Clonogenicity assay for colony number ( B ) and size ( C ) after the first the passage of CLESCs derived from WT vs . Prom1 KO mice in the absence or presence of SHH ( 100ng / ml ) . Cells were seeded in triplicate on 24 - well plates as 5 , 000 cell / per well .

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Clonogenicity assay for colony number (B) and size (C) after the first the passage of CLESCs derived from WT vs. Prom1 KO mice in the absence or presence of SHH (100ng/ml). Cells were seeded in triplicate on 24-well plates as 5,000 cell/per well.


Sphenoid analyses . For these assays , epithelial and mesenchymal cells were mixed at ratio of 5 , 000 : 5 , 000 per well in triplicates on 96 - well plates and grown for 3 days on Matrigel . Representative micrographs of entire well under indicated conditions are shown . Quantification of sphere diameter on samples presented in ( D ) .

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Sphenoid analyses. For these assays, epithelial and mesenchymal cells were mixed at ratio of 5,000:5,000 per well in triplicates on 96-well plates and grown for 3 days on Matrigel. Representative micrographs of entire well under indicated conditions are shown. Quantification of sphere diameter on samples presented in (D).


Representative image of CLESCs harbouring the Ki67 - Fucci system showing colored cells at different cell cycle phases .

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Representative image of CLESCs harbouring the Ki67-Fucci system showing colored cells at different cell cycle phases.


Flow cytometry analysis of Ki67 - Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1 . As a result , > 95 % of the events have been included in the analysis . The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1 . 0 software .

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Flow cytometry analysis of Ki67-Fucci CLESCs at different cell cycle stages in cells transfected with empty vector or full length Prom1. As a result, >95% of the events have been included in the analysis. The accuracy of the strategy has been validated by two independent researchers for IF quantification using Fiji 1.0 software.


Representative IF staining of AcTub ( blue ) on Ki67 - Fucci CLESCs without and with the presence of SHH . Note that the cilium could be visualised ( arrows ) solely when SHH is present .

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Representative IF staining of AcTub (blue) on Ki67-Fucci CLESCs without and with the presence of SHH. Note that the cilium could be visualised (arrows) solely when SHH is present.


Representative IF double staining of either AcTub ( D , red ) or Arl13b ( E , red ) with Sox2 ( green ) or Ki67 ( green ) in CLESCs in the presence of SHH . Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells , respectively .

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Representative IF double staining of either AcTub (D, red) or Arl13b (E, red) with Sox2 (green) or Ki67 (green) in CLESCs in the presence of SHH. Note the expression levels of Sox2 and Ki67 distinguished stem cells and transit amplifying cells, respectively.


Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice ( A ) and their density in the respective regions ( B ) . 3 animals were analyzed for each genotype . The number of quantified cilia is indicated ( n ) .

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Size of individual cilia in stem cell and transit amplifying cell regions of WT and Prom1 KO mice (A) and their density in the respective regions (B). 3 animals were analyzed for each genotype. The number of quantified cilia is indicated (n).


Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor ( Tubacin ) and appropriate controls ( Nitubacin and DMSO , C ) Colonies were stained with crystal

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Clonogenicity analysis of CLESCs treated with either Hdac6 inhibitor (Tubacin) and appropriate controls (Nitubacin and DMSO, C) Colonies were stained with crystal violet


Clonogenicity analysis of CLESCs treated with siArl13b and its siControl . Colonies were stained with crystal

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Clonogenicity analysis of CLESCs treated with siArl13b and its siControl. Colonies were stained with crystal violet


siRNA knockdown efficiency of Arl13b ( E ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for

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siRNA knockdown efficiency of Arl13b (E) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH


siRNA knockdown efficiency of Hdac6 ( F ) in Ki67 FUCCI - CLESCs was evaluated real - time RT - PCR analysis . The results are in arbitrary values after normalization for

siRNA knockdown efficiency of Hdac6 (F) in Ki67 FUCCI-CLESCs was evaluated real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH


Flow cytometry analysis and quantification of samples indicated in ( E and F ) .

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Flow cytometry analysis and quantification of samples indicated in (E and F).


Representative IF double staining and 3D reconstruction of the Prom1 ( green ) and AcTub ( red ) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant ( top panels ) . Black - white inserts show Prom1 signals only . Same cells were analyzed by SEM ( bottom panels ) . Note that Prom1 is asymmetrically distributed along the primary cilium ( white arrows ) as observed in vivo ( see Fig 2C ) and mutation K138Q significantly impacts on the primary cilium length . MV , microvilli .

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Representative IF double staining and 3D reconstruction of the Prom1 (green) and AcTub (red) at the apical domain of polarized MDCK cells expressing Prom1 and its K138Q mutant (top panels). Black-white inserts show Prom1 signals only. Same cells were analyzed by SEM (bottom panels). Note that Prom1 is asymmetrically distributed along the primary cilium (white arrows) as observed in vivo (see Fig 2C) and mutation K138Q significantly impacts on the primary cilium length. MV, microvilli.


IF staining and 3D reconstruction of AcTub - labeled ( red ) primary cilium at the apical membrane of parental MDCK cells ( MDCK ) and those cells transfected with Prom1 or its K138Q mutant . The images were used to quantify the primary cilium length ( Appendix Figs S6B and C ) . Arrows indicate primary cilia with more than 4 μm in length .

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IF staining and 3D reconstruction of AcTub-labeled (red) primary cilium at the apical membrane of parental MDCK cells (MDCK) and those cells transfected with Prom1 or its K138Q mutant. The images were used to quantify the primary cilium length (Appendix Figs S6B and C). Arrows indicate primary cilia with more than 4 μm in length.


Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Arl13b ( K ) Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin .

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Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Arl13b (K) Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.


Co - immunoisolation ( IS ) analyses were performed with paramagnetic beads using antibodies against either Prom1 ( AC133 ) or with anti - FITC antibody as control ( L ) . Recovered materials were analyzed by immunoblotting against the indicated proteins . Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum - associated forms of Prom1 , respectively , while arrows show Arl13b . Arrowhead , immunoglobulin .

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Co-immunoisolation (IS) analyses were performed with paramagnetic beads using antibodies against either Prom1 (AC133) or with anti-FITC antibody as control (L). Recovered materials were analyzed by immunoblotting against the indicated proteins. Brackets and asterisks indicate the plasma membrane and endoplasmic reticulum-associated forms of Prom1, respectively, while arrows show Arl13b. Arrowhead, immunoglobulin.


The ratio Arl13b / Prom1 immunoreactivities was quantified ( n = 3 ) ( M ) .

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The ratio Arl13b/Prom1 immunoreactivities was quantified (n = 3) (M).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase - contrast microscopy ( A ) Box - whisker plots show the number of domes per 600 mm2 ( A ) . Three images were analyzed per experiment ( n = 4 ) . 4X magnification ( inset in panel A ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by phase-contrast microscopy (A) Box-whisker plots show the number of domes per 600 mm2 (A). Three images were analyzed per experiment (n = 4). 4X magnification (inset in panel A).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM ( B , top panels ) or confocal laser scanning microscopy after DAPI staining ( B , bottom panels , Individual cells within a given dome are indicated ( B , top panels ) and were quantified using three different planes ( bottom , centre and top ) of DAPI - labeled nuclei ( B , bottom panels ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc Cells were observed by SEM (B, top panels) or confocal laser scanning microscopy after DAPI staining (B, bottom panels, Individual cells within a given dome are indicated (B, top panels) and were quantified using three different planes (bottom, centre and top) of DAPI-labeled nuclei (B, bottom panels).


Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc ( A - C ) or as indicated ( D ) . confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated ( C ) . Means and standard deviations are displayed . 15 domes per independent experiment were examined ( n = 3 ) . Cell density at a given time of culture ( 7 , 10 and 14 days ) was determined by counting DAPI - labeled nuclei per 0 . 1 mm2 ( D , n = 4 ) .

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Parental MDCK cells or those transfected with Prom1 or K138Q mutant were grown for 7 dpc (A-C) or as indicated (D). confocal laser scanning microscopy after DAPI staining Domes of similar size were evaluated (C). Means and standard deviations are displayed. 15 domes per independent experiment were examined (n = 3). Cell density at a given time of culture (7, 10 and 14 days) was determined by counting DAPI-labeled nuclei per 0.1 mm2 (D, n = 4).


Cilia with a length of > 4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM ( arrows ) .

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Cilia with a length of >4 µm were rarely observed in K138Q mutant and displayed an irregular shape with constrictions along their distal part as observed by SEM (arrows).


Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The entire pellets and 1 / 40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1 , AcTub and β - actin ( F ) . Molecular mass markers are indicated . All three proteins were detected in the vesicle fractions ( P3 and P4 ) suggesting Prom1 + vesicles are released from primary cilia and microvilli .

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Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The entire pellets and 1/40 of cell lysates were analyzed by immunoblotting using antibodies against human Prom1, AcTub and β-actin (F). Molecular mass markers are indicated. All three proteins were detected in the vesicle fractions (P3 and P4) suggesting Prom1+ vesicles are released from primary cilia and microvilli.


Mutation in the Hdac6 - binding site of Prom1 increases the release of Prom1 + vesicles . 16 h - conditioned medium was subjected to differential centrifugation for 5 min at 300 x g ( P1 ) ; 20 min at 1 , 200 x g ( P2 ) ; 30 min at 10 , 000 x g ( P3 ) and 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent cell lysates were prepared . The release of Prom1 + vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions ( G )

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Mutation in the Hdac6-binding site of Prom1 increases the release of Prom1+ vesicles. 16 h-conditioned medium was subjected to differential centrifugation for 5 min at 300 x g (P1); 20 min at 1,200 x g (P2); 30 min at 10,000 x g (P3) and 1 h at 200,000 x g (P4). In parallel, detergent cell lysates were prepared. The release of Prom1+ vesicles was quantified using its immunoreactivities associated with cell and vesicle fractions (G)


The mRNA expression of SHH effector genes ( Gli1 / 2 and Glis1 - 3 ) on laser microdissection captured samples were evaluated by real - time RT - PCR analysis . The results are in arbitrary values after normalization for GapDH . Each sample was isolated from distinct mice ( n = 3 ) .

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The mRNA expression of SHH effector genes (Gli1/2 and Glis1-3) on laser microdissection captured samples were evaluated by real-time RT-PCR analysis. The results are in arbitrary values after normalization for GapDH. Each sample was isolated from distinct mice (n = 3).


Representative IF staining of Gli1 ( left panel , red ) and Glis2 ( right panel , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Dotted lines , basement membrane .

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Representative IF staining of Gli1 (left panel, red) and Glis2 (right panel, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Dotted lines, basement membrane.


3D reconstruction of Glis2 staining ( see B , red ) and its association with DAPI - labeled nuclei ( blue ) . Nuclear transparency was set at 0 or 70 % as indicated .

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3D reconstruction of Glis2 staining (see B, red) and its association with DAPI-labeled nuclei (blue). Nuclear transparency was set at 0 or 70% as indicated.


Representative IF double staining of Glis2 ( red , bottom panel ) and AcTub ( green , top and bottom panels ) along stem cell - transit amplifying cell axis in lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions .

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Representative IF double staining of Glis2 (red, bottom panel) and AcTub (green, top and bottom panels) along stem cell-transit amplifying cell axis in lower incisor CLE at P7. Samples were counterstained with DAPI (blue). Note the association of Glis2 with primary cilia particularly in the transition zone between stem cell and transit amplifying cell regions.


Volume of Sox2 - positive region on the lower incisor CLE at P7 was evaluated in WT vs . Glis2 KO mice upon IF staining . Number of animals analyzed is indicated ( n ) .

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Volume of Sox2-positive region on the lower incisor CLE at P7 was evaluated in WT vs. Glis2 KO mice upon IF staining. Number of animals analyzed is indicated (n).


Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop ( top panels , green ) or cytoplasmic C - terminal end ( bottom panels , red ) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice . Samples are counterstained with DAPI ( blue ) . Dotted lines , basement membrane . Arrowheads marks approximate boundary of stem cell regions .

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Representative IF staining of Prom1 using specific antibodies targeting either its extracellular loop (top panels, green) or cytoplasmic C-terminal end (bottom panels, red) on the stem cell and transit amplifying cell regions of lower incisor CLE at P7 WT and Glis2 KO mice. Samples are counterstained with DAPI (blue). Dotted lines, basement membrane. Arrowheads marks approximate boundary of stem cell regions.


Representative IF double staining of Glis2 ( red ) and AcTub ( green ) as a marker of primary cilia in stem cell region of WT vs . Prom1 KO mice . Note the reduction of Glis2 expression in Prom1 - deficient animals .

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Representative IF double staining of Glis2 (red) and AcTub (green) as a marker of primary cilia in stem cell region of WT vs. Prom1 KO mice. Note the reduction of Glis2 expression in Prom1-deficient animals.


Proximity ligation assay analysis of Prom1 and Glis2 association ( red ) in stem cell and transit amplifying cell regions of lower incisor CLE at P7 . As negative control , one conjugated primary antibody ( anti - Glis2 ) was omitted . SCs , stem cells ; TACs , transit amplifying cells .

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Proximity ligation assay analysis of Prom1 and Glis2 association (red) in stem cell and transit amplifying cell regions of lower incisor CLE at P7. As negative control, one conjugated primary antibody (anti-Glis2) was omitted. SCs, stem cells; TACs, transit amplifying cells.


Time dependent Prom1 and Glis2 co - expression ( proximity ligation assay ) in the cultured CLESCs starved for 4 days , and then treated with SHH ( 100 ng / ml ) and importazole ( 40 µM ) , an inhibitor of importin β1function . DMSO was used as vehicle control . Data are represented triplicated samples as mean and standard deviation .

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Time dependent Prom1 and Glis2 co-expression (proximity ligation assay) in the cultured CLESCs starved for 4 days, and then treated with SHH (100 ng/ml) and importazole (40 µM), an inhibitor of importin β1function. DMSO was used as vehicle control. Data are represented triplicated samples as mean and standard deviation.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . Arrows indicated genes with expression changed for more than 2 folder increase or decrease .

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PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). Arrows indicated genes with expression changed for more than 2 folder increase or decrease.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the PCR array ( in ( C ) ) ( D ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH .

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PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the PCR array (in (C)) (D) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.


PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs . WT mice ( n = 3 ) . The mRNA expression of gene significantly changed in the Stat pathway members ( E ) was evaluated in WT , Prom1 and Glis2 KO mice by real - time RT - PCR analysis . Each sample was isolated from distinct mice ( n = 3 ) . The results are in arbitrary values after normalization for GapDH .

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PCR array analyses were performed on LCM captured P7 CLE of Prom1 and Glis2 KO vs. WT mice (n = 3). The mRNA expression of gene significantly changed in the Stat pathway members (E) was evaluated in WT, Prom1 and Glis2 KO mice by real-time RT-PCR analysis. Each sample was isolated from distinct mice (n = 3). The results are in arbitrary values after normalization for GapDH.


Chromatin IP analysis of binding of Glis2 to Stat3 promoter ( see Appendix Fig S9D ) in the absence or presence of SHH at different concentration as indicated on triplicated samples , using specific anti - Glis2 antibodies and rabbit IgG as control .

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Chromatin IP analysis of binding of Glis2 to Stat3 promoter (see Appendix Fig S9D) in the absence or presence of SHH at different concentration as indicated on triplicated samples, using specific anti-Glis2 antibodies and rabbit IgG as control.


( A ) Three glycosylated forms of endogenous SMO protein are shown . NIH3T3 cells were harvested and treated with indicated glycosidases . Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form . The representative results of three independent experiments are shown .

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(A) Three glycosylated forms of endogenous SMO protein are shown. NIH3T3 cells were harvested and treated with indicated glycosidases. Black bracket indicates mature glycosylated form and grey bracket indicates core glycosylated form. The representative results of three independent experiments are shown.


( C ) PTCH1 inhibited the Endo H - resistant glycosylation of SMO . HEK293T cells were co - transfected with the SMO and PTCH1 expression plasmids . After 48 h , the cells were harvested , lysed , and treated with indicated glycosidases . Results shown are representative of three independent experiments .

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(C) PTCH1 inhibited the Endo H-resistant glycosylation of SMO. HEK293T cells were co-transfected with the SMO and PTCH1 expression plasmids. After 48 h, the cells were harvested, lysed, and treated with indicated glycosidases. Results shown are representative of three independent experiments.


( D ) Quantification of Endo H - resistant form of SMO shown in ( C ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

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(D) Quantification of Endo H-resistant form of SMO shown in (C). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( E ) Shh increased the Endo H - resistant form of SMO . Cells were transfected as indicated for 24 h , and incubated with ShhN conditioned medium for an additional 24 h . Cells were harvested and analyzed as in ( C ) . Results shown are representative of three independent experiments .

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(E) Shh increased the Endo H-resistant form of SMO. Cells were transfected as indicated for 24 h, and incubated with ShhN conditioned medium for an additional 24 h. Cells were harvested and analyzed as in (C). Results shown are representative of three independent experiments.


( F ) Quantification of Endo H - resistant form of SMO shown in ( E ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

(F) Quantification of Endo H-resistant form of SMO shown in (E). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( G ) Shh increased the Endo H - resistant form of endogenous SMO in MEF cells . Results shown are representative of three independent experiments .

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(G) Shh increased the Endo H-resistant form of endogenous SMO in MEF cells. Results shown are representative of three independent experiments.


( H ) Densitometric analysis of Endo H - resistant form of SMO shown in ( G ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein .

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(H) Densitometric analysis of Endo H-resistant form of SMO shown in (G). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO protein.


( A ) Biotinylation of SMO by SAR1B - BirA * ( asterisk denotes the R118G mutation in BirA ) in a proximity - dependent biotinylation assay . The cells were transfected with plasmids encoding SMO and SAR1B - BirA * - FLAG . After 48 h , cells were treated with different doses of biotin for 4 h . Cells were harvested and lysates were dialyzed to remove free biotin . Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis . ( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

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(A) Biotinylation of SMO by SAR1B-BirA* (asterisk denotes the R118G mutation in BirA) in a proximity-dependent biotinylation assay. The cells were transfected with plasmids encoding SMO and SAR1B-BirA*-FLAG. After 48 h, cells were treated with different doses of biotin for 4 h. Cells were harvested and lysates were dialyzed to remove free biotin. Biotinylated proteins were pulled down by streptavidin beads and subjected to immunoblotting analysis. (B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( B ) The cells were treated with different doses of biotin for 24 h . Cells were harvested , lysed , and subjected to procedure as shown in Fig . 2A .

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(B) The cells were treated with different doses of biotin for 24 h. Cells were harvested, lysed, and subjected to procedure as shown in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A .

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(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A.


( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A .

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(D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A.


( C ) The GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while the GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . The cells were transfected with plasmids expressing SMO and indicated SAR1B - BirA * . After 48 h , cells were treated with 15 μM biotin for 4 h . Then experiments were carried out as described in Fig . 2A . ( D ) PTCH1 decreased SAR1B - BirA * - mediated biotinylation of SMO , but not LMAN1 . The experiments were done similarly in Fig . 2A . ( E ) The Shh increased the binding of SAR1B to SMO . After transfection for 24 h , cells were incubated with the ShhN conditioned medium for an additional 24 h . Then 15 μM biotin was added into the culture medium for 4 h . The cells were harvested and analyzed as described in Fig . 2A .

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(C) The GDP-locked SAR1B(G37A)-BirA* failed to label SMO, while the GTP-locked SAR1B(H79G)-BirA* could efficiently biotinylate SMO. The cells were transfected with plasmids expressing SMO and indicated SAR1B-BirA*. After 48 h, cells were treated with 15 μM biotin for 4 h. Then experiments were carried out as described in Fig. 2A. (D) PTCH1 decreased SAR1B-BirA*-mediated biotinylation of SMO, but not LMAN1. The experiments were done similarly in Fig. 2A. (E) The Shh increased the binding of SAR1B to SMO. After transfection for 24 h, cells were incubated with the ShhN conditioned medium for an additional 24 h. Then 15 μM biotin was added into the culture medium for 4 h. The cells were harvested and analyzed as described in Fig. 2A.


( B ) Treatment with glycosidase demonstrated that SMO ( 4A , in which RRTWCR was mutated to AATACA ) was sensitive to Endo H . The experiments were done similarly in Fig . 2A . Black bracket indicates mature glycosylated form .

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(B) Treatment with glycosidase demonstrated that SMO (4A, in which RRTWCR was mutated to AATACA) was sensitive to Endo H. The experiments were done similarly in Fig. 2A. Black bracket indicates mature glycosylated form.


( C ) SAR1B - BirA * - mediated biotinylation of the SMO variants . The experiments were done similarly in Fig . 2A .

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(C) SAR1B-BirA*-mediated biotinylation of the SMO variants. The experiments were done similarly in Fig. 2A.


( D ) In vitro budding of SMO . Membranes were prepared and incubated in vitro in the presence of cytosol , ATP , GTP , and an ATP - regenerating system as described in experimental procedures . Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis .

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(D) In vitro budding of SMO. Membranes were prepared and incubated in vitro in the presence of cytosol, ATP, GTP, and an ATP-regenerating system as described in experimental procedures. Then vesicle and membrane fractions were separated by centrifugation and subjected to immunoblotting analysis.


( E ) In vitro budding of the SMO variants . The experiments were done as same as in ( D ) .

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(E) In vitro budding of the SMO variants. The experiments were done as same as in (D).


( F ) The ciliary localization of SMO variants . Smo - KO NIH3T3 cells were transfected with the EGFP fused SMO variants , incubated with serum starvation medium for 24 h to fully induce cilia formation , and treated with or without ShhN conditioned medium for 4 h . Cells were immunostained with the acetylated tubulin ( red ) . Results shown are representative of three independent experiments . Scale bars : 5μm .

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(F) The ciliary localization of SMO variants. Smo-KO NIH3T3 cells were transfected with the EGFP fused SMO variants, incubated with serum starvation medium for 24 h to fully induce cilia formation, and treated with or without ShhN conditioned medium for 4 h. Cells were immunostained with the acetylated tubulin (red). Results shown are representative of three independent experiments. Scale bars: 5μm.


( G ) Quantitative assessment of the intensity of ciliary SMO in ( F ) by Image J . More than 45 cells were randomly selected and counted for each group .

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(G) Quantitative assessment of the intensity of ciliary SMO in (F) by Image J. More than 45 cells were randomly selected and counted for each group.


( H ) Quantification of the percentage of cells with ciliary SMO . 80 - 90 cilia were analyzed in each group in each experiment .

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(H) Quantification of the percentage of cells with ciliary SMO. 80-90 cilia were analyzed in each group in each experiment.


( I ) Gli - luciferase reporter assay in Smo - KO NIH3T3 cells . The cells were transfected with plasmids encoding SMO or SMO variants , GLI - promoted firefly luciferase and renilla luciferase . After 24 h , cells were incubated with a medium containing different amounts of ShhN for another 24 h .

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(I) Gli-luciferase reporter assay in Smo-KO NIH3T3 cells. The cells were transfected with plasmids encoding SMO or SMO variants, GLI-promoted firefly luciferase and renilla luciferase. After 24 h, cells were incubated with a medium containing different amounts of ShhN for another 24 h.


( A ) Shh increased cholesterylation of SMO . HEK293T cells were transfected , harvested , and

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(A) Shh increased cholesterylation of SMO. HEK293T cells were transfected, harvested, and analyzed


( B ) The SMO cholesterylation of Ptch1 - KO NIH3T3 cells were increased comparing with WT cells .

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(B) The SMO cholesterylation of Ptch1-KO NIH3T3 cells were increased comparing with WT cells.


( C ) The CP - modified SMO was mainly resistant to Endo H . The CP - modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃ . Then proteins were eluted with SDS - PAGE sample buffer and were subjected to immunoblotting analysis . Results shown are representative of two independent experiments .

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(C) The CP-modified SMO was mainly resistant to Endo H. The CP-modified SMO proteins were pulled down by streptavidin beads and digested with Endo H or PNGase F at 37 ℃. Then proteins were eluted with SDS-PAGE sample buffer and were subjected to immunoblotting analysis. Results shown are representative of two independent experiments.


( D ) Cholesterylation of SMO ( 4A ) was inhibited by PTCH1 and increased by ShhN .

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(D) Cholesterylation of SMO(4A) was inhibited by PTCH1 and increased by ShhN.


( E ) The CP - modified SMO ( 4A ) was sensitive to Endo H treatment .

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(E) The CP-modified SMO(4A) was sensitive to Endo H treatment.


( F ) The levels of CP - modified WT SMO and SMO ( 4A ) were compared . 1 . 39 % of SMO ( WT ) pellet samples and 11 . 1 % of SMO ( 4A ) pellet samples were loaded . Results shown are representative of three independent experiments .

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(F) The levels of CP-modified WT SMO and SMO(4A) were compared. 1.39% of SMO(WT) pellet samples and 11.1% of SMO(4A) pellet samples were loaded. Results shown are representative of three independent experiments.


( G ) Quantification of CP - modified SMO shown in ( F ) . Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO .

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(G) Quantification of CP-modified SMO shown in (F). Three independent experiments were quantified by Image J and expressed as a percentage of the total SMO.


( H , I ) The stimulative / inhibitory effect of Shh / PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation . The experiments were done the same as Fig . 2E .

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(H, I) The stimulative /inhibitory effect of Shh/PTCH1 on SMO and SAR1B interaction was compromised by D95E mutation. The experiments were done the same as Fig. 2E.


ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h , and then incubated with a medium containing ShhN and CP for 2 and 3 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO Cells were treated with ALOD4 for 1 h, and then incubated with a medium containing ShhN and CP for 2 and 3 h. Cells were harvested and analyzed


ALOD4 inhibited the cholesterol modification of SMO ( 4A ) . Cells were treated with ALOD4 for 1 h In C , ShhN and CP added for 2 h . Cells were harvested and

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ALOD4 inhibited the cholesterol modification of SMO(4A). Cells were treated with ALOD4 for 1 h In C, ShhN and CP added for 2 h. Cells were harvested and analyzed


( D ) Ciliary localization of endogenous SMO treated with or without ALOD4 . Cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

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(D) Ciliary localization of endogenous SMO treated with or without ALOD4. Cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( E ) Quantitative assessment of the intensity of ciliary SMO in ( D ) . More than 80 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

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(E) Quantitative assessment of the intensity of ciliary SMO in (D). More than 80 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( F ) Quantification of the percentage of cells with ciliary SMO in ( D ) . Data are presented as mean ± SEM from three biological replicates ( * * * * p < 0 . 0001 , one - way ANOVA with Dunnett ' s multiple comparison test ) . 90 - 100 cilia were analyzed in each group in each experiment .

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(F) Quantification of the percentage of cells with ciliary SMO in (D). Data are presented as mean ± SEM from three biological replicates (****p < 0.0001, one-way ANOVA with Dunnett's multiple comparison test). 90-100 cilia were analyzed in each group in each experiment.


( G ) Effect of ALOD4 on the expression of Gli1 was measured by qRT - PCR . The cells were treated with ALOD4 for 1 h , and then incubated with ShhN conditioned medium for 5 h .

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(G) Effect of ALOD4 on the expression of Gli1 was measured by qRT-PCR. The cells were treated with ALOD4 for 1 h, and then incubated with ShhN conditioned medium for 5 h.


( A , B ) GRAMD1B increased cholesterylation of SMO . HEK293T cells were co - transfected with the SMO or SMO ( 4A ) and GRAMD1B protein expression plasmids for 24 h , and then incubated with a cholesterol depletion medium ( DMEM supplemented with 5 % lipoprotein - deficient serum , 1 μM lovastatin and 10 μM mevalonate ) for 12 h , followed by 2 μg / mL of CP treatment . The cells were harvested and analyzed as Results shown are representative of two independent experiments .

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(A, B) GRAMD1B increased cholesterylation of SMO. HEK293T cells were co-transfected with the SMO or SMO (4A) and GRAMD1B protein expression plasmids for 24 h, and then incubated with a cholesterol depletion medium (DMEM supplemented with 5% lipoprotein-deficient serum, 1 μM lovastatin and 10 μM mevalonate) for 12 h, followed by 2 μg/mL of CP treatment. The cells were harvested and analyzed as Results shown are representative of two independent experiments.


( C , D ) GRAMD1B promoted SMO maturation . The expression plasmids of SMO or SMO ( D95E ) and GRAMD1B were transfected into SMO - KO HEK293T cells . After 12 h , cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD - cholesterol for 8 h . Black bracket indicates mature glycosylated form .

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(C, D) GRAMD1B promoted SMO maturation. The expression plasmids of SMO or SMO(D95E) and GRAMD1B were transfected into SMO-KO HEK293T cells. After 12 h, cells were switched to a cholesterol depletion medium for 16 h and then added 50 μM MβCD-cholesterol for 8 h. Black bracket indicates mature glycosylated form.


( E ) Expression of Gli1 mRNAs measured by qRT - PCR in NIH3T3 cells stably expressing GRAMD1B . After incubating in serum starvation medium for 16 h , cells were depleted of cholesterol by treatment with 1 . 5 % HPCD for 0 . 5 h and then incubated in serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 10 μM MβCD - cholesterol , ShhN as indicated for 24 h .

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(E) Expression of Gli1 mRNAs measured by qRT-PCR in NIH3T3 cells stably expressing GRAMD1B. After incubating in serum starvation medium for 16 h, cells were depleted of cholesterol by treatment with 1.5% HPCD for 0.5 h and then incubated in serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 10 μM MβCD-cholesterol, ShhN as indicated for 24 h.


( F ) Confocal images of NIH3T3 cells stably expressing mCherry - GRAMD1B . PM was labeled with wheat germ agglutinin ( WGA ) - CF ® 488A conjugate . Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD - cholesterol for 1 h . Cells were then treated with or without ShhN conditioned medium for another 2 h . Results shown are representative of three independent experiments .

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(F) Confocal images of NIH3T3 cells stably expressing mCherry-GRAMD1B. PM was labeled with wheat germ agglutinin (WGA)-CF®488A conjugate. Cells were incubated in a cholesterol depletion medium for 16 h and switched to the same medium containing 50 μM MβCD-cholesterol for 1 h. Cells were then treated with or without ShhN conditioned medium for another 2 h. Results shown are representative of three independent experiments.


( G ) The percentage of cells with GRAMD1B localization on PM . 60 - 70 cells were analyzed in each group in each experiment .

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(G) The percentage of cells with GRAMD1B localization on PM. 60-70 cells were analyzed in each group in each experiment.


( A ) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a , 1b , 1c , 2 , and 3 ( Gramd - 5KO ) . The cells were incubated in a cholesterol depletion medium for 12 h , followed by 2 μg / mL of CP and ShhN for an additional 12h .

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(A) The cholesterol modification of endogenous SMO was decreased in NIH3T3 cells lacking Gramd1a, 1b, 1c, 2, and 3 (Gramd-5KO). The cells were incubated in a cholesterol depletion medium for 12 h, followed by 2 μg/mL of CP and ShhN for an additional 12h.


( B ) The glycosylation of endogenous SMO was decreased in Gramd - 5KO NIH3T3 cells . The ShhN conditioned medium was applied for 24 h . Black bracket indicates mature glycosylated form .

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(B) The glycosylation of endogenous SMO was decreased in Gramd-5KO NIH3T3 cells. The ShhN conditioned medium was applied for 24 h. Black bracket indicates mature glycosylated form.


( C ) The ciliary localization of endogenous SMO in WT or Gramd - 5KO NIH3T3 cells . Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h . Results shown are representative of three independent experiments .

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(C) The ciliary localization of endogenous SMO in WT or Gramd-5KO NIH3T3 cells. Cells were incubated in serum starvation medium for 24 h to fully induce cilia formation and then incubated with or without ShhN conditioned medium for 2 h. Results shown are representative of three independent experiments.


( D ) Quantitative assessment of the intensity of ciliary SMO in ( C ) . More than 100 cells were randomly selected and counted for each group . Data are presented as median with interquartile .

(D) Quantitative assessment of the intensity of ciliary SMO in (C). More than 100 cells were randomly selected and counted for each group. Data are presented as median with interquartile.


( E ) Quantification of the percentage of cells with ciliary SMO . 86 - 100 cilia were analyzed in each group in each experiment .

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(E) Quantification of the percentage of cells with ciliary SMO. 86-100 cilia were analyzed in each group in each experiment.


( F ) The protein levels of Gli1 was significantly reduced in Gramd - 5KO cells . The cells were incubated in a serum starvation medium with or without ShhN for 24 h . Results shown are representative of two independent experiments .

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(F) The protein levels of Gli1 was significantly reduced in Gramd-5KO cells. The cells were incubated in a serum starvation medium with or without ShhN for 24 h. Results shown are representative of two independent experiments.


( G ) GLI - luciferase reporter assay in WT or Gramd - 5KO NIH3T3 cells . Data are presented as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; * p < 0 . 05 , two - way ANOVA with Sidak ' s multiple comparison test ) .

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(G) GLI-luciferase reporter assay in WT or Gramd-5KO NIH3T3 cells. Data are presented as mean ± SEM from three technical replicates (****p < 0.0001; ***p < 0.001; *p < 0.05, two-way ANOVA with Sidak's multiple comparison test).


( H ) The Gli1 mRNA levels were measured in WT , Gramd - 5KO , Gramd - 5KO overexpression GRAMD1A , 1B , 1C NIH3T3 cells by qRT - PCR after treatment with or without ShhN conditioned medium for 8 h . Data are expressed as mean ± SEM from three technical replicates ( * * * * p < 0 . 0001 , two - way ANOVA with Tukey ' s multiple comparisons test ) .

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(H) The Gli1 mRNA levels were measured in WT, Gramd-5KO, Gramd-5KO overexpression GRAMD1A, 1B, 1C NIH3T3 cells by qRT-PCR after treatment with or without ShhN conditioned medium for 8 h. Data are expressed as mean ± SEM from three technical replicates (****p < 0.0001, two-way ANOVA with Tukey's multiple comparisons test).


( I ) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells . The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 2 μg / mL of CP for an additional 2 h .

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(I) The GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 cells. The cells were incubated in cholesterol depletion medium for 16 h and then treated with 3 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 2 μg/mL of CP for an additional 2 h.


( J ) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells . Cells were incubated in serum starvation medium for 16 h , depleted of cholesterol by 1 . 5 % HPCD for 0 . 5 h , and then treated with serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate and 1 μM AI3d for 6 h . Next , cells were incubated in the same medium containing ShhN and 10 μM MβCD - cholesterol for an additional

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(J) The GRAMD inhibitor AI3d decreased the mRNA levels of Gli1 in NIH3T3 cells. Cells were incubated in serum starvation medium for 16 h, depleted of cholesterol by 1.5% HPCD for 0.5 h, and then treated with serum starvation medium containing 1 μM lovastatin, 10 μM mevalonate and 1 μM AI3d for 6 h. Next, cells were incubated in the same medium containing ShhN and 10 μM MβCD-cholesterol for an additional 16


Nucleosomal array oligomers are globular . A Nucleosomal array oligomers were stained with DAPI and examined using FM ( fluorescence microscopy ) as described in the Materials and Methods section . Shown are representative images obtained in 4 . 5 mM and 10mM MgCl2 . B Control FM images obtained in 0 , 1 and 2 . 5 mM MgCl2 .

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Nucleosomal array oligomers are globular.A Nucleosomal array oligomers were stained with DAPI and examined using FM (fluorescence microscopy) as described in the Materials and Methods section. Shown are representative images obtained in 4.5 mM and 10mM MgCl2.B Control FM images obtained in 0, 1 and 2.5 mM MgCl2.


C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods . Shown in the left panels are representative images obtained in 4 . 5 mM , and 10 mM MgCl2 . Shown in the right panels are images of the interior of the oligomers ( white arrows , left panels ) after cropping and re - scaling .

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C Nucleosomal array oligomers were negatively stained and visualized by TEM as described under Materials and Methods. Shown in the left panels are representative images obtained in 4.5 mM, and 10 mM MgCl2. Shown in the right panels are images of the interior of the oligomers (white arrows, left panels) after cropping and re-scaling.


Figure 2Sedimentation velocity analysis of the salt - dependent assembly of nucleosomal array oligomers . A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2 . The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample ( see Materials and Methods ) . The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) .

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Figure 2Sedimentation velocity analysis of the salt-dependent assembly of nucleosomal array oligomers.A Representative experiment showing the second moment sedimentation coefficients of the oligomeric nucleosomal arrays as a function of MgCl2. The second moment sedimentation coefficient is equivalent to the mass average sedimentation coefficient for the entire sample (see Materials and Methods). The inset shows the mean second moment sedimentation coefficient the standard error for three replicated experiments.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).


A , B SAXS profiles of the nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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A, B SAXS profiles of the nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


A 601 - nucleosomal arrays were incubated in 0 . 5 , 5 mM MgCl2 , 5 mM MgCl2 + MNase , and analyzed by the differential centrifugation assay to determine the fraction oligomeric . The amounts of DNA in the supernatant fraction were measured . Note that for the MNase - digested oligomers the supernatant fraction also includes the digested free linker DNA . Each value is the mean of three measurements and the error bars represent the standard deviation .

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A 601-nucleosomal arrays were incubated in 0.5, 5 mM MgCl2, 5 mM MgCl2 + MNase, and analyzed by the differential centrifugation assay to determine the fraction oligomeric. The amounts of DNA in the supernatant fraction were measured. Note that for the MNase-digested oligomers the supernatant fraction also includes the digested free linker DNA. Each value is the mean of three measurements and the error bars represent the standard deviation.


B Verification of complete MNase digestion . DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase , and then electrophoresed on agarose gel . The position of mono nucleosome is marked with a star symbol .

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B Verification of complete MNase digestion. DNA was purified from the nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase, and then electrophoresed on agarose gel. The position of mono nucleosome is marked with a star symbol.


C Nucleosomal array oligomers ( left ) or with MNase treatment ( right ) were stained with DAPI and examined using FM . Shown are representative images obtained . Note that the sizes of MNase - treated oligomers are much smaller than those of the control oligomers ( left and Fig . 1A ) .

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C Nucleosomal array oligomers (left) or with MNase treatment (right) were stained with DAPI and examined using FM. Shown are representative images obtained. Note that the sizes of MNase-treated oligomers are much smaller than those of the control oligomers (left and Fig. 1A).


A Oligomers assembled from H1 - nucleosomal arrays were stained with DAPI and examined using FM . Shown are representative images obtained in 4 mM and 5 mM MgCl2 . B Control FM images obtained in 0 , 1 and 3 mM MgCl2 .

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A Oligomers assembled from H1-nucleosomal arrays were stained with DAPI and examined using FM. Shown are representative images obtained in 4 mM and 5 mM MgCl2.B Control FM images obtained in 0, 1 and 3 mM MgCl2.


C H1 - nucleosomal array oligomers were negatively stained and visualized by TEM . Shown in the left panel is a representative image obtained in 4 mM MgCl2 . Shown in the right panel is an image of the interior of the oligomer ( white arrow , left panel ) after cropping and re - scaling .

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C H1-nucleosomal array oligomers were negatively stained and visualized by TEM. Shown in the left panel is a representative image obtained in 4 mM MgCl2. Shown in the right panel is an image of the interior of the oligomer (white arrow, left panel) after cropping and re-scaling.


Figure 6Sedimentation analysis of salt - dependent H1 - oligomer assembly . A Representative experiment showing the second moment sedimentation coefficients of the H1 - oligomers as a function of MgCl2 . The dashed line indicates the upper limit of measurable sedimentation coefficients ( ~ 106 S ) . The white symbol is intended to show that the sedimentation coefficient of the H1 - oligomers in 5 mM MgCl2 is beyond the detectable limit . B Analysis of the same raw data as in panel ( A ) by the time derivative method to yield the sedimentation coefficient distribution , g ( s * ) . C The g ( s * ) profiles in 2 . 5 mM , 3 mM , and 3 . 5 mM from panel ( B ) are re - plotted on a smaller scale .

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Figure 6Sedimentation analysis of salt-dependent H1-oligomer assembly.A Representative experiment showing the second moment sedimentation coefficients of the H1-oligomers as a function of MgCl2. The dashed line indicates the upper limit of measurable sedimentation coefficients (~106 S). The white symbol is intended to show that the sedimentation coefficient of the H1-oligomers in 5 mM MgCl2 is beyond the detectable limit.B Analysis of the same raw data as in panel (A) by the time derivative method to yield the sedimentation coefficient distribution, g(s*).C The g(s*) profiles in 2.5 mM, 3 mM, and 3.5 mM from panel (B) are re-plotted on a smaller scale.


A , B SAXS profiles of the H1 - nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) and 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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A, B SAXS profiles of the H1-nuclesomal arrays in 0 (TE), 1, 2.5 mM MgCl2 (A) and 5, 10 mM MgCl2 (B) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


C , D SAXS profiles of the native chicken chromatin in 0 ( TE ) , 0 . 5 , 1 mM MgCl2 ( C ) and 2 . 5 , 5 mM MgCl2 ( D ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , intensity ; S , scattering vector ( 1 / nm ) ) .

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C, D SAXS profiles of the native chicken chromatin in 0 (TE), 0.5, 1 mM MgCl2 (C) and 2.5, 5 mM MgCl2 (D) are shown as plots of log(I × S2) vs. 1/S (I, intensity; S, scattering vector (1/nm)).


A FMimages of chromatin structure in the nuclei with 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) . Insets show the intensity line profiles between the two marked arrow heads in the images .

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A FMimages of chromatin structure in the nuclei with 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right). Insets show the intensity line profiles between the two marked arrow heads in the images.


B SAXS profiles of the isolated HeLa nuclei . SAXS profiles of the nuclei in 0 mM ( left ) , 1 mM ( center ) and 5 mM MgCl2 ( right ) are shown in blue . For comparison , the scattering curves of H1 - arrays from Figs . 7A and B were overlaid as red lines ( left , 0 mM ; center and right , 10 mM MgCl2 ) .

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B SAXS profiles of the isolated HeLa nuclei. SAXS profiles of the nuclei in 0 mM (left), 1 mM (center) and 5 mM MgCl2 (right) are shown in blue. For comparison, the scattering curves of H1-arrays from Figs. 7A and B were overlaid as red lines (left, 0 mM; center and right, 10 mM MgCl2).


( b ) Flow cytometry - based stratification of cells in different cell cycle phases and in G0 ( quiescence , 2n DNA - and low RNA content ) , shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours , respectively . Cells were detached , fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis .

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(b) Flow cytometry-based stratification of cells in different cell cycle phases and in G0 (quiescence, 2n DNA- and low RNA content), shown for SKOV3 cells in serum starvation conditions at 8 and 96 hours, respectively. Cells were detached, fixed with ethanol and stained with Hoechst 33342 and Pyronin Y prior to flow cytometry analysis.


( c ) Distribution of cell cycle phases in six cell lines exposed to quiescence - inducing conditions ( see panel a ) for 8 and 96 hours , respectively . Each stacked bar plot reports the fraction of cells in G0 , G1 , S and G2 / M phases , estimated using the assay depicted in panel b .

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(c) Distribution of cell cycle phases in six cell lines exposed to quiescence-inducing conditions (see panel a) for 8 and 96 hours, respectively. Each stacked bar plot reports the fraction of cells in G0, G1, S and G2/M phases, estimated using the assay depicted in panel b.


( d ) Cell confluence of adherent cell cultures in quiescence - inducing conditions ( i . e . serum starvation and glutamine limitation ) was monitored using time - lapse microscopy in a plate reader ( TECAN Spark 10M ) in 1 . 5 - hour intervals . Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach . Data points and shaded areas indicate mean ± standard deviation ( SD ) over three biological replicates . Approximately 9500 , 4500 , 19500 , 6700 , 19500 and 9500 cells / well were seeded for glutamine limitation and serum starvation of A549 , CCD1070Sk , HCT116 , HFL1 , MCF7 and SKOV3 cells , respectively .

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(d) Cell confluence of adherent cell cultures in quiescence-inducing conditions (i.e. serum starvation and glutamine limitation) was monitored using time-lapse microscopy in a plate reader (TECAN Spark 10M) in 1.5-hour intervals. Contact inhibited cells no longer grow in monolayers and could not be accurately imaged with this approach. Data points and shaded areas indicate mean ± standard deviation (SD) over three biological replicates. Approximately 9500, 4500, 19500, 6700, 19500 and 9500 cells/well were seeded for glutamine limitation and serum starvation of A549, CCD1070Sk, HCT116, HFL1, MCF7 and SKOV3 cells, respectively.


( b ) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium ( RPMI - 1640 with 5 % dialyzed FBS ) . Cell confluence as a proxy of cell numbers was monitored using automated time - lapse microscopy in a plate reader ( TECAN Spark 10M ) . Data points and shaded areas indicate mean ± SD over three biological replicates . ( c ) Growth phenotypic changes induced by TMZ treatment in quiescence - inducing conditions ( serum starvation ) in A549 and HCT116 cancer cells . Growth was monitored via cell confluence as described in panel b . At 96 hours , cells were stimulated to exit quiescence by replacing serum - free media with fresh growth medium without TMZ ( regrowth ) . Data points and shaded areas indicate mean ± SD over three biological replicates .

O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-CELL_LINE O B-CELL_LINE O O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O 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-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O

(b) Growth phenotypic changes induced by TMZ treatment in A549 and HCT116 cells grown in full growth medium (RPMI-1640 with 5% dialyzed FBS). Cell confluence as a proxy of cell numbers was monitored using automated time-lapse microscopy in a plate reader (TECAN Spark 10M). Data points and shaded areas indicate mean ± SD over three biological replicates. (c) Growth phenotypic changes induced by TMZ treatment in quiescence-inducing conditions (serum starvation) in A549 and HCT116 cancer cells. Growth was monitored via cell confluence as described in panel b. At 96 hours, cells were stimulated to exit quiescence by replacing serum-free media with fresh growth medium without TMZ (regrowth). Data points and shaded areas indicate mean ± SD over three biological replicates.


( d ) Viability of quiescence - induced cells treated with TMZ , or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes : Hoechst 33342 ( all nuclei ) and propidium iodide ( dead cells ) . Data points and error bars indicate mean ± SD over three biological replicates .

O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O

(d) Viability of quiescence-induced cells treated with TMZ, or with TMZ plus 10 µM etomoxir was assessed using a fluorescence imaging assay involving two fluorescent dyes: Hoechst 33342 (all nuclei) and propidium iodide (dead cells). Data points and error bars indicate mean ± SD over three biological replicates.


( e ) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ . Fold - changes were estimated relative to steady - state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates .

O O O O O O O O O O B-SMALL_MOLECULE O B-CELL_LINE O B-CELL_LINE O O O O O O B-TISSUE O O O O B-SMALL_MOLECULE O O O O O 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) Dynamic changes in the intracellular levels of citrate in A549 and HCT116 cells induced to enter quiescence by serum starvation with or without TMZ. Fold-changes were estimated relative to steady-state levels in full medium Data points and error bars indicate mean ± SD over three biological replicates.


( f ) Volcano plot showing TMZ - induced metabolic changes ( 1 mM , 48 hours ) in A549 cells in serum starvation Shown are differences in log2 fold - change between TMZ treatment and serum starvation alone ( x - axis ) , and their statistical significance ( y - axis , adjusted p - value , t - test and Benjamini - Hochberg correction ) .

O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_LINE O O B-TISSUE O O O O O O O O O O B-SMALL_MOLECULE O O B-TISSUE O O O O O O O O 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) Volcano plot showing TMZ-induced metabolic changes (1 mM, 48 hours) in A549 cells in serum starvation Shown are differences in log2 fold-change between TMZ treatment and serum starvation alone (x-axis), and their statistical significance (y-axis, adjusted p-value, t-test and Benjamini-Hochberg correction).


( h ) Comparison of relative changes ( log2 fold - change ) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment ( 1 mM , y - axis ) against untreated quiescence - induced A549 cells ( x - axis ) . Fold - changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations ( Spearman , p < 0 . 01 ) with cell death at 96 hours are highlighted in blue and yellow , respectively .

O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(h) Comparison of relative changes (log2 fold-change) in the abundance of 1791 putatively annotated metabolites upon TMZ treatment (1 mM, y-axis) against untreated quiescence-induced A549 cells (x-axis). Fold-changes are calculated relative to the expected ion intensity in full medium at steady state Significant positive and negative correlations (Spearman, p < 0.01) with cell death at 96 hours are highlighted in blue and yellow, respectively.


( i ) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence - inducing conditions ( serum starvation , left panels ) or full growth medium ( 5 % dFBS , right panels ) , treated with TMZ ( 0 . 4 or 1 mM ) , 5 - fluorouracil ( 5 - FU , 0 . 5 mM , positive control ) or medium ( untreated ) . Early apoptotic cells were detected using ApoTracker Green under co - staining with Hoechst 33342 ( nuclei ) and propidium iodide ( dead cells ) . Cell staining was continuously monitored in 3 - hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto . Data points and shaded areas indicate mean ± SD over three biological replicates . Approximately 3400 and 7500 cells / well were seeded of A549 and HCT116 cells , respectively .

O O O O O O O O O B-CELL_LINE O B-CELL_LINE O O O O O O 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-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-CELL_LINE O B-CELL_LINE O O O O

(i) Fractions of early apoptotic cells in A549 or HCT116 cancer cell cultures in quiescence-inducing conditions (serum starvation, left panels) or full growth medium (5% dFBS, right panels), treated with TMZ (0.4 or 1 mM), 5-fluorouracil (5-FU, 0.5 mM, positive control) or medium (untreated). Early apoptotic cells were detected using ApoTracker Green under co-staining with Hoechst 33342 (nuclei) and propidium iodide (dead cells). Cell staining was continuously monitored in 3-hours intervals for 96 hours using automated fluorescence microscopy in a TECAN Spark Cyto. Data points and shaded areas indicate mean ± SD over three biological replicates. Approximately 3400 and 7500 cells/well were seeded of A549 and HCT116 cells, respectively.


( D and E ) qRT - PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence ( D ) or presence ( E ) of translation - blocking GFP MO . Bar charts show the average of three independent biological replicates . Error bars show standard deviation ( SD ) . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O 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-GENEPROD B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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 and E) qRT-PCR analysis of codon tag reporter mRNA levels at 6 hpf relative to 2 hpf in the absence (D) or presence (E) of translation-blocking GFP MO. Bar charts show the average of three independent biological replicates. Error bars show standard deviation (SD). Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( B - E ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the codon effects measured by PACE ( y - axis ) . Each dot represents a single codon . Graphs for all codons ( B ) , codons for polar amino acids ( C ) , nonpolar amino acids ( D ) , and charged amino acids ( E ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-E) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the codon effects measured by PACE (y-axis). Each dot represents a single codon. Graphs for all codons (B), codons for polar amino acids (C), nonpolar amino acids (D), and charged amino acids (E) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( F - I ) Scatter plots showing a correlation between relative ribosome density ( 28 - 30 - nt footprints ; x - axis ) and the tRNA levels ( y - axis , Each dot represents a single codon . Graphs for all codons ( F ) , codons for polar amino acids ( G ) , nonpolar amino acids ( H ) , and charged amino acids ( I ) are shown . Data information : the regression line is shown in blue and the 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - test .

O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O 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-I) Scatter plots showing a correlation between relative ribosome density (28-30-nt footprints; x-axis) and the tRNA levels (y-axis, Each dot represents a single codon. Graphs for all codons (F), codons for polar amino acids (G), nonpolar amino acids (H), and charged amino acids (I) are shown. Data information: the regression line is shown in blue and the 95% confidence interval is shown in gray. P-values were calculated using Student's t-test.


( B ) Charged tRNA levels in mock - injected and AnsB - overexpressing embryos at 6 hpf . Total and charged tRNAs were measured by qRT - PCR coupled to the OXOPAP assay . The relative charged tRNA level in mock - injected embryos were set to one . Data information bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O 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) Charged tRNA levels in mock-injected and AnsB-overexpressing embryos at 6 hpf. Total and charged tRNAs were measured by qRT-PCR coupled to the OXOPAP assay. The relative charged tRNA level in mock-injected embryos were set to one. Data information bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(D) Poly(A) tail analysis of Asn AAC codon tag reporter mRNA at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( F ) Poly ( A ) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf . ( G ) Quantification of the PAT assay at 6 hpf in ( F ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(F) Poly(A) tail analysis of Leu CUG codon tag reporter mRNA at 2 and 6 hpf. (G) Quantification of the PAT assay at 6 hpf in (F).


( B ) Western blotting to detect the sfGFP levels at 6 hpf . Tubulin ( Tub ) was detected as a loading control .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O

(B) Western blotting to detect the sfGFP levels at 6 hpf. Tubulin (Tub) was detected as a loading control.


( C ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos at 6 hpf relative to 2 hpf . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos at 6 hpf relative to 2 hpf. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( D ) Poly ( A ) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf . The developmental stages are shown above as hpf . The lane labeled A0 shows the 3 ´ UTR fragment without a poly ( A ) tail . ( E ) Quantification of the PAT assay at 6 hpf in ( D ) .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O O O O O O 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-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(D) Poly(A) tail analysis of hCMV uORF2 reporter mRNAs at 2 and 6 hpf. The developmental stages are shown above as hpf. The lane labeled A0 shows the 3´ UTR fragment without a poly(A) tail. (E) Quantification of the PAT assay at 6 hpf in (D).


( G ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf . ( H ) qRT - PCR analysis of hCMV uORF2 reporter mRNA levels in wild - type embryos , MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc - tagged full - length ( full ) or RING domain deleted ( ∆ RING ) Znf598 . mRNA levels at 6 hpf relative to 2 hpf are shown . Data information : bar charts show the average of three independent biological replicates . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O B-TISSUE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-ORGANISM O O O O O O O O B-TISSUE O O B-TISSUE O O B-TISSUE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in MZznf598 embryos at 6 hpf relative to 2 hpf. (H) qRT-PCR analysis of hCMV uORF2 reporter mRNA levels in wild-type embryos, MZznf598 embryos and MZznf598 embryos injected with a mRNA encoding Myc-tagged full-length (full) or RING domain deleted (∆RING) Znf598. mRNA levels at 6 hpf relative to 2 hpf are shown. Data information: bar charts show the average of three independent biological replicates. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( J ) Results of PACE in MZznf598 zebrafish embryos . Black circles show the relative stability of the reporter mRNAs ( averages of two biological replicates ) in MZznf598 embryos . Gray circles show the relative stability of reporter mRNAs in wild - type embryos The stability of a codon - tag reporter with a UGA stop codon was set to one . Error bars represent maximum and minimum data points . The relative effect of each codon on mRNA stability in wild - type embryos is shown as a color gradient from red ( destabilizing ) to blue ( stabilizing ) .

O O O O O B-EXP_ASSAY O O B-ORGANISM B-TISSUE O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O 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-TISSUE O O O O O O O O O O O O O O O O O

(J) Results of PACE in MZznf598 zebrafish embryos. Black circles show the relative stability of the reporter mRNAs (averages of two biological replicates) in MZznf598 embryos. Gray circles show the relative stability of reporter mRNAs in wild-type embryos The stability of a codon-tag reporter with a UGA stop codon was set to one. Error bars represent maximum and minimum data points. The relative effect of each codon on mRNA stability in wild-type embryos is shown as a color gradient from red (destabilizing) to blue (stabilizing).


( B ) Results of the tandem ORF assay with Lys AAG × 8 and Lys AAA × 8 sequences . ( C ) Results of the tandem ORF assay with Gly × 8 and Pro × 8 sequences . ( D ) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags . ( E ) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities . Data information : normalized Fluc activity with no insert was set to one . All experiments were repeated three times , and the average Fluc activity is shown as bar charts . Error bars show SD . Individual data points are shown as dots . P - values were calculated using two - sided Student ' s t - test .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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) Results of the tandem ORF assay with Lys AAG×8 and Lys AAA×8 sequences. (C) Results of the tandem ORF assay with Gly×8 and Pro×8 sequences. (D) Results of the tandem ORF assay with Asn AAC and Asn AAU codon tags. (E) Results of the tandem ORF assay using EGFP ORFs with different codon optimalities. Data information: normalized Fluc activity with no insert was set to one. All experiments were repeated three times, and the average Fluc activity is shown as bar charts. Error bars show SD. Individual data points are shown as dots. P-values were calculated using two-sided Student's t-test.


( B ) SEC trace and SDS - PAGE of purified NHE9 * ( residues 8 to and 574 out of 644 ) as depicted by schematic .

O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O O O O O O O O O O

(B) SEC trace and SDS-PAGE of purified NHE9* (residues 8 to and 574 out of 644) as depicted by schematic.


( C ) Activity of NHE9 * co - reconstituted with ATPase into liposomes to mimic the in vivo situation ( schematic ) . Representative ACMA fluorescence traces of liposome reconstituted NHE9 * ( blue ) , NHE9 * double - mutant N243A - D244A ( red ) , and rat fructose transporter GLUT5 ( black ) . ATP - driven H + pumping establishes a ΔpH ( 0 - 3 min ) . H + efflux is initiated by the addition of 40 mM NaCl , and subsequent addition of NH4Cl ( 4 min ) collapses the proton gradient .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O

(C) Activity of NHE9* co-reconstituted with ATPase into liposomes to mimic the in vivo situation (schematic). Representative ACMA fluorescence traces of liposome reconstituted NHE9* (blue), NHE9* double-mutant N243A-D244A (red), and rat fructose transporter GLUT5 (black). ATP-driven H+ pumping establishes a ΔpH (0-3 min). H+ efflux is initiated by the addition of 40 mM NaCl, and subsequent addition of NH4Cl (4 min) collapses the proton gradient.


( D ) Michaelis - Menten kinetics for NHE9 * ( red ) , NHE9 ΔCTD ( green ) , N243A - D244A ( blue ) and rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In all experiments errors bars , s . e . m . ; n = 3 technical repeats . The apparent KM values are an average from n = 3 separate protein reconstitutions .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O O B-ORGANISM B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O 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) Michaelis-Menten kinetics for NHE9* (red), NHE9 ΔCTD (green), N243A-D244A (blue) and rat GLUT5 (black) as detected by ACMA dequenching following substrate addition. In all experiments errors bars, s.e.m.; n = 3 technical repeats. The apparent KM values are an average from n = 3 separate protein reconstitutions.