3D reconstruction showing association of Prom1 ( green ) with AcTub - labeled ( red ) cilia in transit amplifying regions . Note that expression of Prom1 is not limited cilium but also to microvilli . A representative example of Prom1 association with one cilium at to transit amplifying transition region . Green channel transparency was set up to 70 % .

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3D reconstruction showing the association of Prom1 (green) with AcTub-labeled (red) primary cilia in stem cell and transit amplifying cell regions. Note that the expression of Prom1 is not limited primary cilium but also to microvilli. A representative example of Prom1 association with one primary cilium at the stem cell to transit amplifying cell transition region. Green channel transparency was set up to 70%.


Representative IF of Prom1 using antibodies directed either its extracellular loop ( clone 13A4 , green ) or C - terminal end ( Biorbyt , Orb129549 , red ) on transit amplifying regions of WT vs . Prom1 KO mice . Samples are counterstained with DAPI ( blue ) . Note lack of Prom1 labeling in Prom1 KO mice .

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Representative IF staining of Prom1 using antibodies directed either its extracellular loop (clone 13A4, green) or cytoplasmic C-terminal end (Biorbyt, Orb129549, red) on transit amplifying cell regions of the WT vs. Prom1 KO mice. Samples are counterstained with DAPI (blue). Note the lack of Prom1 labeling in Prom1 KO mice.


mRNA profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 3 ) in cultured CLESCs . qRT - PCR results are in arbitrary values after normalization for GapDH .

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The mRNA profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. qRT- PCR results are in arbitrary values after normalization for GapDH.


profiling on shRNA - meditated Prom1 knockdown ( 3 different shRNAs were used , marked as NO . 1 , 2 3 ) in cultured CLESCs . immunoblotting was performed with anti - Prom1 C - terminal antibody . β - actin was used as loading control ( F ) . Molecular mass markers are indicated .

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protein profiling on shRNA-meditated Prom1 knockdown (3 different shRNAs were used, marked as NO.1, 2 and 3) in cultured CLESCs. The immunoblotting was performed with anti-Prom1 C-terminal antibody. β-actin was used as loading control (F). Molecular mass markers are indicated.


Representative IF of Prom1 ( red ) Sox2 ( green ) in CLESCs in vitro . Sample is counterstained with DAPI ( blue ) . Arrows indicate colocalisation of Prom1 with low - medium level of Sox2 in nuclei .

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Representative IF staining of Prom1 (red) and Sox2 (green) in CLESCs in vitro. Sample is counterstained with DAPI (blue). Arrows indicate colocalisation of Prom1 with low-medium level of Sox2 in the cell nuclei.


Subcellular fractionation of CLESCs into cytoplasm / ( C + M ) ( N ) fractions . Materials were analyzed by immunoblotting for Prom1 ( H ) Arl13b ( I ) . As controls , membranes were blotted for anti - Lamin A / C ( nuclei markers ) GapDH ( cytoplasm marker ) . Molecular mass markers are indicated . Note presence of full - Prom1 as well as Arl13b in fraction .

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Subcellular fractionation of CLESCs into cytoplasm/membrane (C+M) and nuclear (N) fractions. Materials were analyzed by immunoblotting for Prom1 (H) and Arl13b (I). As controls, membranes were blotted for anti-Lamin A/C (nuclei markers) and GapDH (cytoplasm marker). Molecular mass markers are indicated. Note the presence of full-length Prom1 as well as Arl13b in nuclear fraction.


Representative ( A ) quantitative ( B ) of IF 3D reconstruction on Sox2 - positive region for WT vs . Prom1 KO mice . Ant : Anterior ; Post : Posterior ( A ) . Statistics was performed using Student ' s t - : * * , p < 0 . 01 . Data are represented as mean standard deviation .

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Representative images (A) and quantitative analysis (B) of IF staining and 3D reconstruction on Sox2-positive region for WT vs. Prom1 KO mice. Ant: Anterior; Post: Posterior (A). Statistics was performed using Student's t-test: **, p < 0.01. Data are represented as mean and standard deviation.


Representative IF double of Sox 2 ( green ) Ki67 ( red ) on transit amplifying regions of WT vs . Prom1 KO mice on frontal sections . Samples are counterstained with DAPI ( blue ) . of Ki67 + number in Sox2 - positive region on IF data from WT Prom1 KO mice ( C ) .

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Representative IF double staining of Sox 2 (green) and Ki67 (red) on the and transit amplifying cell regions of WT vs. Prom1 KO mice on frontal sections. Samples are counterstained with DAPI (blue). Quantification of Ki67+ cell number in Sox2-positive region based on IF data from WT and Prom1 KO mice (C).


Stereo of three explants cultured with indicated recombination conditions before harvesting at day 3 . Dotted indicates CLE .

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Stereo images of three explants cultured with indicated recombination conditions before harvesting at day 3. Dotted line indicates the CLE.


Passaging capacity of CLESCs derived from P7 WT Prom1 KO mice . Note passages of derived from WT are still ongoing while those from Prom1 KO mice were arrested at first passage . Arrows indicated were still under passaging while blunt bars represent could not be passaged stopped at indicated passage .

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Passaging capacity analysis of the CLESCs derived from P7 WT and Prom1 KO mice. Note the passages of cells derived from WT animals are still ongoing while those from Prom1 KO mice were arrested at the first passage. Arrows indicated cells were still under passaging while blunt bars represent cells could not be passaged and stopped at the indicated passage.


Clonogenicity for colony number ( B ) ( C ) after first passage of CLESCs derived from WT vs . Prom1 KO mice in absence or presence of SHH ( 100ng / ml ) . were seeded in triplicate on 24 - well plates as 5 , 000 / 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 mesenchymal were mixed at of 5 , 000 : 5 , 000 per well in triplicates on 96 - well plates grown for 3 days on Matrigel . Representative of entire well under indicated conditions are shown . 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 Ki67 - Fucci system showing colored at different 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 of Ki67 - Fucci CLESCs at different cycle stages in transfected with empty vector or full Prom1 . As a result , > 95 % of events have been included in . accuracy of strategy has been validated by two independent researchers for IF 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 of AcTub ( blue ) on Ki67 - Fucci CLESCs without with presence of SHH . Note that 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 of either AcTub ( D , red ) or Arl13b ( E , red ) with Sox2 ( green ) or Ki67 ( green ) in CLESCs in presence of SHH . Note expression of Sox2 Ki67 distinguished transit amplifying , 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.


of cilia in transit amplifying regions of WT Prom1 KO mice ( A ) their density in respective regions ( B ) . 3 were analyzed for each genotype . 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 of CLESCs treated with either Hdac6 inhibitor ( Tubacin ) appropriate controls ( Nitubacin 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 of CLESCs treated with siArl13b 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 . 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 . 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 of samples indicated in ( E F ) .

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


Representative IF double 3D reconstruction of Prom1 ( green ) AcTub ( red ) at apical domain of polarized MDCK expressing Prom1 its K138Q mutant ( top panels ) . Black - inserts show Prom1 signals only . Same were analyzed by ( bottom panels ) . Note that Prom1 is asymmetrically distributed along cilium ( arrows ) as observed in vivo ( see Fig 2C ) mutation K138Q significantly impacts on cilium . 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 3D reconstruction of AcTub - labeled ( red ) cilium at apical of parental MDCK ( MDCK ) those transfected with Prom1 or its K138Q mutant . were used to quantify cilium ( Appendix Figs S6B C ) . Arrows indicate cilia with more than 4 μm in .

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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 indicated . Brackets asterisks indicate plasma 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 indicated . Brackets asterisks indicate plasma 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.


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

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


Parental MDCK or those transfected with Prom1 or K138Q mutant were grown for 7 dpc were observed by phase - contrast microscopy ( A ) Box - whisker plots show number of domes per 600 mm2 ( A ) . Three 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 or those transfected with Prom1 or K138Q mutant were grown for 7 dpc were observed by ( B , top panels ) or confocal scanning microscopy after DAPI ( B , bottom panels , within a given dome are indicated ( B , top panels ) were quantified using three different planes ( bottom , centre 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 or those transfected with Prom1 or K138Q mutant were grown for 7 dpc ( A - C ) or as indicated ( D ) . confocal scanning microscopy after DAPI Domes of similar were evaluated ( C ) . Means standard deviations are displayed . 15 domes per independent experiment were examined ( n = 3 ) . density at a given time of culture ( 7 , 10 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 of > 4 µm were rarely observed in K138Q mutant displayed an irregular shape with constrictions along their distal part as observed by ( 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 Hdac6 - binding site of Prom1 increases 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 ) 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent lysates were prepared . entire pellets 1 / 40 of lysates were analyzed by immunoblotting using antibodies against human Prom1 , AcTub β - actin ( F ) . Molecular mass markers are indicated . three were detected in vesicle fractions ( P3 P4 ) suggesting Prom1 + vesicles are released from cilia 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 Hdac6 - binding site of Prom1 increases 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 ) 1 h at 200 , 000 x g ( P4 ) . In parallel , detergent lysates were prepared . release of Prom1 + vesicles was quantified using its immunoreactivities associated with 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)


mRNA expression of SHH effector genes ( Gli1 / 2 Glis1 - 3 ) on microdissection captured samples were evaluated by real - time RT - PCR . 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 of Gli1 ( left panel , red ) Glis2 ( right panel , red ) on transit amplifying regions of lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Dotted lines , basement .

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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 ( see B , red ) its association with DAPI - labeled nuclei ( blue ) . 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 of Glis2 ( red , bottom panel ) AcTub ( green , top bottom panels ) along - transit amplifying axis in lower incisor CLE at P7 . Samples were counterstained with DAPI ( blue ) . Note association of Glis2 with cilia particularly in transition zone between transit amplifying 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 lower incisor CLE at P7 was evaluated in WT vs . Glis2 KO mice upon IF . Number of 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 of Prom1 using specific antibodies targeting either its extracellular loop ( top panels , green ) or C - terminal end ( bottom panels , red ) on transit amplifying regions of lower incisor CLE at P7 WT Glis2 KO mice . Samples are counterstained with DAPI ( blue ) . Dotted lines , basement . Arrowheads marks approximate boundary of 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 of Glis2 ( red ) AcTub ( green ) as a marker of cilia in region of WT vs . Prom1 KO mice . Note reduction of Glis2 expression in Prom1 - deficient .

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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 of Prom1 Glis2 association ( red ) in transit amplifying regions of lower incisor CLE at P7 . As negative control , one conjugated antibody ( anti - Glis2 ) was omitted . SCs , ; TACs , transit amplifying .

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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 Glis2 co - expression ( proximity ligation ) in cultured CLESCs starved for 4 days , then treated with SHH ( 100 ng / ml ) importazole ( 40 µM ) , an inhibitor of importin β1function . DMSO was used as vehicle control . Data are represented triplicated samples as mean 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 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 Glis2 KO vs . WT mice ( n = 3 ) . mRNA expression of gene significantly changed in PCR array ( in ( C ) ) ( D ) was evaluated in WT , Prom1 Glis2 KO mice by real - time RT - PCR . Each sample was isolated from distinct mice ( n = 3 ) . 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 Glis2 KO vs . WT mice ( n = 3 ) . mRNA expression of gene significantly changed in Stat pathway members ( E ) was evaluated in WT , Prom1 Glis2 KO mice by real - time RT - PCR . Each sample was isolated from distinct mice ( n = 3 ) . 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.


IP of binding of Glis2 to Stat3 promoter ( see Appendix Fig S9D ) in absence or presence of SHH at different as indicated on triplicated samples , using specific anti - Glis2 antibodies 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 are shown . NIH3T3 were harvested treated with indicated glycosidases . Black bracket indicates mature glycosylated form grey bracket indicates glycosylated form . 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 Endo H - resistant glycosylation of SMO . HEK293T were co - transfected with SMO PTCH1 expression plasmids . After 48 h , were harvested , lysed , 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 ) of Endo H - resistant form of SMO shown in ( C ) . Three independent experiments were quantified by Image J expressed as a of total SMO .

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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 Endo H - resistant form of SMO . were transfected as indicated for 24 h , incubated with ShhN conditioned medium for an additional 24 h . were harvested 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 ) of Endo H - resistant form of SMO shown in ( E ) . Three independent experiments were quantified by Image J expressed as a of total SMO .

(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 Endo H - resistant form of endogenous SMO in MEF . 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 of Endo H - resistant form of SMO shown in ( G ) . Three independent experiments were quantified by Image J expressed as a of total SMO .

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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 R118G mutation in BirA ) in a proximity - dependent biotinylation . were transfected with plasmids encoding SMO SAR1B - BirA * - FLAG . After 48 h , were treated with different doses of biotin for 4 h . were harvested lysates were dialyzed to remove free biotin . Biotinylated were pulled down by streptavidin beads subjected to immunoblotting . ( B ) were treated with different doses of biotin for 24 h . were harvested , lysed , 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 ) were treated with different doses of biotin for 24 h . were harvested , lysed , 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 ) GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . were transfected with plasmids expressing SMO indicated SAR1B - BirA * . After 48 h , 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 . 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 ) GDP - locked SAR1B ( G37A ) - BirA * failed to label SMO , while GTP - locked SAR1B ( H79G ) - BirA * could efficiently biotinylate SMO . were transfected with plasmids expressing SMO indicated SAR1B - BirA * . After 48 h , 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 . experiments were done similarly in Fig . 2A . ( E ) Shh increased binding of SAR1B to SMO . After transfection for 24 h , were incubated with ShhN conditioned medium for an additional 24 h . Then 15 μM biotin was added into culture medium for 4 h . were harvested 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 . 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 SMO variants . 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 incubated in vitro in presence of cytosol , ATP , GTP , an ATP - regenerating system as described in experimental procedures . Then vesicle fractions were separated by centrifugation subjected to immunoblotting .

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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 SMO variants . 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 ) ciliary localization of SMO variants . Smo - KO NIH3T3 were transfected with EGFP fused SMO variants , incubated with serum starvation medium for 24 h to fully induce cilia formation , treated with or without ShhN conditioned medium for 4 h . were immunostained with 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 of ciliary SMO in ( F ) by Image J . More than 45 were randomly selected 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 ) of of 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 in Smo - KO NIH3T3 . were transfected with plasmids encoding SMO or SMO variants , GLI - promoted firefly luciferase renilla luciferase . After 24 h , 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 were transfected , harvested ,

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


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

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


( C ) CP - modified SMO was mainly resistant to Endo H . CP - modified SMO were pulled down by streptavidin beads digested with Endo H or PNGase F at 37 ℃ . Then were eluted with SDS - PAGE sample buffer were subjected to immunoblotting . 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 increased by ShhN .

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


( E ) 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 ) of CP - modified WT SMO SMO ( 4A ) were compared . 1 . 39 % of SMO ( WT ) pellet samples 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 ) of CP - modified SMO shown in ( F ) . Three independent experiments were quantified by Image J expressed as a of 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 ) stimulative / inhibitory effect of Shh / PTCH1 on SMO SAR1B interaction was compromised by D95E mutation . experiments were done 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 cholesterol modification of SMO were treated with ALOD4 for 1 h , then incubated with a medium containing ShhN CP for 2 3 h . were harvested

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

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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 . were treated with ALOD4 for 1 h , 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 of ciliary SMO in ( D ) . More than 80 were randomly selected 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 ) of of with ciliary SMO in ( D ) . Data are presented as mean ± from three biological replicates ( * * * * p < 0 . 0001 , one - way ANOVA with Dunnett ' s multiple comparison ) . 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 expression of Gli1 was measured by qRT - PCR . were treated with ALOD4 for 1 h , 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 were co - transfected with SMO or SMO ( 4A ) GRAMD1B expression plasmids for 24 h , then incubated with a cholesterol depletion medium ( DMEM supplemented with 5 % lipoprotein - deficient serum , 1 μM lovastatin 10 μM mevalonate ) for 12 h , followed by 2 μg / mL of CP treatment . were harvested 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 . expression plasmids of SMO or SMO ( D95E ) GRAMD1B were transfected into SMO - KO HEK293T . After 12 h , were switched to a cholesterol depletion medium for 16 h 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 stably expressing GRAMD1B . After incubating in serum starvation medium for 16 h , were depleted of cholesterol by treatment with 1 . 5 % HPCD for 0 . 5 h then incubated in serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate 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 of NIH3T3 stably expressing mCherry - GRAMD1B . PM was labeled with wheat germ agglutinin ( WGA ) - CF ® 488A conjugate . were incubated in a cholesterol depletion medium for 16 h switched to same medium containing 50 μM MβCD - cholesterol for 1 h . 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 ) of with GRAMD1B localization on PM . 60 - 70 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 ) cholesterol modification of endogenous SMO was decreased in NIH3T3 lacking Gramd1a , 1b , 1c , 2 , 3 ( Gramd - 5KO ) . were incubated in a cholesterol depletion medium for 12 h , followed by 2 μg / mL of CP 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 ) glycosylation of endogenous SMO was decreased in Gramd - 5KO NIH3T3 . 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 ) ciliary localization of endogenous SMO in WT or Gramd - 5KO NIH3T3 . were incubated in serum starvation medium for 24 h to fully induce cilia formation 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 of ciliary SMO in ( C ) . More than 100 were randomly selected 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 ) of of 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 ) of Gli1 was significantly reduced in Gramd - 5KO . 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 in WT or Gramd - 5KO NIH3T3 . Data are presented as mean ± from three technical replicates ( * * * * p < 0 . 0001 ; * * * p < 0 . 001 ; * p < 0 . 05 , two - way ANOVA with Sidak ' s multiple comparison ) .

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

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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 ) GRAMD inhibitor AI3d inhibited cholesterol modification of SMO in NIH3T3 . were incubated in cholesterol depletion medium for 16 h then treated with 3 μM AI3d for 6 h . Next , were incubated in same medium containing ShhN 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 ) GRAMD inhibitor AI3d decreased mRNA of Gli1 in NIH3T3 . were incubated in serum starvation medium for 16 h , depleted of cholesterol by 1 . 5 % HPCD for 0 . 5 h , then treated with serum starvation medium containing 1 μM lovastatin , 10 μM mevalonate 1 μM AI3d for 6 h . Next , were incubated in same medium containing ShhN 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 examined using FM ( microscopy ) as described in Materials Methods section . Shown are representative obtained in 4 . 5 mM 10mM MgCl2 . B Control FM obtained in 0 , 1 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 visualized by as described under Materials Methods . Shown in left panels are representative obtained in 4 . 5 mM , 10 mM MgCl2 . Shown in right panels are of interior of oligomers ( arrows , left panels ) after cropping 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 of salt - dependent assembly of nucleosomal array oligomers . A Representative experiment showing second moment sedimentation coefficients of oligomeric nucleosomal arrays as a function of MgCl2 . second moment sedimentation coefficient is equivalent to mass average sedimentation coefficient for entire sample ( see Materials Methods ) . inset shows mean second moment sedimentation coefficient standard error for three replicated experiments . B of same raw data as in panel ( A ) by time derivative method to yield 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 nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , ; 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 , analyzed by differential centrifugation to determine fraction oligomeric . amounts of in supernatant fraction were measured . Note that for MNase - digested oligomers supernatant fraction also includes digested free linker . Each value is mean of three measurements error bars represent 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 . was purified from nucleosomal arrays incubated in 5 mM MgCl2 or 5 mM MgCl2 + MNase , then electrophoresed on agarose gel . 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 examined using FM . Shown are representative obtained . Note that sizes of MNase - treated oligomers are much smaller than those of control oligomers ( left 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 examined using FM . Shown are representative obtained in 4 mM 5 mM MgCl2 . B Control FM obtained in 0 , 1 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 visualized by . Shown in left panel is a representative image obtained in 4 mM MgCl2 . Shown in right panel is an image of interior of oligomer ( arrow , left panel ) after cropping 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 of salt - dependent H1 - oligomer assembly . A Representative experiment showing second moment sedimentation coefficients of H1 - oligomers as a function of MgCl2 . dashed indicates upper limit of measurable sedimentation coefficients ( ~ 106 S ) . symbol is intended to show that sedimentation coefficient of H1 - oligomers in 5 mM MgCl2 is beyond detectable limit . B of same raw data as in panel ( A ) by time derivative method to yield sedimentation coefficient distribution , g ( s * ) . C g ( s * ) profiles in 2 . 5 mM , 3 mM , 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 H1 - nuclesomal arrays in 0 ( TE ) , 1 , 2 . 5 mM MgCl2 ( A ) 5 , 10 mM MgCl2 ( B ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , ; 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 native chicken in 0 ( TE ) , 0 . 5 , 1 mM MgCl2 ( C ) 2 . 5 , 5 mM MgCl2 ( D ) are shown as plots of log ( I × S2 ) vs . 1 / S ( I , ; 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 structure in nuclei with 0 mM ( left ) , 1 mM ( center ) 5 mM MgCl2 ( right ) . Insets show profiles between two marked arrow heads in .

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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 isolated HeLa nuclei . SAXS profiles of nuclei in 0 mM ( left ) , 1 mM ( center ) 5 mM MgCl2 ( right ) are shown in blue . For comparison , scattering curves of H1 - arrays from Figs . 7A B were overlaid as red lines ( left , 0 mM ; center 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 - stratification of in different cycle phases in G0 ( quiescence , 2n - low content ) , shown for SKOV3 in serum starvation conditions at 8 96 hours , respectively . were detached , fixed with ethanol stained with Hoechst 33342 Pyronin Y prior to flow cytometry .

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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 cycle phases in six lines exposed to quiescence - inducing conditions ( see panel a ) for 8 96 hours , respectively . Each stacked bar plot reports fraction of in G0 , G1 , S G2 / M phases , estimated using 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 ) confluence of adherent cultures in quiescence - inducing conditions ( i . e . serum starvation glutamine limitation ) was monitored using time - lapse microscopy in a plate reader ( TECAN Spark 10M ) in 1 . 5 - hour intervals . Contact inhibited no longer grow in monolayers could not be accurately imaged with this approach . Data points shaded areas indicate mean ± standard deviation ( SD ) over three biological replicates . Approximately 9500 , 4500 , 19500 , 6700 , 19500 9500 / well were seeded for glutamine limitation serum starvation of A549 , CCD1070Sk , HCT116 , HFL1 , MCF7 SKOV3 , 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 HCT116 grown in full growth medium ( RPMI - 1640 with 5 % dialyzed FBS ) . confluence as a proxy of numbers was monitored using automated time - lapse microscopy in a plate reader ( TECAN Spark 10M ) . Data points 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 HCT116 cancer . Growth was monitored via confluence as described in panel b . At 96 hours , were stimulated to exit quiescence by replacing serum - free media with fresh growth medium without TMZ ( regrowth ) . Data points 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 I-CELL_LINE O O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O O B-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 B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-CELL_LINE I-CELL_LINE O O B-EXP_ASSAY 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 B-SMALL_MOLECULE 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 treated with TMZ , or with TMZ plus 10 µM etomoxir was assessed using a imaging involving two dyes : Hoechst 33342 ( nuclei ) propidium iodide ( dead ) . Data points error bars indicate mean ± SD over three biological replicates .

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

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(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 in serum starvation Shown are differences in log2 fold - change between TMZ treatment serum starvation alone ( x - axis ) , their statistical significance ( y - axis , adjusted p - value , t - Benjamini - Hochberg correction ) .

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

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(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 in A549 or HCT116 cancer 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 were detected using ApoTracker Green under co - with Hoechst 33342 ( nuclei ) propidium iodide ( dead ) . was continuously monitored in 3 - hours intervals for 96 hours using automated microscopy in a TECAN Spark Cyto . Data points shaded areas indicate mean ± SD over three biological replicates . Approximately 3400 7500 / well were seeded of A549 HCT116 , respectively .

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

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(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 ) codon effects measured by PACE ( y - axis ) . Each dot represents a single codon . Graphs for codons ( B ) , codons for polar ( C ) , nonpolar ( D ) , charged ( E ) are shown . Data information : regression is shown in blue 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - .

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(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 ) tRNA ( y - axis , Each dot represents a single codon . Graphs for codons ( F ) , codons for polar ( G ) , nonpolar ( H ) , charged ( I ) are shown . Data information : regression is shown in blue 95 % confidence interval is shown in gray . P - values were calculated using Student ' s t - .

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

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

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(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 of Leu CUG codon tag reporter mRNA at 2 6 hpf . ( G ) of PAT at 6 hpf in ( F ) .

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(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 sfGFP at 6 hpf . Tubulin ( Tub ) was detected as a loading control .

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


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

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

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

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

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

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

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

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

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(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 ) rat GLUT5 ( black ) as detected by ACMA dequenching following substrate addition . In experiments errors bars , s . e . m . ; n = 3 technical repeats . apparent KM values are an average from n = 3 separate reconstitutions .

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(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.