A ) Oocytes were fixed for chromosome spreads 8 hours after GVBD , and stained for PP2A - c ( red ) , Sgo2 ( green ) , CREST ( blue ) and DNA ( Hoechst , shown in grey ) . Spreads were classified into metaphase I and anaphase I , depending on whether chromosome segregation had taken place , or not . Arrowhead indicates fused sister kinetochores , arrows indicate separated sister kinetochores .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oocytes were fixed for chromosome spreads 8 hours after GVBD, and stained for PP2A-c (red), Sgo2 (green), CREST (blue) and DNA (Hoechst, shown in grey). Spreads were classified into metaphase I and anaphase I, depending on whether chromosome segregation had taken place, or not. Arrowhead indicates fused sister kinetochores, arrows indicate separated sister kinetochores.


B ) Example of a whole mount oocyte staining , in early anaphase I after cold - treatment to visualize only cold - stable microtubule fibers , stained with anti - tubulin antibody ( green ) . Kinetochores are stained with CREST serum ( red ) and chromosomes with Hoechst ( blue ) . Shown is an overlay of 20 z - sections . Arrows indicate separated sister kinetochores .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example of a whole mount oocyte staining, in early anaphase I after cold-treatment to visualize only cold-stable microtubule fibers, stained with anti-tubulin antibody (green). Kinetochores are stained with CREST serum (red) and chromosomes with Hoechst (blue). Shown is an overlay of 20 z-sections. Arrows indicate separated sister kinetochores.


C ) Metaphase I ( 6 hours after GVBD ) and metaphase II ( 16 - 20 hours after GVBD ) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin ( pink ) and the centromere ( green ) , respectively . Chromosomes were stained with Hoechst ( blue ) . Below , schematic representation of a bivalent in metaphase I and a dyad in metaphase II . Enlargement shows the centromere region and a region that we call " chromatid junction " with the corresponding scheme .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Metaphase I (6 hours after GVBD) and metaphase II (16-20 hours after GVBD) chromosome spreads were stained with H3K9me3 antibody and CREST serum to reveal pericentromeric heterochromatin (pink) and the centromere (green), respectively. Chromosomes were stained with Hoechst (blue). Below, schematic representation of a bivalent in metaphase I and a dyad in metaphase II. Enlargement shows the centromere region and a region that we call "chromatid junction" with the corresponding scheme.


D ) Quantification of total Sgo2 signal at the centromere and the chromatid junction , normalized to CREST at centromeres , on chromosome spreads from oocytes at the indicated stages of meiotic maturation . Each dot shows mean per oocyte ; the number of oocytes analyzed is indicated . Below : scheme illustrating how Sgo2 signals ( in red ) were measured at the indicated meiotic stages . Ifluor stands for a mean fluorescence intensity of the area within the box ( dotted lines , see Material and Methods for quantification approach ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of total Sgo2 signal at the centromere and the chromatid junction, normalized to CREST at centromeres, on chromosome spreads from oocytes at the indicated stages of meiotic maturation. Each dot shows mean per oocyte; the number of oocytes analyzed is indicated. Below: scheme illustrating how Sgo2 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines, see Material and Methods for quantification approach).


E ) Chromosome spreads as in A ) , stained for Sgo2 ( red ) , CREST ( green ) , and DNA ( Hoechst , in blue ) . Spreads in early anaphase I and late anaphase I - telophase I were classified , depending on whether dyads of two chromosome sets had been found close to each other as one group , or were scattered apart into two groups . In early anaphase I , Sgo2 is found inbetween sister kinetochores that are already separated . F ) Quantification of E ) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair ( dot plot on the left ) , and distance between sister kinetochores ( dot plot on the right ) at the indicated stages of meiosis I . Meta : metaphase , ana : anaphase , telo : telophase . Each dot represents one kinetochore pair ; the number of kinetochore pairs analysed is indicated . Below : scheme of Sgo2 signal ( in red ) measurements .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated. F) Quantification of E) showing total Sgo2 signal in the centromere region relative to CREST per kinetochore pair (dot plot on the left), and distance between sister kinetochores (dot plot on the right) at the indicated stages of meiosis I. Meta: metaphase, ana: anaphase, telo: telophase. Each dot represents one kinetochore pair; the number of kinetochore pairs analysed is indicated. Below: scheme of Sgo2 signal (in red) measurements.


E ) Chromosome spreads as in A ) , stained for Sgo2 ( red ) , CREST ( green ) , and DNA ( Hoechst , in blue ) . Spreads in early anaphase I and late anaphase I - telophase I were classified , depending on whether dyads of two chromosome sets had been found close to each other as one group , or were scattered apart into two groups . In early anaphase I , Sgo2 is found inbetween sister kinetochores that are already separated .

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

E) Chromosome spreads as in A), stained for Sgo2 (red), CREST (green), and DNA (Hoechst, in blue). Spreads in early anaphase I and late anaphase I - telophase I were classified, depending on whether dyads of two chromosome sets had been found close to each other as one group, or were scattered apart into two groups. In early anaphase I, Sgo2 is found inbetween sister kinetochores that are already separated.


A ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Sgo2 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . On the right , schemes illustrate the chromosome figures observed , and the corresponding Sgo2 staining ( in red ) .

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

A) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Sgo2 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). On the right, schemes illustrate the chromosome figures observed, and the corresponding Sgo2 staining (in red).


B ) Dyads of metaphase II chromosome spreads of control , Bub1KD , Mps1 ∆ N and Reversine - treated oocytes ( from GVBD onwards ) stained with Sgo2 antibody ( red ) , CREST serum ( green ) and Hoechst ( blue ) .

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

B) Dyads of metaphase II chromosome spreads of control, Bub1KD, Mps1∆N and Reversine-treated oocytes (from GVBD onwards) stained with Sgo2 antibody (red), CREST serum (green) and Hoechst (blue).


C ) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards , corresponding to the time of anaphase I onset . PBE : Polar Body Extrusion .

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

C) Dyads of metaphase II chromosome spreads of oocytes treated with Reversine from GVBD + 8 hours onwards, corresponding to the time of anaphase I onset. PBE: Polar Body Extrusion.


D ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Mps1 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later , respectively . E ) Quantification of D ) showing Mps1 signal relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Mps1 signal averaged per oocyte ( right dot plot ) at the indicated time points . The number of kinetochore pairs ( kinetochores in anaphase II ) and oocytes analyzed is indicated . The scheme of measurements is shown on the right .

O O O 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 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

D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively. E) Quantification of D) showing Mps1 signal relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Mps1 signal averaged per oocyte (right dot plot) at the indicated time points. The number of kinetochore pairs (kinetochores in anaphase II) and oocytes analyzed is indicated. The scheme of measurements is shown on the right.


D ) Chromosome spreads at the indicated stages of meiosis , stained for endogenous Mps1 ( red ) , centromeres with CREST serum ( green ) and DNA with Hoechst ( blue ) . For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later , 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 B-MEASURED_VAR 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

D) Chromosome spreads at the indicated stages of meiosis, stained for endogenous Mps1 (red), centromeres with CREST serum (green) and DNA with Hoechst (blue). For early and late anaphase II spreads oocytes were chemically activated with Strontium and fixed 25min or 1h later, respectively.


F ) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst . Where indicated , oocytes were treated with Reversine from GVBD onwards , and treatment was renewed every 8 hours . Early CSF corresponds to 8 hours after GVBD ( Reversine treated oocytes undergo anaphase I on average 4 - 5 hours after GVBD ) , and late CSF corresponds to 36 hours after GVBD ( > 29 hours in CSF arrest ) . PSCS : precocious sister chromatid segregation .

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

F) Percentage of oocytes harbouring one or more single sister chromatids when analysed by chromosome spreads stained with CREST and Hoechst. Where indicated, oocytes were treated with Reversine from GVBD onwards, and treatment was renewed every 8 hours. Early CSF corresponds to 8 hours after GVBD (Reversine treated oocytes undergo anaphase I on average 4-5 hours after GVBD), and late CSF corresponds to 36 hours after GVBD (>29 hours in CSF arrest). PSCS: precocious sister chromatid segregation.


G ) Quantification of total Sgo2 signal on spreads in early or late CSF arrest ( 16 hours or 30 hours after GVBD , respectively ) relative to CREST per kinetochore pair ( per single kinetochore in anaphase II ) ( left dot plot ) , and Sgo2 signal averaged per oocyte ( right dot plot ) at the indicated stages . The number of kinetochore pairs and oocytes analyzed is indicated . The scheme of measurements is shown on the right . Meta : metaphase , ana : anaphase .

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

G) Quantification of total Sgo2 signal on spreads in early or late CSF arrest (16 hours or 30 hours after GVBD, respectively) relative to CREST per kinetochore pair (per single kinetochore in anaphase II) (left dot plot), and Sgo2 signal averaged per oocyte (right dot plot) at the indicated stages. The number of kinetochore pairs and oocytes analyzed is indicated. The scheme of measurements is shown on the right. Meta: metaphase, ana: anaphase.


( A and D ) Oocytes in meiosis I ( 6 hours after GVBD ) or meiosis II ( left and right panels , respectively ) were treated with STLC for 2 . 5 hours and fixed for whole mount immunofluorescence . Cold stable microtubules were stained with an antibody against tubulin ( green ) , centromeres with CREST ( red ) and DNA with Hoechst ( blue ) . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(A and D) Oocytes in meiosis I (6 hours after GVBD) or meiosis II (left and right panels, respectively) were treated with STLC for 2.5 hours and fixed for whole mount immunofluorescence. Cold stable microtubules were stained with an antibody against tubulin (green), centromeres with CREST (red) and DNA with Hoechst (blue). Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


( B and E ) Chromosome spreads at metaphase and anaphase of meiosis I ( 6 and 9 hours after GVBD ) or meiosis II ( left and right panels , respectively ) , in the presence or absence of STLC ( for 2 . 5 hours ) and Reversine ( + Rev . , for 1 hour ) as indicated . Spreads were stained with CREST serum ( red ) and Hoechst ( grey ) . Single chromosomes are present in anaphase I and single sister chromatids in anaphase II . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(B and E) Chromosome spreads at metaphase and anaphase of meiosis I (6 and 9 hours after GVBD) or meiosis II (left and right panels, respectively), in the presence or absence of STLC (for 2.5 hours) and Reversine (+Rev., for 1 hour) as indicated. Spreads were stained with CREST serum (red) and Hoechst (grey). Single chromosomes are present in anaphase I and single sister chromatids in anaphase II. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


( C and F ) Chromosome spreads exactly as described in B and E ) , except that spreads were additionally stained with an antibody against Rec8 ( green ) . The CREST signal appears in red and DNA in blue . Rev : Reversine . Scale bar indicates 5 µm in ( A and D ) , and 10 µm in ( B , C , E and F ) . In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed .

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

(C and F) Chromosome spreads exactly as described in B and E), except that spreads were additionally stained with an antibody against Rec8 (green). The CREST signal appears in red and DNA in blue. Rev: Reversine. Scale bar indicates 5 µm in (A and D), and 10 µm in (B, C, E and F). In all panels n indicates the number of oocytes with the shown phenotype and the total number of oocytes analyzed.


A ) Metaphase II oocytes from mice carrying a conditional , oocyte - specific invalidation of separase ( Separaseflox / flox Zp3 Cre + , or Sep - / - ) and oocytes from litter mates ( Separaseflox / flox , or Sep + / + ) were obtained after superovulation . In vivo matured control ( Sep + / + ) and Sep - / - oocytes are found in oviducts at comparable numbers , enclosed by cumulus cells . Example of cumulus enclosed oocytes from the oviduct is shown below . In ( A ) , n indicates the number of mice and a mean number of oocytes per mouse of each genotype , error bars are + s . e . m .

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A) Metaphase II oocytes from mice carrying a conditional, oocyte-specific invalidation of separase (Separaseflox/flox Zp3 Cre+, or Sep-/-) and oocytes from litter mates (Separaseflox/flox, or Sep+/+) were obtained after superovulation. In vivo matured control (Sep+/+) and Sep-/- oocytes are found in oviducts at comparable numbers, enclosed by cumulus cells. Example of cumulus enclosed oocytes from the oviduct is shown below. In (A), n indicates the number of mice and a mean number of oocytes per mouse of each genotype, error bars are + s.e.m.


B ) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation . Microtubules were stained with anti - tubulin antibody ( green ) , centromeres with CREST serum ( red ) and DNA with Hoechst ( blue ) . Bivalents are attached in a monopolar fashion . Approximate number of z - sections used for overlays to visualize the whole spindle ( on the left ) or individual chromosomes ( magnifications on the right ) are indicated . C ) Same as B ) , except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards . In ( B and C ) n indicates number of oocytes analyzed .

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

B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated. C) Same as B), except that oocytes of the indicated genotypes were obtained after in vitro maturation from GV onwards. In (B and C) n indicates number of oocytes analyzed.


B ) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation . Microtubules were stained with anti - tubulin antibody ( green ) , centromeres with CREST serum ( red ) and DNA with Hoechst ( blue ) . Bivalents are attached in a monopolar fashion . Approximate number of z - sections used for overlays to visualize the whole spindle ( on the left ) or individual chromosomes ( magnifications on the right ) are indicated .

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

B) Whole mount immunofluorescence to stain cold stable microtubule fibers after in vivo maturation. Microtubules were stained with anti-tubulin antibody (green), centromeres with CREST serum (red) and DNA with Hoechst (blue). Bivalents are attached in a monopolar fashion. Approximate number of z-sections used for overlays to visualize the whole spindle (on the left) or individual chromosomes (magnifications on the right) are indicated.


D ) Metaphase - to - anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated , in GV . The schemes indicate the chromosome figures observed upon activation . Chromosome spreads are stained with CREST serum ( green ) , and Rec8 antibody ( red ) . DNA was stained with DAPI ( blue ) . In ( D ) n indicates the number of oocytes with the shown phenotype ( e . g . , upon successful activation ) and the total number of oocytes analyzed .

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

D) Metaphase-to-anaphase transition of meiosis II in oocytes of the indicated genotype cultured in vitro and injected with the separase constructs as indicated, in GV. The schemes indicate the chromosome figures observed upon activation. Chromosome spreads are stained with CREST serum (green), and Rec8 antibody (red). DNA was stained with DAPI (blue). In (D) n indicates the number of oocytes with the shown phenotype (e.g., upon successful activation) and the total number of oocytes analyzed.


A ) Sep + / + or Sep - / - oocytes were matured in vitro until metaphase II . Sep - / - oocytes were injected with wild - type separase or cleavage mutant separase encoding mRNAs in metaphase II . Oocytes were either fixed in metaphase II or activated and fixed in anaphase II . Spreads were stained with Rec8 antibody ( red ) , CREST serum ( green ) , and DAPI ( blue ) to label DNA . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed

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

A) Sep+/+ or Sep-/- oocytes were matured in vitro until metaphase II. Sep-/- oocytes were injected with wild-type separase or cleavage mutant separase encoding mRNAs in metaphase II. Oocytes were either fixed in metaphase II or activated and fixed in anaphase II. Spreads were stained with Rec8 antibody (red), CREST serum (green), and DAPI (blue) to label DNA. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed


A ) Sep + / + or Sep - / - oocytes were matured in vitro until metaphase II . Sep - / - oocytes were injected with wild - type separase or cleavage mutant separase encoding mRNAs in metaphase II . Oocytes were either fixed in metaphase II or activated and fixed in anaphase II . Spreads were stained with Rec8 antibody ( red ) , CREST serum ( green ) , and DAPI ( blue ) to label DNA . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes

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


B ) Representative image of kinetochore individualization in Sep - / - oocytes injected with wild - type separase in metaphase II and fixed after activation , from A ) . Shown is staining with CREST serum ( green ) , and DAPI ( blue ) to label DNA . Arrows indicate separated sister kinetochores . The scheme on the left illustrates the individualization of sister kinetochores ( green arrows ) at anaphase II . n indicates the number of oocytes with the shown phenotype ( e . g . in anaphase II , after successful activation ) and the total number of oocytes analysed Scale bars : 10 µm , except bottom panel in B ) , where the scale bar is 2 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B) Representative image of kinetochore individualization in Sep-/- oocytes injected with wild-type separase in metaphase II and fixed after activation, from A). Shown is staining with CREST serum (green), and DAPI (blue) to label DNA. Arrows indicate separated sister kinetochores. The scheme on the left illustrates the individualization of sister kinetochores (green arrows) at anaphase II. n indicates the number of oocytes with the shown phenotype (e.g. in anaphase II, after successful activation) and the total number of oocytes analysed Scale bars: 10 µm, except bottom panel in B), where the scale bar is 2 μm.


A ) Live imaging movie of GFP - Sgo2 expressing oocytes ( from GV onwards ) undergoing meiosis I . The green channel to visualize GFP - Sgo2 localized to chromosomes is shown . Timepoints were taken every 10min , shown is a montage of selected frames such as indicated ( hours : time after GVBD ) at the metaphase - to - anaphase transition of meiosis I and in metaphase II . Each timepoint is comprised of an overlay of 11 z - sections of 3μm for GFP . The arrow heads indicate bivalents . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Live imaging movie of GFP-Sgo2 expressing oocytes (from GV onwards) undergoing meiosis I. The green channel to visualize GFP-Sgo2 localized to chromosomes is shown. Timepoints were taken every 10min, shown is a montage of selected frames such as indicated (hours: time after GVBD) at the metaphase-to-anaphase transition of meiosis I and in metaphase II. Each timepoint is comprised of an overlay of 11 z-sections of 3μm for GFP. The arrow heads indicate bivalents. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed


A ) Live imaging movie of GFP - Sgo2 expressing oocytes ( from GV onwards ) undergoing meiosis I . The green channel to visualize GFP - Sgo2 localized to chromosomes is shown . Timepoints were taken every 10min , shown is a montage of selected frames such as indicated ( hours : time after GVBD ) at the metaphase - to - anaphase transition of meiosis I and in metaphase II . Each timepoint is comprised of an overlay of 11 z - sections of 3μm for GFP . The arrow heads indicate bivalents . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes

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B ) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP - Sgo2 expressing oocyte . Spreads were stained with Rec8 antibody ( pink ) , CREST serum ( green ) , Hoechst for DNA ( blue ) , GFP - Sgo2 was visualized by GFP fluorescence ( grey ) . The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed

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

B) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP-Sgo2 expressing oocyte. Spreads were stained with Rec8 antibody (pink), CREST serum (green), Hoechst for DNA (blue), GFP-Sgo2 was visualized by GFP fluorescence (grey). The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms. In (A-C) n indicates the number of control non-injected and GFP-Sgo2-expressing oocytes analyzed


B ) Representative images of a chromosome spread showing Rec8 localization on a bivalent chromosome in metaphase II of a GFP - Sgo2 expressing oocyte . Spreads were stained with Rec8 antibody ( pink ) , CREST serum ( green ) , Hoechst for DNA ( blue ) , GFP - Sgo2 was visualized by GFP fluorescence ( grey ) . The scheme on the right shows a bivalent with individualized kinetochores and some Rec8 staining on arms . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes

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


C ) Metaphase I ( 4hrs after GVBD ) and II spreads after GFP - Sgo2 expression ( from GV onwards ) . Spreads were stained with CREST serum ( red ) , Hoechst for DNA ( grey ) , GFP - Sgo2 was visualized by GFP fluorescence ( green ) . Arrowheads mark separated sister kinetochores . On the right , graph showing percentage of metaphase II spreads containing bivalent chromosomes . In ( A - C ) n indicates the number of control non - injected and GFP - Sgo2 - expressing oocytes analyzed The number of analysed oocytes in C )

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

C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP -Sgo2-expressing oocytes analyzed The number of analysed oocytes in C)


C ) Metaphase I ( 4hrs after GVBD ) and II spreads after GFP - Sgo2 expression ( from GV onwards ) . Spreads were stained with CREST serum ( red ) , Hoechst for DNA ( grey ) , GFP - Sgo2 was visualized by GFP fluorescence ( green ) . Arrowheads mark separated sister kinetochores . On the right , graph showing percentage of metaphase II spreads containing bivalent chromosomes . In ( A - C ) n indicates the number of control non - injected 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 O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Metaphase I (4hrs after GVBD) and II spreads after GFP-Sgo2 expression (from GV onwards). Spreads were stained with CREST serum (red), Hoechst for DNA (grey), GFP-Sgo2 was visualized by GFP fluorescence (green). Arrowheads mark separated sister kinetochores. On the right, graph showing percentage of metaphase II spreads containing bivalent chromosomes. In (A-C) n indicates the number of control non-injected and GFP


D ) Chromosome spreads of oocytes expressing GFP - Sgo2 ( from GV onwards ) in metaphase II , and upon activation . Spreads were labeled with Rec8 antibody ( shown in red , far - red secondary antibody was used ) , CREST serum ( pseudo - colored in green ) , and Hoechst ( blue ) . GFP direct fluorescence was imaged for GFP - Sgo2 detection . Below the corresponding chromosome figures are represented in the schemes . E ) Quantification of Rec8 signal found around and in between CREST signals that were close to each other . Below : scheme illustrating how Rec8 signals ( in red ) were measured at the indicated meiotic stages . Ifluor stands for a mean fluorescence intensity of the area within the box ( dotted lines ) . Meta : metaphase , ana : anaphase . D ) , the number of oocytes with the shown phenotype and the total number of oocytes analyzed . On each graph , mean is shown , error bars are + s . e . m . in C ) and and ± SD in E ) , asterisks indicate significant difference ( * * * * = p < 0 , 0001 ) according to Mann - Whitney U test . AU : arbitrary units . The number of analysed kinetochore pairs in E ) is indicated .

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D) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes. E) Quantification of Rec8 signal found around and in between CREST signals that were close to each other. Below: scheme illustrating how Rec8 signals (in red) were measured at the indicated meiotic stages. Ifluor stands for a mean fluorescence intensity of the area within the box (dotted lines). Meta: metaphase, ana: anaphase. D), the number of oocytes with the shown phenotype and the total number of oocytes analyzed. On each graph, mean is shown, error bars are +s.e.m. in C) and and ± SD in E), asterisks indicate significant difference (**** = p<0,0001) according to Mann-Whitney U test. AU: arbitrary units. The number of analysed kinetochore pairs in E) is indicated.


D ) Chromosome spreads of oocytes expressing GFP - Sgo2 ( from GV onwards ) in metaphase II , and upon activation . Spreads were labeled with Rec8 antibody ( shown in red , far - red secondary antibody was used ) , CREST serum ( pseudo - colored in green ) , and Hoechst ( blue ) . GFP direct fluorescence was imaged for GFP - Sgo2 detection . Below the corresponding chromosome figures are represented in the schemes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chromosome spreads of oocytes expressing GFP-Sgo2 (from GV onwards) in metaphase II, and upon activation. Spreads were labeled with Rec8 antibody (shown in red, far-red secondary antibody was used), CREST serum (pseudo-colored in green), and Hoechst (blue). GFP direct fluorescence was imaged for GFP-Sgo2 detection. Below the corresponding chromosome figures are represented in the schemes.


Giemsa staining of a femoral section from 8 - day - old mice showing endochondral ossification defects . Scale bar : 100 μm .

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Giemsa staining of a femoral section from 8-day-old mice showing endochondral ossification defects. Scale bar: 100 μm.


Representative femur sections from 8 - day - old wild - type and Dnmt2 − / − mice . The staining of perivascular cells ( GLS , green ) is strongly reduced in Dnmt2 − / − bone marrow . Nuclei are counterstained with DAPI . Scale bar : 100 μm .

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Representative femur sections from 8-day-old wild-type and Dnmt2−/− mice. The staining of perivascular cells (GLS, green) is strongly reduced in Dnmt2−/− bone marrow. Nuclei are counterstained with DAPI. Scale bar: 100 μm.


Quantitative analysis of capillary numbers . GLS - positive capillaries were counted in wild - type ( wt ) and Dnmt2 − / − ( D2 - ) mice , in three independent high - power fields ( HPF ) per mouse ( n = 7 ) .

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Quantitative analysis of capillary numbers. GLS-positive capillaries were counted in wild-type (wt) and Dnmt2−/− (D2-) mice, in three independent high-power fields (HPF) per mouse (n = 7).


Quantification of cell numbers in haematopoietic tissues of 8 - day - old mice ( n = 8 ) .

Quantification of cell numbers in haematopoietic tissues of 8-day-old mice (n = 8).


Representative FACS plots of wild - type and Dnmt2 − / − BM stained for Lin − Sca1 + and cKit + cells .

Representative FACS plots of wild-type and Dnmt2−/− BM stained for Lin− Sca1+ and cKit+ cells.


Quantification of bone marrow LSK cells in wild - type ( three independent experiments , each with pooled BM samples from 4 to 6 mice ) and Dnmt2 − / − bone marrow ( three independent experiments , each with pooled BM samples of 5 - 9 mice ) .

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Quantification of bone marrow LSK cells in wild-type (three independent experiments, each with pooled BM samples from 4 to 6 mice) and Dnmt2−/−bone marrow (three independent experiments, each with pooled BM samples of 5-9 mice).


Restoration of the LSK cell population in adult Dnmt2 − / − mice . Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild - type littermates at the indicated age ( 2 - 3 independent experiments , each with pooled bone marrow samples from 3 to 9 mice ) .

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Restoration of the LSK cell population in adult Dnmt2−/− mice. Frequencies of LSK cells were normalized to the frequencies of LSK cells from the bone marrow of wild-type littermates at the indicated age (2-3 independent experiments, each with pooled bone marrow samples from 3 to 9 mice).


Increased LSK CD34 − and reduced LSK CD34 + cell populations in 8 - day - and 1 - year - old Dnmt2 − / − mice compared to wild - type ( two independent experiments , each with pooled bone marrow samples from 3 - 9 mice ) .

Increased LSK CD34− and reduced LSK CD34+cell populations in 8-day- and 1-year-old Dnmt2−/− mice compared to wild-type (two independent experiments, each with pooled bone marrow samples from 3-9 mice).


qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues , normalized to GAPDH .

qPCR analysis of Dnmt2 mRNA expression in haematopoietic tissues, normalized to GAPDH.


Western blot analysis of Dnmt2 protein expression in haematopoietic tissues .

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 .

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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 .

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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 .

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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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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.


Flow cytometry analysis of bone marrow primary mesenchymal cells .

Flow cytometry analysis of bone marrow primary mesenchymal cells.


Quantification of fibroblast colony - forming units ( CFU - F ) .

Quantification of fibroblast colony-forming units (CFU-F).


Quantification of alkaline phosphatase colony - forming units ( CFU - ALP ) .

Quantification of alkaline phosphatase colony-forming units (CFU-ALP).


Dnmt2 expression after 3 days of mesenchymal cell differentiation .

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 .

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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 .

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 .

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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 .

qPCR analysis of the adipocyte markers CEBp alpha and Glut4.


Bisulfite 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 ) .

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Bisulfite 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 .

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

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 .

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

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.


Analysis 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 .

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

Analysis 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 .

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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 .

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

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 ) .

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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 .

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.


Frequency 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 ) .

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

Frequency 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).


( 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 .

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(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 ) .

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(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 ) .

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(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 .

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(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 .

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(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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(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.


( 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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 ) .

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(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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(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).


( 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 .

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(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 .

(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 .

(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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(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.


( 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 .

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(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 ) .

(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 .

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(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 .

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(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 .

(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.


( A ) Colonic epithelial monolayers from a wild ‐ type and p22 mut mouse stained with LC3β ( green ) and EEA1 ( red ) . Bar = 5 μm .

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(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 ) .

(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 .

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(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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 ) .

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(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 .

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(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 .

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(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.


Exogeneous 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

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Exogeneous 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 .

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(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 .

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(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 )

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(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 ) .

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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).


( 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 ) .

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(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 ) .

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(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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(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).


( 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 .

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(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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(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.


Effects 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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Effects 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 ●.


( 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 ) .

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(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 .

(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.


( 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 .

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(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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(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 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 .

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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 .

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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 ) .

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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 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 .

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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 2b . CDK12 kinase activity is needed for G1 / S progression in cells arrested by serum

Figure 2b. CDK12 kinase activity is needed for G1/S progression in cells arrested by serum starvation


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 .

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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 .

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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 .

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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 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 .

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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 .

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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 .

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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 .

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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.


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 .

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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 .

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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 .

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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 .

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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 .

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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.