periostin staining is comparable in the periosteum of wild - type and Dnmt2 − / − mice , but is reduced in the bone marrow cells ( compare the highlighted areas ) . Scale bar : 100 μm . Nuclei are counterstained with DAPI ( blue ) . Quantitative differences in the length of the nestin - positive trabecular zone ( upper panel ) between wild - type ( 30 measurements , three mice ) and Dnmt2 − / − ( 21 measurements , two mice ) and in bone periostin - positive marrow areas ( lower panel ) between wild - type ( 26 measurements , three mice ) and Dnmt2 − / − ( 45 measurements , three mice ) in femur sections from 8 - day - old mice . Data are presented as mean ± SD .

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Figure 5Analysis of protein synthesis by dynamic SILAC. The top 10% of proteins that are translated slower in Dnmt2−/− versus wild-type cells are indicated in orange. The top 10% of proteins that are translated faster in Dnmt2−/− versus wild-type cells are shown in blue. A change of >  twofold is indicated by the black lines.Dimethyl-labeling analysis of 8-day-old mouse bone marrow tissue. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. Nestin and periostin are marked with arrows.Correlation between protein and mRNA expression for differentially expressed proteins. Proteins that were down-regulated (< twofold change) in Dnmt2-deficient cells are indicated in red, and up-regulated proteins (> twofold change) are shown in green. The Pearson correlation coefficient is −0.03.Representative staining of nestin and periostin in femur sections from 8-day-old wild-type and Dnmt2−/− mice. The anti-periostin staining is comparable in the periosteum of wild-type and Dnmt2−/− mice, but is reduced in the bone marrow cells (compare the highlighted areas). Scale bar: 100 μm. Nuclei are counterstained with DAPI (blue).Quantitative differences in the length of the nestin-positive trabecular zone (upper panel) between wild-type (30 measurements, three mice) and Dnmt2−/− (21 measurements, two mice) and in bone periostin-positive marrow areas (lower panel) between wild-type (26 measurements, three mice) and Dnmt2−/− (45 measurements, three mice) in femur sections from 8-day-old mice. Data are presented as mean ± SD.


Figure 6Frequency of amino acid substitutions in the dSILAC dataset . Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant ( P < 0 . 01 , Fisher ' s exact test ) .

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Figure 6Frequency of amino acid substitutions in the dSILAC dataset. Differences between Asp > Glu and Glu > Asp transitions versus Asn > Lys and His > Gln were found to be highly significant (P < 0.01, Fisher's exact test).


Figure 1 ( A ) Alcian blue ‐ stained sections of descending colons from control and Atg5VC mice . The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside . Bars = 200 μm . ( B ) Quantification of average mucin area / goblet cell in control and Atg5VC mice ( n = 5 - 7 mice / group from 3 independent experiments ; 100 goblet cells measured / mouse ) . * * P0 . 01 as determined by the Student ' s t ‐ test . This analysis was performed on upper crypt goblet cells ( above the upper dashed line in A ) . ( C ) Quantification of the average number of goblet cells per crypt ( n = 3 mice / group ; over 100 crypts measured / mouse ) . ( D ) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons . The yellow dashed lines outline the mucin granules in the apex of the cells . Bars = 2 μm . ( E , F ) Quantification of ( E ) the average granule density and ( F ) the average number of mucin granules / goblet cell ( n = 3 mice / group and 20 goblet cells evaluated / mouse ) . * * * P0 . 001 as determined by the Student ' s t ‐ test . ( G , H ) Whole ‐ mount images of the mucosal surface from control and Atg5VC transverse colons . Whole ‐ mount colonic samples were either stained with Ulex europaeus agglutinin ‐ 1 ( UEA ) that labelled mucus ( G ) or analysed by the scanning electron microscopy ( H ) . Yellow arrows indicate the crypt opening orifices . Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice ( insets in H show higher power image of the latter ) . Bars = 100 μm .

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Figure 1(A) Alcian blue‐stained sections of descending colons from control and Atg5VC mice. The area between the two black dashed lines indicates the crypt base where epithelial progenitors and nascent goblet cells reside. Bars=200 μm.(B) Quantification of average mucin area/goblet cell in control and Atg5VC mice (n=5-7 mice/group from 3 independent experiments; 100 goblet cells measured/mouse). **P0.01 as determined by the Student's t‐test. This analysis was performed on upper crypt goblet cells (above the upper dashed line in A).(C) Quantification of the average number of goblet cells per crypt (n=3 mice/group; over 100 crypts measured/mouse).(D) TEM images of upper crypt goblet cells from control and Atg5VC transverse colons. The yellow dashed lines outline the mucin granules in the apex of the cells. Bars=2 μm.(E, F) Quantification of (E) the average granule density and (F) the average number of mucin granules/goblet cell (n=3 mice/group and 20 goblet cells evaluated/mouse). ***P0.001 as determined by the Student's t‐test.(G, H) Whole‐mount images of the mucosal surface from control and Atg5VC transverse colons. Whole‐mount colonic samples were either stained with Ulex europaeus agglutinin‐1 (UEA) that labelled mucus (G) or analysed by the scanning electron microscopy (H). Yellow arrows indicate the crypt opening orifices. Green arrowheads indicate goblet cells protruding from the surface epithelium of Atg5VC mice (insets in H show higher power image of the latter). Bars=100 μm.


Figure 2 ( B ) Representative whole ‐ mount images of single spheroids cultured in either 50 % conditioned media ( CM ) or 5 % CM + 5 μM DAPT and stained for Muc2 ( green ) to visualize goblet cells . Bars = 20 μm . ( C , D ) Graph of relative expression ( as measured by qRT ‐ PCR ) of Atoh1 ( C ) and Muc2 ( D ) mRNAs comparing spheroids grown in either 5 % CM or 5 % CM + DAPT to spheroids grown in 50 % CM ( n = 4 - 5 samples / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 as determined by a two ‐ tailed Student ' s t ‐ test . ( E , F ) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5 % CM + DAPT and stained for Muc2 ( green : E ) or with the lectin , UEA ( red : F ) . Bars = 20 μm . ( G ) Quantification of average mucin area / goblet cell . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 cells were quantified / sample ; * * P0 . 01 as determined by a two ‐ tailed Student ' s t ‐ test ) . ( H ) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT - PCR ( n = 4 - 5 samples / group ) . No statistically significant differences were found in Muc2 expression as determined by the Student ' s t ‐ test . ( I , K ) Images of FIP200f / f ( I ) and Atg14f / f ( K ) colonic epithelial spheroids treated with recombinant Tat ‐ Cre and stained with UEA ( red ) . ( J , L ) Quantification of average mucin area / goblet cell . Error bars indicate s . e . m . ( n = 6 samples / group ; 100 cells were quantified / sample ; * * P0 . 01 and * * * P0 . 001 as determined by a two ‐ tailed Student ' s t ‐ test ) .

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Figure 2(B) Representative whole‐mount images of single spheroids cultured in either 50% conditioned media (CM) or 5% CM+5 μM DAPT and stained for Muc2 (green) to visualize goblet cells. Bars=20 μm.(C, D) Graph of relative expression (as measured by qRT‐PCR) of Atoh1 (C) and Muc2 (D) mRNAs comparing spheroids grown in either 5% CM or 5% CM+ DAPT to spheroids grown in 50% CM (n=4-5 samples/group). Error bars indicate s.e.m. *P0.05; **P0.01 as determined by a two‐tailed Student's t‐test.(E, F) Images of colonic epithelial spheroids isolated from control and Atg5VC mice grown in 5% CM+DAPT and stained for Muc2 (green: E) or with the lectin, UEA (red: F). Bars=20 μm.(G) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 as determined by a two‐tailed Student's t‐test).(H) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice as measured by qRT-PCR (n=4-5 samples/group). No statistically significant differences were found in Muc2 expression as determined by the Student's t‐test.(I, K) Images of FIP200f/f (I) and Atg14f/f (K) colonic epithelial spheroids treated with recombinant Tat‐Cre and stained with UEA (red).(J, L) Quantification of average mucin area/goblet cell. Error bars indicate s.e.m. (n=6 samples/group; 100 cells were quantified/sample; **P0.01 and ***P0.001 as determined by a two‐tailed Student's t‐test).


Figure 3 ( A ) Images of colonic sections stained for LC3β ( green ) from control and Atg5VC mice . The white ‐ dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme . White asterisks denote non ‐ specific plasma cell staining from the mouse monoclonal anti ‐ LC3β antibody . Bars = 50 μm . ( B ) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild ‐ type mouse . The inset is a low power image of the entire goblet cell . The black box within the inset indicates the area shown in this panel . Note this area is not associated with mucin granules . Bar = 500 nm . ( C ) Enriched KEGG pathways for Atg5 ‐ deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium . P ‐ values were determined by modified Fisher ' s exact test . ( D - G ) Double label immunogold transmission electron microscopic images from wild ‐ type mice for LC3β ( 12 nm particles ) and EEA1 ( D ) , Rab7 ( E ) , Rab11 ( F ) , or Lamp1 ( G ) ( 18 nm particles ) . Bars = 200 nm .

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Figure 3(A) Images of colonic sections stained for LC3β (green) from control and Atg5VC mice. The white‐dashed lines indicate the border between the basal surface of the crypt epitheliumand the mesenchyme. White asterisks denote non‐specific plasma cell staining from the mouse monoclonal anti‐LC3β antibody. Bars=50 μm.(B) Immunogold transmission electron microscopic image for LC3β in a colonic goblet cell from a wild‐type mouse. The inset is a low power image of the entire goblet cell. The black box within the inset indicates the area shown in this panel. Note this area is not associated with mucin granules. Bar=500 nm.(C) Enriched KEGG pathways for Atg5‐deficient epithelial cells obtained from a microarray comparing control and Atg5VC colonic crypt base epithelium. P‐values were determined by modified Fisher's exact test.(D-G) Double label immunogold transmission electron microscopic images from wild‐type mice for LC3β (12 nm particles) and EEA1 (D), Rab7 (E), Rab11 (F), or Lamp1 (G) (18 nm particles). Bars=200 nm.


Figure 4 ( B ) Colonic epithelial monolayers from wild ‐ type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β ( green ) and EEA1 ( red ) . Bars = 5 μm . ( C , D ) Quantification of average number of LC3β dots per cell ( C ) and the percentage of LC3β dots that co ‐ localized with EEA1 per cell ( D ) ( n = 3 experiments / group ; 25 - 30 cells / experiment ) . ( E ) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated . GAPDH is a loading control . Representative image is shown from n = 3 experiments . ( F ) Images of wild ‐ type colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 100 μM Dynasore . Bars = 20 μm . ( G ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * P0 . 001 as determined by the Student ' s t ‐ test .

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Figure 4(B) Colonic epithelial monolayers from wild‐type mice treated with either vehicle or 100 μM Dynasore and stained for LC3β (green) and EEA1 (red). Bars=5 μm.(C, D) Quantification of average number of LC3β dots per cell (C) and the percentage of LC3β dots that co‐localized with EEA1 per cell (D) (n=3 experiments/group; 25-30 cells/experiment).(E) LC3 immunoblots of epithelial spheroids treated with 100 μM Dynasore and 100 nM BafA1 as indicated. GAPDH is a loading control. Representative image is shown from n=3 experiments.(F) Images of wild‐type colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 100 μM Dynasore. Bars=20 μm.(G) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ***P0.001 as determined by the Student's t‐test.


Figure 5 ( A ) Colonic epithelial monolayers from a wild ‐ type and p22 mut mouse stained with LC3β ( green ) and EEA1 ( red ) . Bar = 5 μm . ( B , C ) Quantification of average number of LC3β dots per cell ( B ) and the percentage of LC3β dots that co ‐ localized with EEA1 per cell ( C ) ( n = 3 experiments / group ; 25 - 30 cells / experiment ) . ( D ) LC3 immunoblots of either wild ‐ type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated . Representative image is shown from n = 3 experiments . ( E ) Double label immunogold transmission electron microscopic images for LC3β ( 12μnm particles ) and p22phox ( 18 nm particles ) showing a p22phox ‐ positive single membrane cytoplasmic vesicle ( D ) and a p22phox ‐ , LC3β ‐ positive vacuole ( E ) . Bars = 200μnm . ( F ) Colonic epithelial monolayers from a wild ‐ type mouse stained for LC3β ( green ) and p22phox ( red ) and quantification of percent LC3β dots co ‐ localized with p22phox per cell , Bar = 5 μm . ( G ) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC ‐ UEA to label goblet cellmucin . Bars = 50 μm . ( H ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test .

O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Colonic epithelial monolayers from a wild‐type and p22 mut mouse stained with LC3β (green) and EEA1 (red). Bar=5 μm.(B, C) Quantification of average number of LC3β dots per cell (B) and the percentage of LC3β dots that co‐localized with EEA1 per cell (C) (n=3 experiments/group; 25-30 cells/experiment).(D) LC3 immunoblots of either wild‐type or p22 mut epithelial spheroids treated with 100 nM BafA1 as indicated. Representative image is shown from n=3 experiments.(E) Double label immunogold transmission electron microscopic images for LC3β (12μnm particles) and p22phox (18 nm particles) showing a p22phox‐positive single membrane cytoplasmic vesicle (D) and a p22phox‐, LC3β‐positive vacuole (E). Bars=200μnm.(F) Colonic epithelial monolayers from a wild‐type mouse stained for LC3β (green) and p22phox (red) and quantification of percent LC3β dots co‐localized with p22phox per cell, Bar=5 μm.(G) Colonic epithelial cellspheroids from control and p22 mut mice stained with TRITC‐UEA to label goblet cellmucin. Bars=50 μm.(H) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). Error bars indicate s.e.m. **P0.01; ****P0.0001 as determined by the Student's t‐test.


Figure 6 ( A ) Colonic epithelial cell spheroids from p22phox − / − , Atg5VC and wild ‐ type mice . Wild ‐ type spheroids were treated with vehicle ( control ) , 10 μM DPI ( NADPH oxidase inhibitor ) or 100 μM Dynasore . Intracellular ROS in live cells were labelled by 5 μM DCF . Bars = 100 μm . ( B ) Quantification of fluorescent DCF normalized to control ( n = 3 independent experiments / group ; 30 spheroids quantified / experiment ) . ( C ) Quantification of average area of cytoplasmic mucin from wild ‐ type mice treated with 3 % NAC as compared to controls ( n = 6 - 7 mice / group ) . Error bars indicate s . e . m . * P0 . 05 ; * * P0 . 01 ; * * * * P0 . 0001 as determined by the Student ' s t ‐ test . ( D ) TEM images of colonic epithelial monolayers treated with cerium chloride . The presence of electron dense precipitate is indicative of ROS production . Bar = 500 nm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A) Colonic epithelial cell spheroids from p22phox−/−, Atg5VC and wild‐type mice. Wild‐type spheroids were treated with vehicle (control), 10 μM DPI (NADPH oxidase inhibitor) or 100 μM Dynasore. Intracellular ROS in live cells were labelled by 5 μM DCF. Bars=100 μm.(B) Quantification of fluorescent DCF normalized to control (n=3 independent experiments/group; 30 spheroids quantified/experiment).(C) Quantification of average area of cytoplasmic mucin from wild‐type mice treated with 3% NAC as compared to controls (n=6-7 mice/group). Error bars indicate s.e.m. *P0.05; **P0.01; ****P0.0001 as determined by the Student's t‐test.(D) TEM images of colonic epithelial monolayers treated with cerium chloride. The presence of electron dense precipitate is indicative of ROS production. Bar=500 nm.


Figure 7Exogeneous ROS can restore mucingranule accumulation defects . ( A ) Quantification of fluorescent DCF normalized to controls ( n = 3 independent experiments / group ; 10 - 30 spheroids quantified / culture ) . * P0 . 05 as determined by ANOVA with Tukey ' s multiple post ‐ test comparisons ( B ) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC ‐ UEA ( red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( C ) Quantification of average mucin area / goblet cell ( n = 6 samples / group ; 150 cells were quantified / sample ) . * * * P0 . 001 , * * * * P . 0001 as determined by ANOVA with Tukey ' s multiple post ‐ test comparisons . ( D ) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT - PCR . No statistically significant differences were found in either group as determined by ANOVA . ( E , G ) Images of p22 mut or Dynasore ‐ treated colonic epithelial spheroids ( mucin is labelled with TRITC ‐ UEA ; red ) after treatment with either vehicle or 300 μM H2O2 . Bars = 20 μm . ( F , H ) Quantification of average mucin area / goblet cell ( n = 6 sample / group ; 30 - 60 cells were quantified / sample ) corresponding to experiments performed using p22 mut ( E ) and Dynasore ‐ treated wild ‐ type spheroids ( G ) Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA ‐ AM ( I ) or PMA / ionomycin ( J ) . ( I , J ) Quantification of average mucin area / goblet cell ( n = 5 sample / group ; 150 cells were quantified / sample ) . Error bars indicate s . e . m . * * * * P0 . 0001 as determined by the Student ' s t ‐ test ( I ) or ANOVA with Tukey ' s multiple post ‐ test comparisons ( J ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7Exogeneous ROS can restore mucingranule accumulation defects. (A) Quantification of fluorescent DCF normalized to controls (n=3 independent experiments/group; 10-30 spheroids quantified/culture). *P0.05 as determined by ANOVA with Tukey's multiple post‐test comparisons(B) Images of control and Atg5VC colonic epithelial spheroids labelled with TRITC‐UEA (red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (C) Quantification of average mucin area/goblet cell (n=6 samples/group; 150 cells were quantified/sample). ***P0.001, ****P.0001 as determined by ANOVA with Tukey's multiple post‐test comparisons.(D) Relative expression of Muc2 mRNA in differentiated spheroids from control and Atg5VC mice treated with either vehicle or 300 μM H2O2 as measured by qRT-PCR. No statistically significant differences were found in either group as determined by ANOVA.(E, G) Images of p22 mut or Dynasore‐treated colonic epithelial spheroids (mucin is labelled with TRITC‐UEA; red) after treatment with either vehicle or 300 μM H2O2. Bars=20 μm. (F, H) Quantification of average mucin area/goblet cell (n=6 sample/group; 30-60 cells were quantified/sample) corresponding to experiments performed using p22 mut (E) and Dynasore‐treated wild‐type spheroids (G)Atg5VC and p22 mut spheroids were treated with 300 μM H2O2 plus or minus 100 μM BAPTA‐AM (I) or PMA/ionomycin (J). (I, J) Quantification of average mucin area/goblet cell (n=5 sample/group; 150 cells were quantified/sample). Error bars indicate s.e.m. ****P0.0001 as determined by the Student's t‐test (I) or ANOVA with Tukey's multiple post‐test comparisons (J).


Figure 1 ( A ) Wild ‐ type ( YW5 ‐ 1B ) , apg10 ‐ 1 mutant ( MT91 ‐ 4 ‐ 2 ) and Δapg10 ( TFD10 ‐ L1 ) cells were incubated in SD ( − N ) medium containing 1 mM PMSF at 30 ° C . After incubation for 6 h , cells were observed under light microscopy ( Nomarski images ) . ( B ) Wild ‐ type ( closed circles ) , apg10 ‐ 1 mutant ( closed triangles ) and Δapg10 ( open circles ) cells were grown to 1 OD600 / ml in YPD and then transferred to SD ( − N ) medium . After incubation at 30 ° C for the times indicated , their viability was determined by phloxine B staining ( Tsukada and Ohsumi , 1993 ) . ( C ) Cell lysates from wild ‐ type cells with empty vector pRS316 ( lane 1 ) or HAAPG12 ( lane 2 ) , and also apg10 ‐ 1 ( lane 3 ) and Δapg10 cells with HAAPG12 ( lane 4 ) were subjected to Western blotting analysis with anti ‐ HA antibody ( 16B12 ) .

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Figure 1(A) Wild‐type (YW5‐1B), apg10‐1 mutant (MT91‐4‐2) and Δapg10 (TFD10‐L1) cells were incubated in SD(−N) medium containing 1 mM PMSF at 30°C. After incubation for 6 h, cells were observed under light microscopy (Nomarski images).(B) Wild‐type (closed circles),apg10‐1 mutant (closed triangles) and Δapg10 (open circles) cells were grown to 1 OD600/ml in YPD and then transferred to SD(−N) medium. After incubation at 30°C for the times indicated, their viability was determined by phloxine B staining ( Tsukada and Ohsumi, 1993).(C) Cell lysates from wild‐type cells with empty vector pRS316 (lane 1) or HAAPG12 (lane 2), and also apg10‐1 (lane 3) and Δapg10 cells with HAAPG12 (lane 4) were subjected to Western blotting analysis with anti‐HA antibody (16B12).


Figure 2 ( A ) Two ‐ hybrid assay for interactions of Apg10p with Apg5p , Apg7p and Apg12p . Yeast cells ( PJ69 ‐ 4A ) harboring the indicated plasmids were streaked out on plates with medium lacking adenine to assay for interaction ‐ dependent activation of the ADE2 gene . ( B ) Total lysates from Δapg5 cells with HAAPG10 and / or MycAPG12 on 2μ plasmid were immunoprecipitated with anti ‐ HA ( mAb 16B12 ) or anti ‐ Myc ( mAb 9E10 ) antibody . Precipitates were analyzed by Western blotting using anti ‐ HA or anti ‐ Myc antibody . Apg12p adduct of Apg10p is indicated by ● and an unidentified complex of 67 kDa by ● ● . The positions of cross ‐ reacting IgG heavy chain ( H ) and light chain ( L ) are indicated by arrowheads .

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Figure 2(A) Two‐hybrid assay for interactions of Apg10p with Apg5p, Apg7p and Apg12p. Yeast cells (PJ69‐4A) harboring the indicated plasmids were streaked out on plates with medium lacking adenine to assay for interaction‐dependent activation of the ADE2 gene.(B) Total lysates from Δapg5 cells with HAAPG10 and/or MycAPG12 on 2μ plasmid were immunoprecipitated with anti‐HA (mAb 16B12) or anti‐Myc (mAb 9E10) antibody. Precipitates were analyzed by Western blotting using anti‐HA or anti‐Myc antibody. Apg12p adduct of Apg10p is indicated by ● and an unidentified complex of 67 kDa by ●●. The positions of cross‐reacting IgG heavy chain (H) and light chain (L) are indicated by arrowheads.


Figure 3Effects of the deletion of APG5 or APG7 on Apg12p - Apg10p thioester formation . Coimmunoprecipitation was performed with wild ‐ type ( KA311B ) , Δapg5 ( YNM122 ) or Δapg7 ( YTS12 ) cells harboring HAAPG10 and MycAPG12 on 2μ plasmids . The resulting precipitates were analyzed by Western blotting . The DTT ‐ resistant Apg12p - Apg5p conjugate is indicated by ● .

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Figure 3Effects of the deletion of APG5 or APG7 on Apg12p-Apg10p thioester formation. Coimmunoprecipitation was performed with wild‐type (KA311B), Δapg5 (YNM122) or Δapg7 (YTS12) cells harboring HAAPG10 and MycAPG12 on 2μ plasmids. The resulting precipitates were analyzed by Western blotting. The DTT‐resistant Apg12p-Apg5p conjugate is indicated by ●.


Figure 4 ( A ) Cell lysates from Δapg10 cells ( TFD10 ‐ L1 ) harboring HAAPG12 and each mutant form of HAAPG10 on CEN plasmids were subjected to Western blotting analysis with anti ‐ HA antibody ( mAb 16B12 ) . ( B ) Cell extracts from the wild ‐ type cells ( KA311B ) harboring MycAPG12 and mutated HAAPG10 were subjected to immunoprecipitation and the resulting precipitates were analyzed by Western blotting with the rabbit anti ‐ HA antibody .

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Figure 4(A) Cell lysates from Δapg10 cells (TFD10‐L1) harboring HAAPG12 and each mutant form of HAAPG10 on CEN plasmids were subjected to Western blotting analysis with anti‐HA antibody (mAb 16B12).(B) Cell extracts from the wild‐type cells (KA311B) harboring MycAPG12 and mutated HAAPG10 were subjected to immunoprecipitation and the resulting precipitates were analyzed by Western blotting with the rabbit anti‐HA antibody.


Figure 5 ( A ) Autophagic activities were measured by an ALP assay ( Noda and Ohsumi , 1998 ) . YTS3 cells ( Δapg10PHO8 : : pho8Δ60 ) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600 / ml in SC medium lacking tryptophan and then transferred to SD ( − N ) medium . Lysates from the cells after incubation for 0 and 4 h were used for assay . Error bars indicate the SD of three independent experiments . ( B ) Cell lysates from Δapg10 cells ( TFD10 ‐ L1 ) harboring empty vector , pRS316 ( lane 1 ) , wild ‐ type HAAPG10 ( lane 2 ) , HAAPG10C26S ( lane 3 ) , HAAPG10C133S ( lane 4 ) or HAAPG10C137S ( lane 5 ) on CEN plasmids were subjected to Western blotting analysis with anti ‐ API antiserum .

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Figure 5(A) Autophagic activities were measured by an ALP assay ( Noda and Ohsumi, 1998). YTS3 cells (Δapg10PHO8::pho8Δ60) harboring each mutant APG10 on CEN plasmids were grown to 1 OD600/ml in SC medium lacking tryptophan and then transferred to SD(−N) medium. Lysates from the cells after incubation for 0 and 4 h were used for assay. Error bars indicate the SD of three independent experiments.(B) Cell lysates from Δapg10 cells (TFD10‐L1) harboring empty vector, pRS316 (lane 1), wild‐type HAAPG10 (lane 2), HAAPG10C26S (lane 3), HAAPG10C133S (lane 4) or HAAPG10C137S (lane 5) on CEN plasmids were subjected to Western blotting analysis with anti‐API antiserum.


Figure 1 Figure 1b . Genotyping of AS and WT CDK12 clones . Ethidium bromide - stained agarose gel visualizing PCR products from genomic DNA of AS ( AS - PCR ) and WT ( WT - PCR ) CDK12 HCT116 cells and their digest with BslI enzyme ( indicated as AS - BslI and WT - BslI ) . Primer positions and BslI restriction sites are depicted at Expanded View Fig . 1a . Numbers on the left and right indicate DNA marker and DNA fragment sizes , respectively . Figure 1d . Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII . Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3 - MB - PP1 for indicated times . Long and short exp . = long ( 4 - 14 min ) and short ( 10 - 60 s ) exposures , respectively . FUS and tubulin are loading controls . A representative image from 3 replicates is shown . Figure 1e , f . Inhibition of CDK12 in AS CDK12 HCT116 cells results in down - regulation of CDK12 - dependent HR genes . Graph shows RT - qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 ( e ) and WT CDK12 HCT116 ( f ) cells treated for indicated times with 3 - MB - PP1 . mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control ( CTRL ) cells were set to 1 . n = 3 replicates , error bars indicate standard error of the mean ( SEM ) .

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

Figure 1 Figure 1b. Genotyping of AS and WT CDK12 clones. Ethidium bromide-stained agarose gel visualizing PCR products from genomic DNA of AS (AS-PCR) and WT (WT-PCR) CDK12 HCT116 cells and their digest with BslI enzyme (indicated as AS- BslI and WT- BslI). Primer positions and BslI restriction sites are depicted at Expanded View Fig. 1a. Numbers on the left and right indicate DNA marker and DNA fragment sizes, respectively.  Figure 1d. Effect of CDK12 inhibition on phosphorylation of the CTD of RNAPII. Western blot analyses of protein levels by the indicated antibodies in AS CDK12 HCT116 cells treated with 5 µM 3-MB-PP1 for indicated times. Long and short exp.= long (4-14 min) and short (10-60 s) exposures, respectively. FUS and tubulin are loading controls. A representative image from 3 replicates is shown.  Figure 1e, f. Inhibition of CDK12 in AS CDK12 HCT116 cells results in down-regulation of CDK12-dependent HR genes. Graph shows RT-qPCR analysis of relative levels of mRNAs of described genes in AS CDK12 HCT116 (e) and WT CDK12 HCT116 (f) cells treated for indicated times with 3-MB-PP1. mRNA levels were normalized to HPRT1 mRNA expression and the mRNA levels of untreated control (CTRL) cells were set to 1. n=3 replicates, error bars indicate standard error of the mean (SEM).


Figure 2 Figure 2b . CDK12 kinase activity is needed for G1 / S progression in cells arrested by serum starvation . Flow cytometry profiles of control ( - 3 - MB - PP1 ) or inhibitor ( + 3 - MB - PP1 ) treated cells from the experiment depicted in Fig . 2a . The red arrow points to the onset of the G1 / S progression defect in 3 - MB - PP1 - treated cells . To better visualize the G1 / S delay in the presence of the inhibitor the 24 h time point is also shown . n = 3 replicates ; representative result is shown . Figure 2d . CDK12 protein levels peak in the G0 / G1 phase of the cell cycle . Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells . Corresponding cell cycle phases are depicted above time points . A representative Western blot from 3 replicates is shown . Figure 2f , g . Inhibition of CDK12 in early G1 perturbs normal cell cycle progression . Quantification of cells ( % ) in cell cycle phases from flow cytometry profiles of propidium iodide ( f ) and BrdU ( g ) labeled cells upon addition of 3 - MB - PP1 at indicated time points after serum addition in the experiment depicted in Fig . 2e . CTRL in Fig . 2g = control sample without 3 - MB - PP1 . n = 3 replicates , representative result is shown . Figure 2h . Short - term CDK12 inhibition does not activate DNA damage checkpoints . Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times . CPT corresponds to 5 µM camptothecin . A representative Western blot from 3 replicates is shown . FUS is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2 Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation . Flow cytometry profiles of control (-3-MB-PP1) or inhibitor (+3-MB-PP1) treated cells from the experiment depicted in Fig. 2a. The red arrow points to the onset of the G1/S progression defect in 3-MB-PP1-treated cells. To better visualize the G1/S delay in the presence of the inhibitor the 24 h time point is also shown. n=3 replicates; representative result is shown. Figure 2d. CDK12 protein levels peak in the G0/G1 phase of the cell cycle. Western blots show levels of proteins at indicated time points after the release of serum starved AS CDK12 HCT116 cells. Corresponding cell cycle phases are depicted above time points. A representative Western blot from 3 replicates is shown.  Figure 2f, g. Inhibition of CDK12 in early G1 perturbs normal cell cycle progression. Quantification of cells (%) in cell cycle phases from flow cytometry profiles of propidium iodide (f) and BrdU (g) labeled cells upon addition of 3-MB-PP1 at indicated time points after serum addition in the experiment depicted in Fig. 2e. CTRL in Fig. 2g = control sample without 3-MB-PP1. n=3 replicates, representative result is shown.  Figure 2h. Short-term CDK12 inhibition does not activate DNA damage checkpoints. Western blot analyses of phosphorylation of depicted DNA damage response markers upon inhibition of CDK12 for indicated times. CPT corresponds to 5 µM camptothecin. A representative Western blot from 3 replicates is shown. FUS is a loading control.


Figure 3 Figure 3a . CDK12 inhibition results in differential expression of a subset of genes . Comparison of log2 fold - changes versus log2 mean expression in 3 ' end RNA - seq data shows differentially regulated genes after inhibition of CDK12 . Down - ( log2 fold - change < - 1 ) and up - regulated ( log2 fold - change > 1 ) genes are shown in blue and red , respectively . Figure 3d . Validation of RNA - seq for select DNA replication genes by RT - qPCR . Graph shows relative levels of mRNAs of described genes in serum arrested and released ( 0 h G0 / G1 ) AS CDK12 HCT116 cells either treated ( 3 - MB - PP1 ) or not ( CTRL ) with the inhibitor for indicated times after the release . mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release ( 0 h ) was set as 1 . n = 3 replicates , error bars indicate SEM . Figure 3e . Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity . Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released ( 0 h ) cells either treated or not with 3 - MB - PP1 for the indicated times after the release . FUS is a loading control . A representative Western blot of 3 replicates is shown . Figure 3f . CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin . Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3 - MB - PP1 for the indicated times . Histone H2A serves as a loading control of chromatin fractions . A = asynchronous cells , 0 h = time of release . A representative Western blot of 3 replicates is shown .

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

Figure 3 Figure 3a. CDK12 inhibition results in differential expression of a subset of genes. Comparison of log2 fold-changes versus log2 mean expression in 3'end RNA-seq data shows differentially regulated genes after inhibition of CDK12. Down- (log2 fold-change < -1) and up-regulated (log2 fold-change >1) genes are shown in blue and red, respectively.  Figure 3d. Validation of RNA-seq for select DNA replication genes by RT-qPCR. Graph shows relative levels of mRNAs of described genes in serum arrested and released (0 h G0/G1) AS CDK12 HCT116 cells either treated (3-MB-PP1) or not (CTRL) with the inhibitor for indicated times after the release. mRNA levels were normalized to B2M mRNA expression and mRNA levels for each gene at the time of release (0 h) was set as 1. n=3 replicates, error bars indicate SEM.  Figure 3e. Protein levels of core DNA replication factors are dependent on the CDK12 kinase activity. Western blot analyses of protein expression by the depicted antibodies in serum synchronized and released (0 h) cells either treated or not with 3-MB-PP1 for the indicated times after the release. FUS is a loading control. A representative Western blot of 3 replicates is shown.  Figure 3f. CDK12 inhibition affects loading of CDC6 and CDT1 DNA replication factors to chromatin. Western blotting analyses of chromatin association of the indicated DNA replication factors in serum synchronized and released AS CDK12 HCT116 cells treated or not with 3-MB-PP1 for the indicated times. Histone H2A serves as a loading control of chromatin fractions. A=asynchronous cells, 0 h=time of release. A representative Western blot of 3 replicates is shown.


Figure 4 Removal of CDK12 inhibitor in early G1 / S rescues replication gene expression and cell cycle progression . RT - qPCR of replication gene mRNA RT - qPCR were performed 7 , 12 and 15 h post - release , respectively . CTRL = control samples without the 3 - MB - PP1 . In b , n = 3 and error bars indicate SEM . In c , d representative images from 3 biological replicates are shown . Removal of CDK12 inhibitor in early G1 / S rescues cell cycle progression . Western blotting of , protein levels Western blotting were performed 7 , 12 and 15 h post - release , respectively . CTRL = control samples without the 3 - MB - PP1 . In b , n = 3 and error bars indicate SEM . In c , d representative images from 3 biological replicates are shown . Removal of CDK12 inhibitor in early G1 / S rescues replication and cell cycle progression . flow cytometry analyses of cell cycle progression , flow cytometry analyses were performed 7 , 12 and 15 h post - release , respectively . CTRL = control samples without the 3 - MB - PP1 . In b , n = 3 and error bars indicate SEM . In c , d representative images from 3 biological replicates are shown . Figure 4e . Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor . Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3 - MB - PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment . CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition . All cells were harvested either 6 or 9 h after the serum addition . Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated . A representative image of three replicates is shown . Figure 4f . Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells . Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3 - MB - PP1 and replicating BrdU - stained cells were quantified by FACS analyses . CTRL = control samples without the 3 - MB - PP1 . A representative image of 3 replicates is shown . Figure 4g , h . Prolonged CDK12 inhibition causes chromosomal aberrations in cells . Specific chromosomal aberrations in cells treated with 3 - MB - PP1 ( 24 or 48 h ) , 4 mM hydroxyurea ( 5 h ) or control solvent ( CTRL ) were identified by microscopy . A representative image from 3 biological replicates is shown ( g ) . Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified ( h ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 Removal of CDK12 inhibitor in early G1/S rescues replication gene expression and cell cycle progression. RT-qPCR of replication gene mRNA RT-qPCR were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Removal of CDK12 inhibitor in early G1/S rescues cell cycle progression. Western blotting of , protein levels Western blotting were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Removal of CDK12 inhibitor in early G1/S rescues replication and cell cycle progression. flow cytometry analyses of cell cycle progression, flow cytometry analyses were performed 7, 12 and 15 h post-release, respectively. CTRL=control samples without the 3-MB-PP1. In b, n=3 and error bars indicate SEM. In c, d representative images from 3 biological replicates are shown.  Figure 4e. Rescued loading of CDC6 and CDT1 on chromatin after removal of CDK12 inhibitor. Western blot analyses of chromatin fractions of serum starved AS CDK12 HCT116 cells treated with 3-MB-PP1 for 6 or 9 h or with the inhibitor washed off after 1 h of treatment. CTRL corresponds to cells not treated with the inhibitor at the time of the serum addition. All cells were harvested either 6 or 9 h after the serum addition. Histone H2A serves as a loading control of chromatin fractions and studied DNA replication factors are indicated. A representative image of three replicates is shown.  Figure 4f. Inhibition of CDK12 kinase activity in cycling cells leads to decreased numbers of actively replicating cells. Asynchronous AS CDK12 HCT116 cells were grown for 24 and 48 h in the presence or absence of 3-MB-PP1 and replicating BrdU-stained cells were quantified by FACS analyses. CTRL=control samples without the 3-MB-PP1. A representative image of 3 replicates is shown. Figure 4g, h. Prolonged CDK12 inhibition causes chromosomal aberrations in cells. Specific chromosomal aberrations in cells treated with 3-MB-PP1 (24 or 48 h), 4 mM hydroxyurea (5 h) or control solvent (CTRL) were identified by microscopy. A representative image from 3 biological replicates is shown (g). Total numbers of chromosomal aberrations per hundred cells of the representative replicate in g is quantified (h).


Figure 5 Figure 5b Genes down - regulated in nuclear RNA - seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3 ´ ends Metagene analyses of RNAPII ChIP - seq data Each transcript was divided into two parts with fixed length ( transcription start site ( TSS ) - 3kb to + 1 . 5kb and transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) and a central part with variable length corresponding to the rest of gene body ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) and average coverage for each bin was calculated for each transcript in each sample . The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS and TTS . The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated ( CTRL ) cells and cells treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with the Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 . Genes down - regulated in nuclear RNA - seq after CDK12 inhibition have higher relative occupancy of P - Ser2 in their gene bodies . Metagene analyses of P - Ser2 ChIP - seq data Each transcript was divided into two parts with fixed length ( transcription start site ( TSS ) - 3kb to + 1 . 5kb and transcription termination site ( TTS ) - 1 . 5kb to + 3kb ) and a central part with variable length corresponding to the rest of gene body ( shown in % ) . Each part was binned into a fixed number of bins ( 90 / 180 / 90 ) and average coverage for each bin was calculated for each transcript in each sample . The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples . Dotted lines indicate : TSS , 1500 nucleotides downstream of TSS , 1500 nucleotides upstream of TTS and TTS . The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated ( CTRL ) cells and cells treated with 3 - MB - PP1 . P - values are adjusted for multiple testing with the Bonferroni method within each subfigure ; color code : red = adjusted p - value ≤ 10 - 15 , orange = adjusted p - value ≤ 10 - 10 , yellow = adjusted p - value ≤ 10 - 3 . Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity . Nuclear RNA - seq data on the respective strand and RNAPII , P - Ser2 , P - Ser5 and SPT6 ChIP - seq data for MED13 genes from cells either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read counts were normalized to the total number of mapped reads per sample and averaged between replicates . Blue and red boxes below the RNA - seq data indicate the 90 % distance in control and CDK12 inhibited samples , respectively . Figure 5d e . Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity . Nuclear RNA - seq data on the respective strand and RNAPII , P - Ser2 , P - Ser5 and SPT6 ChIP - seq data for UBE3C genes from cells either treated ( red ) or not ( blue , CTRL ) with 3 - MB - PP1 were visualized with Gviz . Read counts were normalized to the total number of mapped reads per sample and averaged between replicates . Blue and red boxes below the RNA - seq data indicate the 90 % distance ( see Fig . 7d and 7e and corresponding text ) in control and CDK12 inhibited samples , respectively .

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

Figure 5 Figure 5b Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have diminished relative occupancy of RNAPII at their 3´ ends Metagene analyses of RNAPII ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.  Genes down-regulated in nuclear RNA-seq after CDK12 inhibition have higher relative occupancy of P-Ser2 in their gene bodies. Metagene analyses of P-Ser2 ChIP-seq data Each transcript was divided into two parts with fixed length (transcription start site (TSS) -3kb to +1.5kb and transcription termination site (TTS) -1.5kb to +3kb) and a central part with variable length corresponding to the rest of gene body (shown in %). Each part was binned into a fixed number of bins (90/180/90) and average coverage for each bin was calculated for each transcript in each sample. The curve for each transcript was normalized to a sum of one and then averaged first across genes and second across samples. Dotted lines indicate: TSS, 1500 nucleotides downstream of TSS, 1500 nucleotides upstream of TTS and TTS. The color track at the bottom of each subfigure indicate the significance of paired Wilcoxon tests comparing the normalized transcript coverages for each bin between untreated (CTRL) cells and cells treated with 3-MB-PP1. P-values are adjusted for multiple testing with the Bonferroni method within each subfigure; color code: red=adjusted p-value≤10-15, orange=adjusted p-value≤10-10, yellow=adjusted p-value≤10-3.  Figure 5d Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for MED13 genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance in control and CDK12 inhibited samples, respectively.  Figure 5d e. Examples of genes whose transcription processivity and expression is dependent on the CDK12 kinase activity. Nuclear RNA-seq data on the respective strand and RNAPII, P-Ser2, P-Ser5 and SPT6 ChIP-seq data for UBE3C genes from cells either treated (red) or not (blue, CTRL) with 3-MB-PP1 were visualized with Gviz. Read counts were normalized to the total number of mapped reads per sample and averaged between replicates. Blue and red boxes below the RNA-seq data indicate the 90% distance (see Fig. 7d and 7e and corresponding text) in control and CDK12 inhibited samples, respectively.


Figure 7 Figure 7a . Longer genes tend to have a larger fraction of differentially used exons . Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length ( including exons and introns ) of their longest transcripts . n = 3 replicates .

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

Figure 7 Figure 7a. Longer genes tend to have a larger fraction of differentially used exons. Box plot shows the fraction of exons significantly differentially used for 9026 expressed genes grouped into deciles based on the genomic length (including exons and introns) of their longest transcripts. n=3 replicates.


Figure 8 Figure 8b . Transcription elongation rate decreases in bodies of CDK12 - dependent but not CDK12 - independent genes after CDK12 inhibition . Graphs show relative levels of pre - mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3 - MB - PP1 or not ( CTRL ) for indicated times after DRB wash off . Pre - mRNA levels were normalized to the samples not treated with DRB ( Unt ) for which the value was set as 1 . n = 3 independent experiments , error bars correspond to SEM . Positions of primers ( designed to span exon - intron junctions ) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs . Figure 8c . Proposed model . Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes . The situation in cells with normal and aberrant CDK12 kinase activity is depicted . CDK12 ( green oval ) phosphorylates ( P ) unknown substrate ( s ) ( orange oval ) , possibly including the CTD ( blue line ) , which results in optimal elongation and processivity ( blue arrow ) of RNAPII ( blue oval ) for CDK12 - sensitive genes . Full length , functional mRNAs are synthesized ( upper panel ) . Inhibition of CDK12 leads to hyperphosphorylation ( capital P ) of Ser2 ( S2 ) in bodies of CDK12 - sensitive genes which is associated with slower elongation and premature termination . Shorter , aberrant mRNAs are made ( lower panel ) . mRNAs are depicted as black lines .

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

Figure 8 Figure 8b. Transcription elongation rate decreases in bodies of CDK12-dependent but not CDK12-independent genes after CDK12 inhibition. Graphs show relative levels of pre-mRNAs of described genes in AS CDK12 HCT116 cells either treated with 3-MB-PP1 or not (CTRL) for indicated times after DRB wash off. Pre-mRNA levels were normalized to the samples not treated with DRB (Unt) for which the value was set as 1. n=3 independent experiments, error bars correspond to SEM. Positions of primers (designed to span exon-intron junctions) and their distance from the transcription start site in kb are indicated in the gene structures shown above the graphs. Figure 8c. Proposed model. Schema shows groups of genes whose RNAPII processivity is particularly sensitive to CDK12 catalytic activity and cellular functions that are especially dependent on optimal expression of these genes. The situation in cells with normal and aberrant CDK12 kinase activity is depicted. CDK12 (green oval) phosphorylates (P) unknown substrate(s) (orange oval), possibly including the CTD (blue line), which results in optimal elongation and processivity (blue arrow) of RNAPII (blue oval) for CDK12-sensitive genes. Full length, functional mRNAs are synthesized (upper panel). Inhibition of CDK12 leads to hyperphosphorylation (capital P) of Ser2 (S2) in bodies of CDK12-sensitive genes which is associated with slower elongation and premature termination. Shorter, aberrant mRNAs are made (lower panel). mRNAs are depicted as black lines.


Figure 1U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , anti - human centromere autoantibody ( ACA ) , and antibodies for Bub1 and TOP2A . Example images are shown ( A ) . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( B ) , centromeric TOP2A / arm TOP2A ( C ) , and arm TOP2A / DNA ( D ) , were determined on around 600 ( B and C ) or 400 ( D ) chromosomes in 20 cells . ns , no statistical significance . Data information : Means and error bars representing S . D . are shown unpaired t test ) . Scale bars , 10 μm . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining with DAPI , and antibodies for Bub1 and TOP2A . Lysates of nocodazole - arrested mitotic cells were immunoblotted with the indicated antibodies ( E ) . ns , no statistical significance . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . Example images are shown ( F ) . U2OS cells were transfected with siRNA , and subjected to immunofluorescence staining . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 500 chromosomes in 15 cells ( G ) . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . Example images of the immunofluorescence staining are shown ( H ) . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric Bub1 / ACA ( I ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . U2OS cells treated with STLC and MG132 for 0 . 5 hr were subjected to further treatment with Reversine , AZ3146 or dimethyl sulfoxide ( DMSO , vehicle control ) for 1 hr in the continued presence of STLC and MG132 . The immunofluorescence intensity ratios of centromeric TOP2A / ACA ( J ) , and centromeric TOP2A / arm TOP2A ( K ) were determined on around 300 chromosomes in 15 cells . Data information : Means and error bars representing S . D . are shown Scale bars , 10 μm . L U2OS cells stably expressing CB - GFP or CB - Bub1 - K - GFP were treated as in ( H ) . Example images of the immunofluorescence staining are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, anti-human centromere autoantibody (ACA), and antibodies for Bub1 and TOP2A. Example images are shown (A). The immunofluorescence intensity ratios of centromeric TOP2A/ACA (B), centromeric TOP2A/arm TOP2A (C), and arm TOP2A/DNA (D), were determined on around 600 (B and C) or 400 (D) chromosomes in 20 cells. ns, no statistical significance. Data information: Means and error bars representing S.D. are shown unpaired t test). Scale bars, 10 μm.U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining with DAPI, and antibodies for Bub1 and TOP2A. Lysates of nocodazole-arrested mitotic cells were immunoblotted with the indicated antibodies (E). ns, no statistical significance.U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. Example images are shown (F).U2OS cells were transfected with siRNA, and subjected to immunofluorescence staining. The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 500 chromosomes in 15 cells (G). Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. Example images of the immunofluorescence staining are shown (H).U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric Bub1/ACA (I) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.U2OS cells treated with STLC and MG132 for 0.5 hr were subjected to further treatment with Reversine, AZ3146 or dimethyl sulfoxide (DMSO, vehicle control) for 1 hr in the continued presence of STLC and MG132. The immunofluorescence intensity ratios of centromeric TOP2A/ACA (J), and centromeric TOP2A/arm TOP2A (K) were determined on around 300 chromosomes in 15 cells. Data information: Means and error bars representing S.D. are shown Scale bars, 10 μm.L U2OS cells stably expressing CB-GFP or CB-Bub1-K-GFP were treated as in (H). Example images of the immunofluorescence staining are shown.


Figure 2U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA , and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A ( A ) . Data information : Scale bars , 10 μm . U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were transfected with siRNA Lysates of nocodazole - arrested mitotic cells were immunoblotted ( B ) . * represents non - specific bands . U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . Example images are shown ( C ) . Data information : Scale bars , 10 μm . U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr , and subjected to immunofluorescence staining . The immunofluorescence intensity ratios of centromeric H2ApT120 / ACA ( D ) , centromeric TOP2A / ACA ( E ) , and centromeric TOP2A / arm TOP2A ( F ) were determined on around 450 chromosomes in 15 cells . Means and error bars representing S . D . are shown ( D - F ; unpaired t test ) .

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Figure 2U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA, and subjected to immunofluorescence staining with DAPI and the antibodies for H2ApT120 and TOP2A (A). Data information: Scale bars, 10 μm.U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were transfected with siRNA Lysates of nocodazole-arrested mitotic cells were immunoblotted (B). * represents non-specific bands.U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. Example images are shown (C). Data information: Scale bars, 10 μm.U2OS cells were treated with DMSO or BAY 1816032 at the indicated concentrations for 1 hr, and subjected to immunofluorescence staining. The immunofluorescence intensity ratios of centromeric H2ApT120/ACA (D), centromeric TOP2A/ACA (E), and centromeric TOP2A/arm TOP2A (F) were determined on around 450 chromosomes in 15 cells. Means and error bars representing S.D. are shown (D-F; unpaired t test).


Figure 3A U2OS cells were subjected to immunofluorescence staining . Example images of mitotic cells at various stages are shown . Data information : Scale bars , 10 μm . B U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining . Example images of anaphase cells are shown . Data information : Scale bars , 10 μm . C U2OS cells stably expressing CB - GFP , CB - Bub1 - K - GFP or CB - Bub1 - K - D946N - GFP were subjected to immunofluorescence staining with DAPI , and antibodies for TOP2A and H2ApT120 or H3pS10 . Example images of interphase cells are shown . The cells in the top line , which is H3pS10 - positive , are in early prophase / late G2 . Data information : Scale bars , 10 μm . D U2OS - LacO cells transiently expressing EGFP - LacI , EGFP - LacI - Bub1 - K ( WT or D946N ) were subjected to immunofluorescence staining . Arrows point to the transgene loci . Data information : Scale bars , 10 μm . E , F MBP - TOP2A ( 429 - 1531 ) - 10xHis was subjected to pulldown by GST , GST - H2A , or GST - H2ApS120 , followed by immunoblotting with antibodies for MBP , GST or H2ApT120 ( E ) . The relative pulldown efficiency was determined ( F ) . Means and error bars representing S . D . from three independent experiments are shown ( unpaired t - test ) .

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Figure 3A U2OS cells were subjected to immunofluorescence staining. Example images of mitotic cells at various stages are shown. Data information: Scale bars, 10 μm.B U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining. Example images of anaphase cells are shown. Data information: Scale bars, 10 μm.C U2OS cells stably expressing CB-GFP, CB-Bub1-K-GFP or CB-Bub1-K-D946N-GFP were subjected to immunofluorescence staining with DAPI, and antibodies for TOP2A and H2ApT120 or H3pS10. Example images of interphase cells are shown. The cells in the top line, which is H3pS10-positive, are in early prophase/late G2. Data information: Scale bars, 10 μm.D U2OS-LacO cells transiently expressing EGFP-LacI, EGFP-LacI-Bub1-K (WT or D946N) were subjected to immunofluorescence staining. Arrows point to the transgene loci. Data information: Scale bars, 10 μm.E, F MBP-TOP2A (429-1531)-10xHis was subjected to pulldown by GST, GST-H2A, or GST-H2ApS120, followed by immunoblotting with antibodies for MBP, GST or H2ApT120 (E). The relative pulldown efficiency was determined (F). Means and error bars representing S.D. from three independent experiments are shown (unpaired t-test).


Figure 4A , B HeLa cells and the Sgo1 - K492A mutant cells were immunostained with the indicated antibodies . Example images are shown ( A ) . The immunofluorescence intensity ratio of centromeric INCENP / ACA was determined on around 250 chromosomes in 10 cells ( B ) . Data information : Means and error bars representing S . D . are shown ; unpaired t - test ) . Scale bars , 10 μm . C , D HeLa and the Sgo1 - K492A cells were exposed to nocodazole for 4 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( C ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 150 chromosomes in 10 cells ( D ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm . E , F HeLa and the Sgo1 - K492A cells were transfected with siRNA , and immunostained with the indicated antibodies . Example images are shown ( E ) . The immunofluorescence intensity ratio of centromeric Aurora B / ACA was determined on around 250 chromosomes in 10 cells ( F ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm . G , H HeLa and the Sgo1 - K492A cells were transfected with siRNA , and treated with nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and fixed for immunostaining . Example images are shown ( G ) . The immunofluorescence intensity ratio of centromeric TOP2A / arm TOP2A was determined on around 280 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . Scale bars , 10 μm .

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Figure 4A, B HeLa cells and the Sgo1-K492A mutant cells were immunostained with the indicated antibodies. Example images are shown (A). The immunofluorescence intensity ratio of centromeric INCENP/ACA was determined on around 250 chromosomes in 10 cells (B). Data information: Means and error bars representing S.D. are shown ; unpaired t-test). Scale bars, 10 μm.C, D HeLa and the Sgo1-K492A cells were exposed to nocodazole for 4 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (C). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 150 chromosomes in 10 cells (D). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.E, F HeLa and the Sgo1-K492A cells were transfected with siRNA, and immunostained with the indicated antibodies. Example images are shown (E). The immunofluorescence intensity ratio of centromeric Aurora B/ACA was determined on around 250 chromosomes in 10 cells (F). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.G, H HeLa and the Sgo1-K492A cells were transfected with siRNA, and treated with nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining. Example images are shown (G). The immunofluorescence intensity ratio of centromeric TOP2A/arm TOP2A was determined on around 280 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test). Scale bars, 10 μm.


Figure 5B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins , and then exposed to nocodazole for 5 hr . Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI , ACA and anti - GFP antibodies . Example images are shown . Scale bars , 10 μm . HeLa cells were transfected and treated Example images are shown ( C ) . Scale bars , 10 μm . HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP - TOP2A / ACA ( D ) , centromeric EGFP - TOP2A / arm EGFP - TOP2A ( E ) , and arm EGFP - TOP2A / DNA ( F ) , were determined on around 160 chromosomes in 8 cells . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained Example images are shown ( G ) . Scale bars , 10 μm . HeLa cells transiently expressing EGFP - TOP2A ( 970 - 1531 ) or EGFP - TOP2A ( 970 - 1531 ) - Δ ( 1015 - 1032 ) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP - TOP2A / arm EGFP - TOP2A was determined on around 250 chromosomes in 10 cells ( H ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Example images are shown ( I ) . Scale bars , 10 μm . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . The relative enrichment of EGFP - TOP2A at the LacO repeats was quantified in 130 cells ( J ) . Data information : Means and error bars representing S . D . are shown unpaired t - test ) . U2OS - LacO cells transiently expressing Myc - LacI - Bub1 and EGFP - TOP2A ( 970 - 1531 ) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc - tag and GFP . Cell lysates were immunoblotted ( K ) .

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Figure 5B HeLa cells were transfected with siRNA and the plasmids encoding the indicated proteins, and then exposed to nocodazole for 5 hr. Mitotic cells were cytospun onto coverslips and subjected to immunofluorescence staining with DAPI, ACA and anti-GFP antibodies. Example images are shown. Scale bars, 10 μm.HeLa cells were transfected and treated Example images are shown (C). Scale bars, 10 μm.HeLa cells were transfected and treated The immunofluorescence intensity ratios of centromeric EGFP-TOP2A/ACA (D), centromeric EGFP-TOP2A/arm EGFP-TOP2A (E), and arm EGFP-TOP2A/DNA (F), were determined on around 160 chromosomes in 8 cells. Data information: Means and error bars representing S.D. are shown unpaired t-test).HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained Example images are shown (G). Scale bars, 10 μm.HeLa cells transiently expressing EGFP-TOP2A (970-1531) or EGFP-TOP2A (970-1531)-Δ(1015-1032) were treated and immunostained The immunofluorescence intensity ratio of centromeric EGFP-TOP2A/arm EGFP-TOP2A was determined on around 250 chromosomes in 10 cells (H). Data information: Means and error bars representing S.D. are shown unpaired t-test).U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Example images are shown (I). Scale bars, 10 μm.U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. The relative enrichment of EGFP-TOP2A at the LacO repeats was quantified in 130 cells (J). Data information: Means and error bars representing S.D. are shown unpaired t-test).U2OS-LacO cells transiently expressing Myc-LacI-Bub1 and EGFP-TOP2A (970-1531) were subjected to immunofluorescence staining with DAPI and antibodies for the Myc-tag and GFP. Cell lysates were immunoblotted (K).