( a ) GFP - LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO ( left ) or with 1 μM SAR405 ( right ) . Cells were fixed , and nuclei were stained using Hoechst 33342 ( blue ) . One representative image of cells out of three independent experiments , acquired using an iCyte imaging cytometer , is shown ( X40 ) . White arrows indicate the GFP - LC3 spots , which correspond to the autophagosomes . Scale bars , 10 μm . ( b ) Histograms show the percentage of positive cells ( top ) and the GFP intensity ( bottom ) , as measured using an imaging cytometer . Treatment of GFP - LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated . For GFP intensity , the values were normalized to 1 for fed conditions . Data show mean ± s . d . for three independent experiments .

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(a) GFP-LC3 H1299 cells were treated with 1 μM of the catalytic mTOR inhibitor AZD8055 in fed conditions with DMSO (left) or with 1 μM SAR405 (right). Cells were fixed, and nuclei were stained using Hoechst 33342 (blue). One representative image of cells out of three independent experiments, acquired using an iCyte imaging cytometer, is shown (X40). White arrows indicate the GFP-LC3 spots, which correspond to the autophagosomes. Scale bars, 10 μm. (b) Histograms show the percentage of positive cells (top) and the GFP intensity (bottom), as measured using an imaging cytometer. Treatment of GFP-LC3 H1299 in fed conditions with 1 μM of AZD8055 and different concentrations of SAR405 is indicated. For GFP intensity, the values were normalized to 1 for fed conditions. Data show mean ± s.d. for three independent experiments.


( c ) The concentration needed to reach 40 % inhibition ( IC40 ) of cell proliferation is indicated . The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines . The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405 . The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼ 0 . 5 , corresponding to compounds that are in equipotent proportion in the mixture ( Supplementary Fig . 20 ) . The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments .

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(c) The concentration needed to reach 40% inhibition (IC40) of cell proliferation is indicated. The top histograms indicate the IC40 of SAR405 as a single agent and in combination with everolimus in the three cell lines. The bottom histograms indicate the IC40 of everolimus as a single agent and in combination with SAR405. The IC40 values for the combination of the two compounds were determined using rays with an effective fraction ∼0.5, corresponding to compounds that are in equipotent proportion in the mixture (Supplementary Fig. 20). The absolute IC40 determination of both compounds is represented as the geometric mean from two or three independent experiments.


Figure 2 . SPOP localizes to nuclear speckles or other nuclear bodies . Confocal microscopy images of fixed NIH 3T3 cells transiently expressing ( A ) full length HA - SPOP , ( B ) GFP - Gli31455 , and ( C ) HA - SPOP + GFP - Gli31 - 455 are shown . DAPI was used to stain the nucleus , SPOP localization was identified using a anti - HA antibody , nuclear speckle localization was identified using a anti - SC - 35 antibody , and Gli31 - 455 localization was identified via GFP fluorescence . SPOP co - localizes with a marker for nuclear speckles or with the substrate Gli31455 . The areas with overlapping HA - and GFP signal contain 75 % of the punctate HA signal , and 100 % of the punctate GFP signal .

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Figure 2. SPOP localizes to nuclear speckles or other nuclear bodies. Confocal microscopy images of fixed NIH 3T3 cells transiently expressing (A) full length HA-SPOP, (B) GFP-Gli31455, and (C) HA-SPOP+GFP-Gli31-455 are shown. DAPI was used to stain the nucleus, SPOP localization was identified using a anti-HA antibody, nuclear speckle localization was identified using a anti-SC-35 antibody, and Gli31-455 localization was identified via GFP fluorescence. SPOP co-localizes with a marker for nuclear speckles or with the substrate Gli31455. The areas with overlapping HA- and GFP signal contain 75% of the punctate HA signal, and 100% of the punctate GFP signal.


( A - B ) NIH 3T3 cells were ( A ) co - transfected with constructs expressing full length SPOP and GFP - Gli31455 or ( B ) transfected with a construct expressing SC - 35 - GFP , and GFP fluorescence was monitored in live cells . ( A ) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching ( at 0 s ) , recovery , and nuclear body fusion events . The arrow in each panel points to the body that was photobleached and subsequently fuses with another body . These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery . ( B ) Snapshots at indicated timepoints show a nuclear speckle fusion event . Additional images are shown in Fig EV1 .

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(A-B) NIH 3T3 cells were (A) co-transfected with constructs expressing full length SPOP and GFP-Gli31455 or (B) transfected with a construct expressing SC-35-GFP, and GFP fluorescence was monitored in live cells. (A) Snapshots were taken from individual timepoints as noted in the Figure to show photobleaching (at 0 s), recovery, and nuclear body fusion events. The arrow in each panel points to the body that was photobleached and subsequently fuses with another body. These data were not used to calculate the average recovery time as the fusion event precludes accurate measurement of fluorescence recovery. (B) Snapshots at indicated timepoints show a nuclear speckle fusion event. Additional images are shown in Fig EV1.


( C ) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec . Data from 45 individual cells and FRAP events were corrected for background , normalized to the minimum and maximum intensity , and the mean is shown with error bars representing the standard error of the mean . The mean characteristic recovery time is indicated ± SEM .

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(C) Individual nuclear speckles were photobleached and FRAP was monitored for 90 sec. Data from 45 individual cells and FRAP events were corrected for background, normalized to the minimum and maximum intensity, and the mean is shown with error bars representing the standard error of the mean. The mean characteristic recovery time is indicated ± SEM.


( D - E ) Nuclear bodies are close to spherical . ( D ) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA - SPOP ( 151 cells ) ( D ) , or GFP - Gli31 - 455 and HA - SPOP ( 155 cells ) ( E ) were calculated as described previously ( Brangwynne et al , 2011 ) . Representative individual images of cells are shown as insets in each panel .

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(D-E) Nuclear bodies are close to spherical. (D) Aspect ratios for speckles observed in NIH 3T3 cells transfected with constructs expressing HA-SPOP (151 cells) (D), or GFP-Gli31-455 and HA-SPOP (155 cells) (E) were calculated as described previously (Brangwynne et al, 2011). Representative individual images of cells are shown as insets in each panel.


( A ) SEC chromatograms of given loading concentrations of SPOP28 - 359 are shown . The concentrations were normalized to the monomer molecular weight , i . e . , identical concentrations of dimeric SPOP ΔBACK ( see Fig EV2 ) and oligomeric SPOP28 - 359 contain identical numbers of protomers . ( B ) SEC chromatograms for SPOP constructs defective in self - association in one or both oligomerization domains are shown . Proteins were injected at the same loading concentration ( 533 µM ) and the elution volume of globular molecular weight standards is noted above the graph . ( C ) SEC chromatograms of SPOP28 - 359 ( 200 µM ) , SPOP mutBACK ( 718 µM ) , and mixtures of the two ( ratios given in the Figure , SPOP at 200 µM ) are shown . The elution volume of globular molecular weight standards is noted above the graph .

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(A) SEC chromatograms of given loading concentrations of SPOP28-359 are shown. The concentrations were normalized to the monomer molecular weight, i.e., identical concentrations of dimeric SPOP ΔBACK (see Fig EV2) and oligomeric SPOP28-359 contain identical numbers of protomers. (B) SEC chromatograms for SPOP constructs defective in self-association in one or both oligomerization domains are shown. Proteins were injected at the same loading concentration (533 µM) and the elution volume of globular molecular weight standards is noted above the graph. (C) SEC chromatograms of SPOP28-359 (200 µM), SPOP mutBACK (718 µM), and mixtures of the two (ratios given in the Figure, SPOP at 200 µM) are shown. The elution volume of globular molecular weight standards is noted above the graph.


( D ) Constructs for expressing full - length HA - SPOP or HA - SPOP mutants capable of oligomerization through only one ( or neither ) of the self - association domains were transfected into NIH 3T3 cells . DAPI was used to stain the nucleus and SPOP localization was identified using an anti - HA antibody . Experiments were performed at least twice on four biological samples . Multiple cells were examined and representative cells are shown . For additional images see Appendix Fig S3 . Scale bar represents 5 µm .

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(D) Constructs for expressing full-length HA-SPOP or HA-SPOP mutants capable of oligomerization through only one (or neither) of the self-association domains were transfected into NIH 3T3 cells. DAPI was used to stain the nucleus and SPOP localization was identified using an anti-HA antibody. Experiments were performed at least twice on four biological samples. Multiple cells were examined and representative cells are shown. For additional images see Appendix Fig S3. Scale bar represents 5 µm.


( A ) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide . All SPOP proteins bound substrate with an affinity similar to that of SPOP28 - 359 ( 4 - 8 µM , see Table 2 ) . Experimental data are shown as circles ; the solid lines are fits to eq . 2 ( Roehrl et al , 2004 ) .

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(A) Substrate binding was assayed by anisotropy using a fluorescently labeled substrate peptide. All SPOP proteins bound substrate with an affinity similar to that of SPOP28-359 (4-8 µM, see Table 2). Experimental data are shown as circles; the solid lines are fits to eq. 2 (Roehrl et al, 2004).


( B ) In vitro crosslinking assays were performed for SPOP28 - 359 and each mutant at 30 μM protein with the amide - specific BS3 crosslinker . Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non - specific crosslinking of protein species . ( C ) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP , SPOP mutBACK , SPOP mutBTB , or SPOP mutBTB - BACK . SPOP1 , SPOP2 and SPOPn identify SPOP monomers , dimers and larger species , respectively . For loading levels see Appendix Fig S4 .

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(B) In vitro crosslinking assays were performed for SPOP28-359 and each mutant at 30 μM protein with the amide-specific BS3 crosslinker. Crosslinking for SPOP ΔBACK and MATH domain are shown to demonstrate that crosslinking conditions do not lead to non-specific crosslinking of protein species. (C) Crosslinking reactions were performed on whole cell lysates from cells expressing wild type SPOP, SPOP mutBACK, SPOP mutBTB, or SPOP mutBTB-BACK. SPOP1, SPOP2 and SPOPn identify SPOP monomers, dimers and larger species, respectively. For loading levels see Appendix Fig S4.


( A ) SV - AUC data for 0 . 2 nM AF488 - SPOP ΔBACK . Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation ( circles ) . The data were subjected to standard c ( s ) analysis in SEDFIT ( Schuck , 2000 ) and the results of this fit are shown ( solid lines , rainbow color scheme ) .

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(A) SV-AUC data for 0.2 nM AF488-SPOP ΔBACK. Fluorescence scans were collected for 12 h and are plotted against distance from the axis of rotation (circles). The data were subjected to standard c(s) analysis in SEDFIT (Schuck, 2000) and the results of this fit are shown (solid lines, rainbow color scheme).


( B ) Sedimentation coefficient distributions for a selection of values from the dilution series are shown .

(B) Sedimentation coefficient distributions for a selection of values from the dilution series are shown.


( C ) The isotherm of weighted - average s - values vs . concentration ( black circles ) , was fit to a dimer self - association model and reveals a KD of 1 . 11 nM , [ 95 % confidence interval 0 . 88 , 1 . 40 nM ] ( solid line ) .

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(C) The isotherm of weighted-average s-values vs. concentration (black circles), was fit to a dimer self-association model and reveals a KD of 1.11 nM, [95% confidence interval 0.88, 1.40 nM] (solid line).


( D ) The BACK domain dimerizes with micromolar affinity . Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP mutBTB ( black circles ) was fitted to a self - association model ( see text for details ) for BACK - BACK dimerization ( black line ) yielding a KD of 36 ± 3 µM ( average and standard deviation from 3 independent experiments ) . Fits to the monomer / dimer equilibrium model and to alternative monomer / trimer , tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines , respectively . Residuals for all models are depicted in the lower panel .

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(D) The BACK domain dimerizes with micromolar affinity. Experimental weight-average molar mass (Mw) from CG-MALS for SPOP mutBTB (black circles) was fitted to a self-association model (see text for details) for BACK-BACK dimerization (black line) yielding a KD of 36 ± 3 µM (average and standard deviation from 3 independent experiments). Fits to the monomer/dimer equilibrium model and to alternative monomer/trimer, tetramer and pentamer equilibrium models are depicted as black solid line and as dashed and grey lines, respectively. Residuals for all models are depicted in the lower panel.


( B ) Experimental weight - average molar mass ( Mw ) from CG - MALS for SPOP28 - 359 ( orange circles ) was fitted to an isodesmic self - association model in which SPOP dimers are the self - associating unit ( orange line ) . The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ( ( SPOP2 ) 11 ) . The fits from three independent experiments yielded a KD2 of 2 . 4 ± 0 . 4 µM . Lines for fits of the data to self - association models that assume formation of individual oligomeric species instead of isodesmic self - association are shown for reference ( gray lines ) .

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(B) Experimental weight-average molar mass (Mw) from CG-MALS for SPOP28-359 (orange circles) was fitted to an isodesmic self-association model in which SPOP dimers are the self-associating unit (orange line). The largest SPOP oligomer taken into account was an undecamer of SPOP dimers ((SPOP2)11). The fits from three independent experiments yielded a KD2 of 2.4 ± 0.4 µM. Lines for fits of the data to self-association models that assume formation of individual oligomeric species instead of isodesmic self-association are shown for reference (gray lines).


( C ) Experimental weight - average molar mass ( Mw ) from CG - MALS data ( circles ) and fits ( solid lines ) are depicted for each SPOP construct assayed , showing that a progressive increase in SPOP oligomer size is observed only when both self - association domains are interaction - competent . This Figure is comprised of data shown in Figures 6D , 7B , and Appendix Fig S5A for direct comparison .

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(C) Experimental weight-average molar mass (Mw) from CG-MALS data (circles) and fits (solid lines) are depicted for each SPOP construct assayed, showing that a progressive increase in SPOP oligomer size is observed only when both self-association domains are interaction-competent. This Figure is comprised of data shown in Figures 6D, 7B, and Appendix Fig S5A for direct comparison.


( A ) Native MS spectrum of 30 µM SPOP28 - 359 confirms that SPOP self - associates through addition of dimers . The increase in mass indicates that the dimer is the stable building block within the assemblies .

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(A) Native MS spectrum of 30 µM SPOP28-359 confirms that SPOP self-associates through addition of dimers. The increase in mass indicates that the dimer is the stable building block within the assemblies.


( C ) Concentration - dependence of the SPOP oligomer size distribution ( mole fraction ) from isodesmic modeling of CG - MALS data .

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(C) Concentration-dependence of the SPOP oligomer size distribution (mole fraction) from isodesmic modeling of CG-MALS data.


Figure 9 . The self - association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo . UAS transgenes were expressed under the control of an epithelial driver C765 - Gal4 in Drosophila melanogaster . ( A ) UAS vector served as a control and yielded a normal wing . Longitudinal veins are denoted by numbers 1 - 5 . Expression of ( B ) SPOP WT resulted in a modest Hh loss - of function phenotype with fusion of LV3 and LV4 ( arrow ) . ( C ) mutBTB acts in a dominant - negative manner , as evidenced by a modest Hh gain - of function phenotype with expansion of the LV3 - LV4 intervein space . Expression of ( D ) mutBACK and ( E ) mutBTB - BACK does not induce a wing patterning defect . ~ 50 animals each were analyzed from two independent crosses .

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Figure 9. The self-association deficient SPOP mutant mutBTB has a substrate degradation defect in vivo. UAS transgenes were expressed under the control of an epithelial driver C765-Gal4 in Drosophila melanogaster. (A) UAS vector served as a control and yielded a normal wing. Longitudinal veins are denoted by numbers 1-5. Expression of (B) SPOP WT resulted in a modest Hh loss-of function phenotype with fusion of LV3 and LV4 (arrow). (C) mutBTB acts in a dominant-negative manner, as evidenced by a modest Hh gain-of function phenotype with expansion of the LV3-LV4 intervein space. Expression of (D) mutBACK and (E) mutBTB-BACK does not induce a wing patterning defect. ~50 animals each were analyzed from two independent crosses.


A , Reconstructed MSOT images ( 800 nm ) without and with linear unmixing data in the neck and the upper and lower abdominal areas , showing the ROIs for the liver , kidney , iBAT , rpWAT , aorta , and SV . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value neck / upper abdominal / lower abdominal : Hb : 2 . 5 / 3 . 5 / 0 . 9 ; HbO2 : 2 . 9 / 4 . 9 / 2 . 5 ; Lipid : 24000 / 19000 / 13000 ) Scale bar : 4 mm .

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A, Reconstructed MSOT images (800 nm) without and with linear unmixing data in the neck and the upper and lower abdominal areas, showing the ROIs for the liver, kidney, iBAT, rpWAT, aorta, and SV. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value neck/upper abdominal/lower abdominal: Hb: 2.5/3.5/0.9; HbO2:2.9/4.9/2.5; Lipid: 24000/19000/13000) Scale bar: 4 mm.


A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , and lipid from chow and HFD - fed mice . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( maximum value chow / HFD : Hb : 1 . 3 / 0 . 6 ; HbO2 : 1 . 8 / 0 . 9 ; Lipid : 27000 / 27000 ) . Scale bar : 4 mm .

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A, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (maximum value chow/HFD: Hb: 1.3/0.6; HbO2: 1.8/0.9; Lipid: 27000/27000). Scale bar: 4 mm.


D , Tissue oxygenation ( sO2 ) and total blood volume ( TBV ) results from liver , kidney , iBAT , and rpWAT , Each dot represents data from one animal , in total 8 animals ( n = 8 ) . Data represent the mean ( + / - 95 % confidence ) . The unpaired t test was used to verify the statistical significance . Data information : In the figure , A . U . = arbitrary units .

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D, Tissue oxygenation (sO2) and total blood volume (TBV) results from liver, kidney, iBAT, and rpWAT, Each dot represents data from one animal, in total 8 animals (n = 8). Data represent the mean (+/- 95% confidence). The unpaired t test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units.


A , Oil - red - O staining of control ( healthy ) and steatotic liver . Scale bar : 100 µm .

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A, Oil-red-O staining of control (healthy) and steatotic liver. Scale bar: 100 µm.


B , Reconstructed MSOT image ( 800 nm ) with linear unmixing and difference value of lipid in lower abdominal section . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid , jet for 700 nm - 930 nm difference . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : Hb : 1 . 1 / 0 . 5 ; HbO2 : 1 . 9 / 0 . 8 ; Lipid : 21000 / 24000 ; 700 - 900nm : 3000 / 3000 ) Scale bar : 4 mm .

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B, Reconstructed MSOT image (800 nm) with linear unmixing and difference value of lipid in lower abdominal section. Unmixing result: blue for Hb, red for HbO2, yellow for lipid, jet for 700 nm - 930 nm difference. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: Hb: 1.1/0.5; HbO2: 1.9/0.8; Lipid: 21000/24000; 700-900nm: 3000/3000) Scale bar: 4 mm.


A , HE staining of livers with grade 0 - 3 steatosis . Scale bar : 100 µm .

A, HE staining of livers with grade 0 - 3 steatosis. Scale bar: 100 µm.


E , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of Hb , HbO2 , and lipid from chow and HFD - fed mice . Unmixing result : blue for Hb , red for HbO2 , yellow for lipid . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value chow 1 month / chow 2 month / chow 3 month / HFD 1 month / HFD 2 month / HFD 3 month : Hb : 2 . 5 / 1 . 9 / 2 . 9 / 2 . 7 / 1 . 8 / 1 . 3 ; HbO2 : 2 . 8 / 2 . 6 / 2 . 8 / 3 . 1 / 2 . 1 / 1 . 9 ; Lipid : 27000 / 19000 / 29000 / 32000 / 26000 / 27000 ) . Scale bar : 4 mm .

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E, Reconstructed MSOT image (800 nm) with linear unmixing data of Hb, HbO2, and lipid from chow and HFD-fed mice. Unmixing result: blue for Hb, red for HbO2, yellow for lipid. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value chow 1 month/ chow 2 month/ chow 3 month/HFD 1 month/HFD 2 month/HFD 3 month: Hb: 2.5/1.9/2.9/2.7/1.8/1.3; HbO2: 2.8/2.6/2.8/3.1/2.1/1.9; Lipid: 27000/19000/29000/ 32000/26000/27000). Scale bar: 4 mm.


F , Longitudinal track of lipid in mouse livers . Each line represents data from one animal . Each data point is from a single measurement of each animal at each time point . All animals have grade 3 steatosis at the endpoint confirmed by histology .

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F, Longitudinal track of lipid in mouse livers. Each line represents data from one animal. Each data point is from a single measurement of each animal at each time point. All animals have grade 3 steatosis at the endpoint confirmed by histology.


A , Reconstructed MSOT image ( 800 nm ) with linear unmixing data of ICG from mice with and without steatosis . The colour bar shows the colour coding of MSOT a . u . from 0 to maximum ( bottom to top ) ( Maximum value control / steatosis : 0 . 07 / 0 . 02 ) Scale bar : 4 mm .

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A, Reconstructed MSOT image (800 nm) with linear unmixing data of ICG from mice with and without steatosis. The colour bar shows the colour coding of MSOT a.u. from 0 to maximum (bottom to top) (Maximum value control/steatosis: 0.07/0.02) Scale bar: 4 mm.


E , Representative fluorescence microscopy and HE staining images of control and steatotic liver . Scale bar : 100 µm .

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E, Representative fluorescence microscopy and HE staining images of control and steatotic liver. Scale bar: 100 µm.


A FBN2 mRNA levels in multiple human tissues . Human tissue cDNAs were used as templates for PCR of FBN2 , followed by agarose gel analyses . GAPDH levels served as loading controls ( biological replicates , n = 2 ) .

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A FBN2 mRNA levels in multiple human tissues. Human tissue cDNAs were used as templates for PCR of FBN2, followed by agarose gel analyses. GAPDH levels served as loading controls (biological replicates, n=2).


B FBN2 mRNA levels in mouse tissues based on quantitative RT - PCR . Data were normalized based on levels in adipose tissues .

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B FBN2 mRNA levels in mouse tissues based on quantitative RT-PCR. Data were normalized based on levels in adipose tissues.


C Comparison of FBN2 transcript levels between human and mouse placentas . Common primers for FBN2 and GAPDH in both species were used for RT - PCR analyses ( biological replicates , n = 3 ) .

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C Comparison of FBN2 transcript levels between human and mouse placentas. Common primers for FBN2 and GAPDH in both species were used for RT-PCR analyses (biological replicates, n=3) .


D Immuno - histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy . During early pregnancy , placensin immunostaining was found in cytotrophoblasts ( c ) , whereas syncytiotrophoblasts ( s ) showed weak staining . At 38 weeks of pregnancy , strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts ( arrows ) . Staining using E - cadherin antibodies confirmed the identity of cytotrophoblasts ( Inset showing higher magnification E - cadherin ( E - Cad ) staining ) . Lower panels depict sections stained with nonimmune IgG as negative controls . Scale bars , 100 μm .

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D Immuno-histochemical detection of placensin in human placental villi obtained from women at 9 and 38 weeks of pregnancy. During early pregnancy, placensin immunostaining was found in cytotrophoblasts (c), whereas syncytiotrophoblasts (s) showed weak staining. At 38 weeks of pregnancy, strong placensin signals were detected in both syncytiotrophoblasts and cytotrophoblasts (arrows). Staining using E-cadherin antibodies confirmed the identity of cytotrophoblasts (Inset showing higher magnification E-cadherin (E-Cad) staining). Lower panels depict sections stained with nonimmune IgG as negative controls. Scale bars, 100 μm.


E Immunoblotting of placensin in human placental villi ( term pregnancy ) . Samples from three individual patients were processed with or without N - glycosidase pre - treatment to reveal untreated and deglycosylated placensin .

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E Immunoblotting of placensin in human placental villi (term pregnancy). Samples from three individual patients were processed with or without N-glycosidase pre-treatment to reveal untreated and deglycosylated placensin.


F For prokaryotic cell expression , a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6 - histidine tags and sub - cloned into the PGEX - 6P - 1 vector for expression in E . coli . Left panel : Coomassie blue staining of proteins in the SDS - PAGE gel before and after digestion with the PreScission protease . 1 : Flow through ; 2 : Washes ; 3 : Before PreScission treatment ; 4 : After PreScission treatment . Right panel : Immunoblotting using placensin antibodies .

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F For prokaryotic cell expression, a cDNA fragment corresponding to residues 2779 to 2912 of the human FBN2 coding region was appended with GST and 6-histidine tags and sub-cloned into the PGEX-6P-1 vector for expression in E. coli. Left panel: Coomassie blue staining of proteins in the SDS-PAGE gel before and after digestion with the PreScission protease. 1: Flow through; 2: Washes; 3: Before PreScission treatment; 4: After PreScission treatment. Right panel: Immunoblotting using placensin antibodies.


G Immunoblotting of recombinant placensin generated in E . coli . and CHO cells . For CHO cell expression , placensin cDNA sequence was subcloned into an adenoviral vector pAV [ Exp ] - CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later . Recombinant placensin proteins secreted from CHO cells ( b ) showed higher molecular weight than those from bacteria ( a ) . Following treatment with N - glycosidase to remove N - linked carbohydrate side chains , prokaryotic - and eukaryotic cell - derived placensin proteins showed similar sizes .

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G Immunoblotting of recombinant placensin generated in E. coli. and CHO cells. For CHO cell expression, placensin cDNA sequence was subcloned into an adenoviral vector pAV[Exp]-CMV downstream of the IgK signal peptide under the control of the CMV promoter for infection of cells before immunoblotting of media 2 days later. Recombinant placensin proteins secreted from CHO cells (b) showed higher molecular weight than those from bacteria (a). Following treatment with N-glycosidase to remove N-linked carbohydrate side chains, prokaryotic- and eukaryotic cell-derived placensin proteins showed similar sizes.


A Prokaryotic cell - derived placensin stimulated cAMP production ( at 10 min . ) , and PKA activity ( at 30 min . ) by primary mouse hepatocytes and hepatocyte - derived AML12 cells ( biological replicates , n = 5 ) .

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A Prokaryotic cell-derived placensin stimulated cAMP production (at 10 min.), and PKA activity (at 30 min.) by primary mouse hepatocytes and hepatocyte-derived AML12 cells (biological replicates, n=5).


B Placensin stimulation of glucose secretion ( at 5h ) and key gluconeogenesis gene transcripts ( at 1h ) in both cell types ( biological replicates , n = 4 ) . Transcript levels for PEPCK and G6Pase were determined using quantitative RT - PCR . Treatment with glucagon ( 10 nM ) served as positive controls .

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B Placensin stimulation of glucose secretion (at 5h) and key gluconeogenesis gene transcripts (at 1h) in both cell types (biological replicates, n=4). Transcript levels for PEPCK and G6Pase were determined using quantitative RT-PCR. Treatment with glucagon (10 nM) served as positive controls.


C Liver from adult female mice was perfused in situ with placensin ( 1 . 0 ug / pulse ) or PBS before measurement of hepatic glucose output from inferior vena cava ( biological replicates , n = 3 ) .

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C Liver from adult female mice was perfused in situ with placensin (1.0 ug/pulse) or PBS before measurement of hepatic glucose output from inferior vena cava (biological replicates, n=3).


D Adult female mice were injected intraperitoneally with placensin ( 30 ug / injection ) with blood sampling at different time points for glucose levels ( biological replicates , n = 5 ) .

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D Adult female mice were injected intraperitoneally with placensin (30 ug/injection) with blood sampling at different time points for glucose levels (biological replicates, n=5).


A Left panel : Immunoblotting of placensin content in HTR - 8 / SVneo , but not BeWo , JAR , and JEG3 , cells . Right panel : Time - dependent secretion of placensin by HTR - 8 / SVneo , but not JAR , cells monitored using an ELISA ( biological replicates , n = 4 - 5 ) .

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A Left panel: Immunoblotting of placensin content in HTR-8/SVneo, but not BeWo, JAR, and JEG3, cells. Right panel: Time-dependent secretion of placensin by HTR-8/SVneo, but not JAR, cells monitored using an ELISA (biological replicates, n=4-5).


B Knockdown of FBN2 in HTR - 8 / SVneo cells led to decreases in FBN2 transcripts and placensin secretion . Cells were treated with FBN2 siRNA ( SiFBN2 ; 20 nM ) or control siRNA ( SiC ; 20 nM ) for 6h . Following media change , cells and conditioned media were collected at 48h after incubation . Placensin transcripts and proteins were determined by RT - PCR and ELISA ( biological replicates , n = 8 ) , respectively . Lower panel : immunoblotting of secreted placensin by HTR - 8 / Svneo .

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B Knockdown of FBN2 in HTR-8/SVneo cells led to decreases in FBN2 transcripts and placensin secretion. Cells were treated with FBN2 siRNA (SiFBN2; 20 nM) or control siRNA (SiC; 20 nM) for 6h. Following media change, cells and conditioned media were collected at 48h after incubation. Placensin transcripts and proteins were determined by RT-PCR and ELISA (biological replicates, n=8), respectively. Lower panel: immunoblotting of secreted placensin by HTR-8/Svneo.


C Placensin stimulation of cAMP production and PKA activities by HTR - 8 / SVneo cells . Cells were treated with placensin for 10 min . before cAMP measurement ( biological replicates , n = 5 ) . For PKA activities , cells were treated with placensin ( 50 ng / ml ) for 30 min . Treatment with forskolin ( 10 uM ) served as positive controls .

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C Placensin stimulation of cAMP production and PKA activities by HTR-8/SVneo cells. Cells were treated with placensin for 10 min. before cAMP measurement (biological replicates, n=5). For PKA activities, cells were treated with placensin (50 ng/ml) for 30 min. Treatment with forskolin (10 uM) served as positive controls.


D Placensin stimulation of transcript levels for MMP9 but not MMP2 ( biological replicates , n = 12 ) . Cells were treated with placensin for 24h before RT - PCR analyses of transcript levels .

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D Placensin stimulation of transcript levels for MMP9 but not MMP2(biological replicates, n=12). Cells were treated with placensin for 24h before RT-PCR analyses of transcript levels.


E Placensin stimulation of MMP9 , but not MMP2 , activities without affecting cell proliferation . After 24h of treatment with placensin , media were collected for zymographic and densitometric analyses as well as determination of total cell numbers ( biological replicates , n = 4 ) .

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E Placensin stimulation of MMP9, but not MMP2, activities without affecting cell proliferation. After 24h of treatment with placensin, media were collected for zymographic and densitometric analyses as well as determination of total cell numbers (biological replicates, n=4).


F Placensin stimulation of HTR - 8 / SVneo cell invasion . After placensin treatment for 24h , cell invasiveness was determined using the Transwell assay ( left panel : micrographs of cells ; right panel : fold changes in number of migrated cells , biological replicates , n = 4 ) . Cells were incubated with mitomycin C to rule out effects on cell proliferation . Scale bars : 100μm . C : control .

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F Placensin stimulation of HTR-8/SVneo cell invasion. After placensin treatment for 24h, cell invasiveness was determined using the Transwell assay (left panel: micrographs of cells; right panel: fold changes in number of migrated cells, biological replicates, n=4). Cells were incubated with mitomycin C to rule out effects on cell proliferation. Scale bars: 100μm. C: control.


A Immunoblotting of serum placensin from patients at second trimester ( 1 - 3 ) , term pregnancy ( 4 - 6 ) using specific placensin antibodies .

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A Immunoblotting of serum placensin from patients at second trimester (1-3), term pregnancy (4-6) using specific placensin antibodies.


B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients . Numbers in parentheses represent number of patients .

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B Serum levels of placensin and glucagon at different weeks of pregnancy measured using specific ELISA in normal and GDM patients. Numbers in parentheses represent number of patients.


stoichiometry ' s for B56α measured by ITC . Global direct fitting shown for one experiment ( reverse ) . Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value . The experiment was done in both direct ( B56 in cell ) and reverse ( B56 in syringe ) with similar results .

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stoichiometry's for B56α measured by ITC. Global direct fitting shown for one experiment (reverse). Each dot is the integrated heat per injection and the error bars represent uncertainty with this integrated value. The experiment was done in both direct (B56 in cell) and reverse (B56 in syringe) with similar results.


C ) Time from Nuclear envelope breakdown ( NEBD ) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell . Only cells with similar expression levels of the various B56 inhibitor constructs were analysed . Median time is indicated by red line . A representative result from at least three independent experiments is shown . At least 25 cells were counted per condition in the experiment shown . A Mann - Whitney U ‐ test was used for statistical analysis ( ns : non ‐ significant , * * * P ≤ 0 . 001 ) .

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C) Time from Nuclear envelope breakdown (NEBD) to mitotic exit of cells expressing the indicated B56 inhibitors with each circle representing a single cell. Only cells with similar expression levels of the various B56 inhibitor constructs were analysed. Median time is indicated by red line. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. A Mann-Whitney U‐test was used for statistical analysis (ns: non‐significant, ***P ≤ 0.001).


D ) Volcano plot representing mass spectrometry identified proteins co - purifying with B56 inhibitor versus control inhibitor from HeLa cells . PP2A - B56 subunits co - purifying with the B56 inhibitor are indicated .

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D) Volcano plot representing mass spectrometry identified proteins co-purifying with B56 inhibitor versus control inhibitor from HeLa cells. PP2A-B56 subunits co-purifying with the B56 inhibitor are indicated.


E ) and F ) Competition assay in HeLa cells stably expressing RFP - tagged B56 inhibitor ( LxxIxE ) or control inhibitor ( AxxAxA ) . YFP - B56α was transfected into and subsequently purified from these cell lines . Loss of binding of indicated proteins determined by either mass spectrometry ( pink - B56 SLIM containing protein and known B56 interactor ; blue - known B56 interactor , green - B56 SLIM containing protein ) ( E ) or Western blotting ( F ) .

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E) and F) Competition assay in HeLa cells stably expressing RFP-tagged B56 inhibitor (LxxIxE) or control inhibitor (AxxAxA). YFP-B56α was transfected into and subsequently purified from these cell lines. Loss of binding of indicated proteins determined by either mass spectrometry (pink - B56 SLIM containing protein and known B56 interactor; blue - known B56 interactor, green - B56 SLIM containing protein) (E) or Western blotting (F).


A - B ) Schematic of synchronisation protocol for G1 / S ( A ) or mitotic ( B ) arrested cells and accompanying volcano plot of phosphorylation sites quantified . The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey . Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink .

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A-B) Schematic of synchronisation protocol for G1/S (A) or mitotic (B) arrested cells and accompanying volcano plot of phosphorylation sites quantified. The phosphorylation sites showing an increase in the presence of the B56 inhibitor are shown in light grey. Phosphorylation sites in a protein containing an LxxIxE motif are coloured pink.


A ) Michaelis - Menten kinetic parameters of purified PP2A - B56α and PP2A - B55α holoenzymes were determined against the indicated phospho - peptides . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments )

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A) Michaelis-Menten kinetic parameters of purified PP2A-B56α and PP2A-B55α holoenzymes were determined against the indicated phospho-peptides. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)


B - D ) In vitro dephosphorylation by the PP2A - B55α and PP2A - B56α holoenzymes of panels of phosphorylated peptides as indicated . Mean and standard deviation shown in plots as black bars ( n = 3 independent experiments )

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B-D) In vitro dephosphorylation by the PP2A-B55α and PP2A-B56α holoenzymes of panels of phosphorylated peptides as indicated. Mean and standard deviation shown in plots as black bars (n=3 independent experiments)


B ) The indicated YFP - FoxO3 constructs were transfected into HeLa cells , purified using YFP resin and PP2A - B56 binding determined by Western blotting . PP2A - A ; scaffold subunit .

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B) The indicated YFP-FoxO3 constructs were transfected into HeLa cells, purified using YFP resin and PP2A-B56 binding determined by Western blotting. PP2A-A; scaffold subunit.


C ) - D ) YFP - FoxO3 constructs were transfected into HeLa cells and lysates ( pS413 ) or YFP purifications ( pS253 ) were subjected to Western Blotting and probed with the indicated phosphoantibodies . Quantifications arise from five ( pS413 ) or three ( pS253 ) independent experiments . Mean and standard deviation shown in plots as black bars .

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C) - D) YFP-FoxO3 constructs were transfected into HeLa cells and lysates (pS413) or YFP purifications (pS253) were subjected to Western Blotting and probed with the indicated phosphoantibodies. Quantifications arise from five (pS413) or three (pS253) independent experiments. Mean and standard deviation shown in plots as black bars.


E ) Steady - state localization of the indicated YFP - FOXO3 variants in HeLa cells . Each data point represents quantification from a single cell . A representative result from at least three independent experiments is shown . At least 25 cells were counted per condition in the experiment shown . Scale bar is 10 μm . Mann - Whitney test significance values ; ∗ p < 0 . 02 , ∗ ∗ ∗ p < 0 . 0002 .

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E) Steady-state localization of the indicated YFP-FOXO3 variants in HeLa cells. Each data point represents quantification from a single cell. A representative result from at least three independent experiments is shown. At least 25 cells were counted per condition in the experiment shown. Scale bar is 10 μm. Mann-Whitney test significance values; ∗p < 0.02, ∗∗∗p < 0.0002.


F ) Dephosphorylation by the PP2A - B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted . Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . Mean and standard deviation from 3 experiments is shown .

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F) Dephosphorylation by the PP2A-B56α holoenzyme complex of substrates with increasing length between phosphorylation sites and binding motifs as depicted. Engineered substrates containing three TP sites were phosphorylated with radioactive ATP using Cdk1 and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. Mean and standard deviation from 3 experiments is shown.


B ) KD values obtained by ITC measurements with full - length recombinant B56α and indicated ADAM17 variant peptides .

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

B) KD values obtained by ITC measurements with full-length recombinant B56α and indicated ADAM17 variant peptides.


C ) The indicated full - length murine Myc - ADAM17 derivatives were transfected into HeLa cells stably expressing YFP - B56α . YFP - B56α was purified ( IP ) and ADAM17 binding determined by Western blotting . PP2A - C ; catalytic subunit , PP2A - A ; scaffold subunit .

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C) The indicated full-length murine Myc-ADAM17 derivatives were transfected into HeLa cells stably expressing YFP-B56α. YFP-B56α was purified (IP) and ADAM17 binding determined by Western blotting. PP2A-C; catalytic subunit, PP2A-A; scaffold subunit.


D ) Dephosphorylation by the PP2A - B56α holoenzyme complex of the indicated phosphorylated GST - ADAM17 ( V724 - C827 ) fragments . The GST - ADAM17 ( V724 - C827 ) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A - B56α holoenzyme . Removal of radioactive phosphate was monitored over time . The mean and Standard deviation of 4 independent experiments are shown .

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

D) Dephosphorylation by the PP2A-B56α holoenzyme complex of the indicated phosphorylated GST-ADAM17 (V724-C827) fragments. The GST-ADAM17 (V724-C827) substrates was phosphorylated with radioactive ATP using Protein Kinase A and incubated with the PP2A-B56α holoenzyme. Removal of radioactive phosphate was monitored over time. The mean and Standard deviation of 4 independent experiments are shown.


E ) Protein expression of ADAM17 variants in the DLD - 1 Adam17 - / - cell line , determined by Western blot . GAPDH was used as an internal loading control .

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E) Protein expression of ADAM17 variants in the DLD-1 Adam17-/- cell line, determined by Western blot. GAPDH was used as an internal loading control.


F ) Amphiregulin ( AREG ) shedding measured by ELISA of conditioned media from untreated , H2O2 treated or irradiated with x - ray DLD - 1 Adam17 - / - cells ( clone # 1 ) expressing full - length ADAM17 variants ( wt , I762A or LEE ) . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Mean and standard deviation indicated from at least three independent experiments .

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F) Amphiregulin (AREG) shedding measured by ELISA of conditioned media from untreated, H2O2 treated or irradiated with x-ray DLD-1 Adam17-/- cells (clone #1) expressing full-length ADAM17 variants (wt, I762A or LEE). Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01, ***p<0.001. Mean and standard deviation indicated from at least three independent experiments.


G ) Exogenous wt and I762A full length ADAM17 was immuno - purified from Adam17 - / - cells treated with H2O2 and subjected to label - free LC - MS / MS to determine differential phosphorylation status of T735 and S808 ( S811 in murine ADAM17 ) .

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G) Exogenous wt and I762A full length ADAM17 was immuno-purified from Adam17-/- cells treated with H2O2 and subjected to label-free LC-MS/MS to determine differential phosphorylation status of T735 and S808 (S811 in murine ADAM17).


A ) EGFR activation in the ADAM17 variant expressing DLD - 1 Adam17 - / - cells upon H2O2 treatment , determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments .

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A) EGFR activation in the ADAM17 variant expressing DLD-1 Adam17-/- cells upon H2O2 treatment, determined by Western blot and quantified below as the ratio of EGFR autophosphorylated at Tyr1068 to total EGFR. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.


( B , C ) B ) Cell proliferation and C ) Matrigel invasion of the ADAM17 variant expressing DLD - 1 Adam17 - / - cells . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 , * * p < 0 . 01 . Mean and standard deviation indicated from three independent experiments .

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(B, C) B) Cell proliferation and C) Matrigel invasion of the ADAM17 variant expressing DLD-1 Adam17-/- cells. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05, **p<0.01. Mean and standard deviation indicated from three independent experiments.


D ) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17 - / - , determined by Western blot . GAPDH was used as an internal loading control .

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D) Protein expression of the ADAM17 variants in the mouse breast cancer cell line 4T1 Adam17-/-, determined by Western blot. GAPDH was used as an internal loading control.


E ) Average tumor volume ± SEM of 4T1 Adam17 - / - cells expressing the ADAM17 variants injected into the mammary fat pad of Balb / c mice ( n = 10 for each group ) . Indicated significances are between the I762A and LEE tumors . Two sided , unpaired Students t - test test was applied to test for significant differences * p < 0 . 05 . Mean and standard error of the mean indicated .

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E) Average tumor volume ± SEM of 4T1 Adam17-/- cells expressing the ADAM17 variants injected into the mammary fat pad of Balb/c mice (n=10 for each group). Indicated significances are between the I762A and LEE tumors. Two sided, unpaired Students t-test test was applied to test for significant differences *p<0.05. Mean and standard error of the mean indicated.


F ) Survival curve of the tumor bearing mice injected with 4T1 Adam17 - / - cells expressing the ADAM17 variants ( n = 10 for each group ) . The indicated significances are between the LEE and wt tumor bearing mice , and the LEE and I762A tumor bearing mice . Log - rank test was applied to test for significant differences . * p < 0 . 05 .

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F) Survival curve of the tumor bearing mice injected with 4T1 Adam17-/- cells expressing the ADAM17 variants (n=10 for each group). The indicated significances are between the LEE and wt tumor bearing mice, and the LEE and I762A tumor bearing mice. Log-rank test was applied to test for significant differences. *p<0.05.


C . Cells were treated with BMP2 for 1 h . D . Cells were stimulated with increasing ( doubling ) doses of BMP4 / 7 ( 0 . 625 - 20 ng / mL ) for 1 h . Data information : In all cases , whole cell extracts were Western blotted using the indicated antibodies . Representative experiments are shown . The levels of pSMAD1 / 5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample or normalized to the 20 ng / mL ligand dose in parental HEK293T cells Act , Activin A ; Par , parental HEK293T cells , KO1 , ACVR1 KO clone 1 .

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C. Cells were treated with BMP2 for 1 h. D. Cells were stimulated with increasing (doubling) doses of BMP4/7 (0.625-20 ng/mL) for 1 h. Data information: In all cases, whole cell extracts were Western blotted using the indicated antibodies. Representative experiments are shown. The levels of pSMAD1/5 relative to Actin were determined and are expressed as a fold change relative to the parental HEK293T untreated sample or normalized to the 20 ng/mL ligand dose in parental HEK293T cells Act, Activin A; Par, parental HEK293T cells, KO1, ACVR1 KO clone 1.


, B . Parental HEK293T , HET and HOM1 cells were treated with BMP2 for the indicated times . Quantifications are shown on the right . Data information : quantifications are the average normalized intensities ± SEM of independent experiments in B , the quantifications are expressed as fold changes relative to 1 h of ligand stimulation . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . The p - values are from two - way ANOVA with Dunnett ' s post - hoc test . In all panels , Western blots of whole cell lysates were probed with the antibodies indicated .

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, B. Parental HEK293T, HET and HOM1 cells were treated with BMP2 for the indicated times. Quantifications are shown on the right. Data information: quantifications are the average normalized intensities ± SEM of independent experiments in B , the quantifications are expressed as fold changes relative to 1 h of ligand stimulation. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The p-values are from two-way ANOVA with Dunnett's post-hoc test. In all panels, Western blots of whole cell lysates were probed with the antibodies indicated.


D . Parental HEK293T , S2 / 3 dKO1 and HOM1 cells were treated with Activin A for the indicated times . Data information : ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . The p - values are from two - way ANOVA with Dunnett ' s post - hoc test . In D quantifications are from a representative experiment and are the intensities of pSMAD1 / 5 normalized to Actin , expressed as fold change relative to untreated cells for each clone . In all panels , Western blots of whole cell lysates were probed with the antibodies indicated .

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D. Parental HEK293T, S2/3 dKO1 and HOM1 cells were treated with Activin A for the indicated times. Data information: ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. The p-values are from two-way ANOVA with Dunnett's post-hoc test. In D quantifications are from a representative experiment and are the intensities of pSMAD1/5 normalized to Actin, expressed as fold change relative to untreated cells for each clone. In all panels, Western blots of whole cell lysates were probed with the antibodies indicated.


B . Parental HEK293T , HET and HOM1 cells were treated with either Activin A or BMP4 / 7 ( both at 20 ng / mL ) for 1 h , in the presence or absence of either 1 µM LDN - 193189 or 10 µM SB - 431542 . Quantifications are the levels of pSMAD1 / 5 normalized to the loading control ( Actin or Tubulin ) , expressed as fold change relative to untreated for each clone . Quantifications are the average normalized intensities ± SEM of 3 - 5 independent experiments . Note that the Activin A quantifications also includes data generated with high dose Activin A in Fig . 3A as they are independent replicates . Data information : In all panels , Western blots of whole cell lysates were probed with the antibodies indicated . hi , high ; Act , Activin A ; SB , SB - 431542 ; LDN , LDN - 193189 . The p - values ( for A , B and D ) are from a t - test with Holm - Sidak correction , for comparison between indicated pairs . ns , not significant ; * , p < 0 . 05 ; * * , p < 0 . 01 , * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 . The p - values in C are from two - way ANOVA with Tukey ' s post hoc correction .

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B. Parental HEK293T, HET and HOM1 cells were treated with either Activin A or BMP4/7 (both at 20 ng/mL) for 1 h, in the presence or absence of either 1 µM LDN-193189 or 10 µM SB-431542. Quantifications are the levels of pSMAD1/5 normalized to the loading control (Actin or Tubulin), expressed as fold change relative to untreated for each clone. Quantifications are the average normalized intensities ± SEM of 3-5 independent experiments. Note that the Activin A quantifications also includes data generated with high dose Activin A in Fig. 3A as they are independent replicates. Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. hi, high; Act, Activin A; SB, SB-431542; LDN, LDN-193189. The p-values (for A, B and D) are from a t-test with Holm-Sidak correction, for comparison between indicated pairs. ns, not significant; *, p< 0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001. The p-values in C are from two-way ANOVA with Tukey's post hoc correction.


A . Parental HEK293T , ACVR2A KO , ACVR2B KO , ACVR2A / B dKO , HOM1 , HOM1 ACVR2A KO , HOM1 ACVR2B KO , HOM1 ACVR2A / B dKO cells were treated with Activin A or BMP2 for 1 h . Data information : In all panels , Western blots of whole cell lysates were probed with the antibodies indicated . Representative experiments are shown . Par , parental HEK293T cells .

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A. Parental HEK293T, ACVR2A KO, ACVR2B KO, ACVR2A/B dKO, HOM1, HOM1 ACVR2A KO, HOM1 ACVR2B KO, HOM1 ACVR2A/B dKO cells were treated with Activin A or BMP2 for 1 h. Data information: In all panels, Western blots of whole cell lysates were probed with the antibodies indicated. Representative experiments are shown. Par, parental HEK293T cells.


C . NIH - 3T3 cells were either untransfected or transfected with either FLAG - SMAD1 or GFP - SMAD3 together with Opto - ACVR1B * . Post transfection , cells were either kept in the dark or exposed to blue light for 1 h , in the presence or absence of 50 µM SB - 505124 or 1 µM LDN - 193189 . Data information : In all panels , Western blots of whole cell lysates were probed with antibodies against pSMAD1 / 5 ( that detected both endogenous and pFLAG - SMAD1 ) , SMAD1 ( which detects both endogenous and FLAG - SMAD1 ) , HA ( which detects the Opto - receptors ) , pSMAD3 ( which detects pGFP - SMAD3 ) , SMAD3 ( which detects GFP - SMAD3 ) and either Tubulin or Actin as the loading controls . Lane numbers are given below each blot for clarity .

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C. NIH-3T3 cells were either untransfected or transfected with either FLAG-SMAD1 or GFP-SMAD3 together with Opto-ACVR1B*. Post transfection, cells were either kept in the dark or exposed to blue light for 1 h, in the presence or absence of 50 µM SB-505124 or 1 µM LDN-193189. Data information: In all panels, Western blots of whole cell lysates were probed with antibodies against pSMAD1/5 (that detected both endogenous and pFLAG-SMAD1), SMAD1 (which detects both endogenous and FLAG-SMAD1), HA (which detects the Opto-receptors), pSMAD3 (which detects pGFP-SMAD3), SMAD3 (which detects GFP-SMAD3) and either Tubulin or Actin as the loading controls. Lane numbers are given below each blot for clarity.


H . Average brightness of His - Activin A - Atto647N in conditions ( A - G ) . Means ± SEM are plotted . Each point represents one analyzed image , collected from 2 - 3 biological replicates . Data information : In H and I the p values are from one - way ANOVA with Tukey ' s post hoc correction . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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H. Average brightness of His-Activin A-Atto647N in conditions (A-G). Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-3 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


I . Average brightness of His - Activin A - Atto647N in ICR - B169 and HSJD - DIPG - 007 cells alone or with pre - incubation with 1 . 5 µg / mL of rhACVR2A Fc and 15 µg / mL of rhACVR2B Fc . Source data is in Fig . EV4B - F . Means ± SEM are plotted . Each point represents one analyzed image , collected from 2 - 4 biological replicates . Data information : In H and I the p values are from one - way ANOVA with Tukey ' s post hoc correction . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 .

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I. Average brightness of His-Activin A-Atto647N in ICR-B169 and HSJD-DIPG-007 cells alone or with pre-incubation with 1.5 µg/mL of rhACVR2A Fc and 15 µg/mL of rhACVR2B Fc. Source data is in Fig. EV4B-F. Means ± SEM are plotted. Each point represents one analyzed image, collected from 2-4 biological replicates. Data information: In H and I the p values are from one-way ANOVA with Tukey's post hoc correction. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.


A , B . HEK293T HOM1 cells were plated on a planar lipid bilayer containing ( NIP ) 1 - H12 - Hylight647 ( A ) or His - Activin A - Atto647N ( B ) and an automated time - lapse was started , where 2 - 4 positions within a well were imaged every 40 s during a 27 - min time period ; 3 time points from one position are shown . TIRF , upper panels , brightfield , lower panels . The cluster intensities from all positions were quantified at 6 time points indicated ( right panel ) and plotted as individual cluster values . The horizontal gray bar denotes the mean . Cells + bilayer denotes bilayer areas under the cells , as identified from the brightfield images , and bilayer denotes areas where no cells were present . A representative experiment from 2 biological replicates quantified is shown . Scale bar 10 μm . In B , the region under cell 2 is shown enlarged in the top right - hand corner .

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A, B. HEK293T HOM1 cells were plated on a planar lipid bilayer containing (NIP)1-H12-Hylight647 (A) or His-Activin A-Atto647N (B) and an automated time-lapse was started, where 2-4 positions within a well were imaged every 40 s during a 27-min time period; 3 time points from one position are shown. TIRF, upper panels, brightfield, lower panels. The cluster intensities from all positions were quantified at 6 time points indicated (right panel) and plotted as individual cluster values. The horizontal gray bar denotes the mean. Cells + bilayer denotes bilayer areas under the cells, as identified from the brightfield images, and bilayer denotes areas where no cells were present. A representative experiment from 2 biological replicates quantified is shown. Scale bar 10 μm. In B, the region under cell 2 is shown enlarged in the top right-hand corner.


b . In vitro activity . Left y - axis : 13C2 - oxalate in mM , right y - axis : 13C - formate in mM , x - axis : time in minutes . SYNB8802 ( blue , circles , solid line : oxalate , dotted line : formate ) The control EcN ( pink , triangle , solid line : oxalate , dotted line : formate ) . Three biological replicates were run and plotted separately .

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b. In vitro activity. Left y-axis: 13C2-oxalate in mM, right y-axis: 13C-formate in mM, x-axis: time in minutes. SYNB8802 (blue, circles, solid line: oxalate, dotted line: formate) The control EcN (pink, triangle, solid line: oxalate, dotted line: formate). Three biological replicates were run and plotted separately.


c . In vitro simulation ( IVS ) . Left y - axis : Rate of oxalate degradation in μmol / h / 109 cells . X - axis = time in hours . Left X - axis 0 - 4 hours , Right X - axis 6 - 48 h . Three biological replicates were run and plotted separately . Data points in the orange box represent incubation in simulated gastric fluid ( SGF ) . Data points in the light blue box represent incubation in simulated intestinal fluid ( SIF ) . Data points in the pink box represent incubation in simulated colonic fluid ( SCF ) .

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c. In vitro simulation (IVS). Left y-axis: Rate of oxalate degradation in μmol/h/109 cells. X-axis = time in hours. Left X-axis 0 - 4 hours, Right X-axis 6 - 48 h. Three biological replicates were run and plotted separately. Data points in the orange box represent incubation in simulated gastric fluid (SGF). Data points in the light blue box represent incubation in simulated intestinal fluid (SIF). Data points in the pink box represent incubation in simulated colonic fluid (SCF).


b . Acute mouse study of UOx lowering with SYNB8802 . The y - axis shows the urinary 13 C2 - oxalate normalized to creatine . The x - axis shows the two groups , EcN ( control , pink ) vs SYNB8802AbxR ( light blue ; n = 15 mice per group ) . Individual dots represent each metabolic cage ( n = 3 mice per metabolic cage ) . Error bars are calculated as SEM . ( unpaired t - test with Welch ' s correction , * * * * p < 0 . 0001 )

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b. Acute mouse study of UOx lowering with SYNB8802. The y-axis shows the urinary 13 C2-oxalate normalized to creatine. The x-axis shows the two groups, EcN (control, pink) vs SYNB8802AbxR (light blue; n = 15 mice per group). Individual dots represent each metabolic cage (n = 3 mice per metabolic cage). Error bars are calculated as SEM. (unpaired t-test with Welch's correction, **** p < 0.0001)


d . Nonhuman primate model development study . The y - axis shows urinary oxalate normalized to creatine . X - axis shows baseline urinary oxalate levels after overnight fast , and urinary oxalate measured 6 hours post administration of either a spinach preparation ( triangle , pink ) or water ( circle , blue ) ( n = 6 for each group ) . Individual dots represent individual animals . Error bars are calculated as SEM . ( Two - way ANOVA followed by Sidak ' s multiple comparison analysis , * * * * p < 0 . 0001 ) .

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d. Nonhuman primate model development study. The y-axis shows urinary oxalate normalized to creatine. X-axis shows baseline urinary oxalate levels after overnight fast, and urinary oxalate measured 6 hours post administration of either a spinach preparation (triangle, pink) or water (circle, blue) (n=6 for each group). Individual dots represent individual animals. Error bars are calculated as SEM. (Two-way ANOVA followed by Sidak's multiple comparison analysis, **** p < 0.0001).


f . Urinary recovery of oxalate in nonhuman primates . The y - axis shows change in urinary oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatment groups ) . Individual dots represent normalized urinary oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * p < 0 . 05 )

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f. Urinary recovery of oxalate in nonhuman primates. The y-axis shows change in urinary oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatment groups). Individual dots represent normalized urinary oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, * p < 0.05)


g . Urinary recovery of 13C2 oxalate in nonhuman primates . The y - axis shows change in urinary 13C2 - oxalate from vehicle control . The x - axis shows vehicle ( control , grey ) and increasing doses of SYNB8802AbxR ( n = 24 for vehicle , n = 6 for treatmentgroups ) . Individual dots represent normalized urinary 13C2 - oxalate for each individual animal . Error bars are calculated as SEM . ( One - way ANOVA followed by Tukey ' s multiple comparison analysis , * * p < 0 . 01 )

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g. Urinary recovery of 13C2 oxalate in nonhuman primates. The y-axis shows change in urinary 13C2-oxalate from vehicle control. The x-axis shows vehicle (control, grey) and increasing doses of SYNB8802AbxR (n = 24 for vehicle, n = 6 for treatmentgroups). Individual dots represent normalized urinary 13C2-oxalate for each individual animal. Error bars are calculated as SEM. (One-way ANOVA followed by Tukey's multiple comparison analysis, ** p < 0.01)


G ) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates ( n = 3 ) . Β - actin serves as an internal loading control .

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G) Western blot analysis of siRNA knockdown of p53 expression in three biological replicates (n=3). Β-actin serves as an internal loading control.


B ) Images represent localization of KTR for ERK , JNK , or p38 under basal growth conditions . Scales bars = 20µm .

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B) Images represent localization of KTR for ERK, JNK, or p38 under basal growth conditions. Scales bars=20µm.


C - E ) Kinase activity for ERK ( C ) , JNK ( D ) , or p38 ( E ) in three representative single cells ( upper panels ) in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) as measured by C / N ratio . Lower panel shows mean C / N ratio in response to 400 ng / ml NCS ( blue ) or 300 µM H2O2 ( red ) . Thick line represents mean with shaded area = SD . N for each condition and kinase is shown in population average plots .

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C-E) Kinase activity for ERK (C), JNK (D), or p38 (E) in three representative single cells (upper panels) in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red) as measured by C/N ratio. Lower panel shows mean C/N ratio in response to 400 ng/ml NCS (blue) or 300 µM H2O2 (red). Thick line represents mean with shaded area=SD. N for each condition and kinase is shown in population average plots.


C - E ) Mean kinase activation for ERK € , JNK ( D ) , or p38 ( E ) in both live ( grey ) and dead ( red ) cells measured over 24 - hours in response to H2O2 . Shaded areas = SD . N for each condition is shown .

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C-E) Mean kinase activation for ERK €, JNK (D), or p38 (E) in both live (grey) and dead (red) cells measured over 24-hours in response to H2O2. Shaded areas=SD. N for each condition is shown.


D ) Mean JNK activity as measured by C / N ratio ( bold line ) ± SD ( shaded area ) in response to 300 µM H2O2 with or without p38 inhibition ( grey ) . Bar graph shows integrated JNK activity ( C / N ratio ) over 24 - hours . Data represents mean ± SD . The number of analyzed cells , n , is shown for each condition . Statistics performed using 2 - tailed t - test . Data information : * * p < 0 . 01 , * * * p < 0 . 001 , ns = not significant .

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D) Mean JNK activity as measured by C/N ratio (bold line)±SD (shaded area) in response to 300 µM H2O2 with or without p38 inhibition (grey). Bar graph shows integrated JNK activity (C/N ratio) over 24-hours. Data represents mean±SD. The number of analyzed cells, n, is shown for each condition. Statistics performed using 2-tailed t-test. Data information: ** p<0.01, *** p<0.001, ns=not significant.


( B ) Representative images of immunostaining demonstrate that FKRP - iPSCs express specific pluripotency - associated markers , including NANOG , OCT4 , Tra - 1 - 60 and SSEA4 . Data information : Scale bars , 50 μm .

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(B) Representative images of immunostaining demonstrate that FKRP-iPSCs express specific pluripotency-associated markers, including NANOG, OCT4, Tra-1-60 and SSEA4. Data information: Scale bars, 50 μm.


( C ) FKRP - iPSCs have a normal karyotype . Data information : Scale bars , 50 μm .

(C) FKRP-iPSCs have a normal karyotype. Data information: Scale bars, 50 μm.


( D ) In vitro differentiation of FKRP - iPSCs to cells representing ectoderm ( β - III Tubulin , Tuj1 ) , mesoderm ( SMA , smooth muscle actin ) and endoderm ( AFP , α - fetoprotein ) . Data information : Scale bars , 50 μm .

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(D) In vitro differentiation of FKRP-iPSCs to cells representing ectoderm (β-III Tubulin, Tuj1), mesoderm (SMA, smooth muscle actin) and endoderm (AFP, α-fetoprotein). Data information: Scale bars, 50 μm.


( C ) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones ( 5D17 , 5D23 and 3B17 ) , compared with their parental FKRPA455D - iPSCs . Selection cassette excision sites are identified in the corrected - iPSC lines .

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(C) Sequence analysis shows precise biallelic correction of the FKRP mutation in three independently corrected iPSC clones (5D17, 5D23 and 3B17), compared with their parental FKRPA455D-iPSCs. Selection cassette excision sites are identified in the corrected-iPSC lines.


( D ) CRISPR / Cas9 corrected - iPSC lines show a normal karyotype .

(D) CRISPR/Cas9 corrected-iPSC lines show a normal karyotype.


( A and B ) Representative images of NSCs derived from FKRP - and corrected - iPSC lines expressing SOX1 , SOX2 and nestin . ( C and D ) Quantification of percentage of SOX1 + ( C ) and SOX2 + ( D ) cells in culture . The efficiency of neural induction is more than 99 % in FKRP - and corrected - iPSC ( 5D17 , 5D23 and 3B17 ) lines . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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(A and B) Representative images of NSCs derived from FKRP- and corrected-iPSC lines expressing SOX1, SOX2 and nestin. (C and D) Quantification of percentage of SOX1+ (C) and SOX2+ (D) cells in culture. The efficiency of neural induction is more than 99% in FKRP- and corrected-iPSC (5D17, 5D23 and 3B17) lines. Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( E and F ) FKRP - and corrected NSC lines can be further differentiated to cortical neural progenitor cells , expressing PAX6 , OTX2 and vimentin . ( G - I ) Quantification of percentage of PAX6 + ( G ) and OTX2 + ( H ) cells in culture . About 91 - 98 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express PAX6 ( G ) . About 93 - 96 % of cells derived from FKRP , 5D17 , 5D23 and 3B17 NSC lines express OTX2 ( H ) . Of the OTX2 + population , about 60 - 67 % cells are also Ki67 + cycling progenitors ( I ) . Data are mean ± s . d . n = 4 technical replicates . Data information : Scale bars , 50 μm .

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(E and F) FKRP- and corrected NSC lines can be further differentiated to cortical neural progenitor cells, expressing PAX6, OTX2 and vimentin. (G-I) Quantification of percentage of PAX6+ (G) and OTX2+ (H) cells in culture. About 91-98% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express PAX6 (G). About 93-96% of cells derived from FKRP, 5D17, 5D23 and 3B17 NSC lines express OTX2 (H). Of the OTX2+ population, about 60-67% cells are also Ki67+ cycling progenitors (I). Data are mean ± s.d. n = 4 technical replicates. Data information: Scale bars, 50 μm.


( J and K ) Glutamatergic projection neurons derived from FKRP and corrected ( 5D17 , 5D23 and 3B17 ) progenitor cells . The vast majority of neurons contain vGlut1 + punctae in their neurites ( labelled by Tuj1 ) . Right panels are enlarged images from the insets of left panels . Data information : Scale bars , 50 μm .

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(J and K) Glutamatergic projection neurons derived from FKRP and corrected (5D17, 5D23 and 3B17) progenitor cells. The vast majority of neurons contain vGlut1+ punctae in their neurites (labelled by Tuj1). Right panels are enlarged images from the insets of left panels. Data information: Scale bars, 50 μm.


( A ) Molecular weight of glycosylated α - dystroglycan ( IIH6 epitope ) in WT - iPSC derived cortical neurons is similar to that in the mouse brain ( ~ 120 kDa ) and lower than that in the mouse muscle ( ~ 156kDa ) . IIH6 reactivity was almost not detected in cortical neurons derived from FKRP - iPSCs . Note that the β - Actin antibody does not cross - react with the muscle sample .

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(A) Molecular weight of glycosylated α-dystroglycan (IIH6 epitope) in WT-iPSC derived cortical neurons is similar to that in the mouse brain (~120 kDa) and lower than that in the mouse muscle (~156kDa). IIH6 reactivity was almost not detected in cortical neurons derived from FKRP-iPSCs. Note that the β-Actin antibody does not cross-react with the muscle sample.


( B ) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC - derived cortical neurons . Intensities of IIH6 reactivity are normalized to β - Actin expression , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * * p < 0 . 01 )

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(B) Representative immunoblots show that targeted gene correction of FKRP restores IIH6 reactivity in iPSC-derived cortical neurons. Intensities of IIH6 reactivity are normalized to β-Actin expression, indicating mean ± s.d. (n = 3 biological replicates; t-test; ** p<0.01)


( C ) Representative laminin overlays show that targeted gene correction restores laminin - binding activity in iPSC - derived cortical neurons . Intensities of laminin - binding activity are normalized to β - dystroglycan , indicating mean ± s . d . ( n = 3 biological replicates ; t - test ; * p < 0 . 05 ) .

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(C) Representative laminin overlays show that targeted gene correction restores laminin-binding activity in iPSC-derived cortical neurons. Intensities of laminin-binding activity are normalized to β-dystroglycan, indicating mean ± s.d. (n = 3 biological replicates; t-test; * p<0.05).


( C ) Dose response curve . H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed , stained and imaged as described above . The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non - linear regression curve was fitted for the 3 compounds showing the highest increase in α - dystroglycan glycosylation ( IIH6 immunostaining ) . Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown . HPFPTzone : ( 5Z ) - 5 - [ ( 3 - ethoxy - 4 - hydroxy - phenyl ) methylene ] - 3 - ( 4 - fluorophenyl ) - 2 - thioxo - thiazolidin - 4 - one . 4BPPNit : 4 - ( 4 - bromophenyl ) - 6 - ethylsulfanyl - 2 - oxo - 3 , 4 - dihydro - 1H - pyridine - 5 - carbonitrile . DPPTzone : ( 5E ) - 5 - [ ( 2 , 5 - dimethyl - 1 - phenyl - pyrrol - 3 - yl ) methylene ] - 3 - phenyl - 2 - thioxo - thiazolidin - 4 - one . Regression curve and EC50 values were calculated using GraphPad Prism 7 software . In images shown , green represents glycosylated α - dystroglycan and blue Hoechst staining . Scale bar , 50 µm .

(C) Dose response curve. H2K 2B4 cells were treated with serial dilutions of 11 hit compounds for 24 h and fixed, stained and imaged as described above. The average values and standard deviations of the mean fluorescent intensities of technical triplicates were calculated and a non-linear regression curve was fitted for the 3 compounds showing the highest increase in α-dystroglycan glycosylation (IIH6 immunostaining). Representative images of cells treated with 5 µM of the respective compounds as well as EC50 values are shown. HPFPTzone: (5Z)-5-[(3-ethoxy-4-hydroxy-phenyl)methylene]-3-(4-fluorophenyl)-2-thioxo-thiazolidin-4-one. 4BPPNit: 4-(4-bromophenyl)-6-ethylsulfanyl-2-oxo-3,4-dihydro-1H-pyridine-5-carbonitrile. DPPTzone: (5E)-5-[(2,5-dimethyl-1-phenyl-pyrrol-3-yl)methylene]-3-phenyl-2-thioxo-thiazolidin-4-one. Regression curve and EC50 values were calculated using GraphPad Prism 7 software. In images shown, green represents glycosylated α-dystroglycan and blue Hoechst staining. Scale bar, 50 µm.


( E ) Flow cytometry analysis confirmed that 4BPPNit treatment ( 20 µM ) significantly increased IIH6 - reactive glycan in mouse H2K 2B4 cells , compared with untreated cells . There was no significant difference in the percentage of IIH6 - positive cells . Values indicate mean ± s . e . m . ( n = 6 , triplicates of 2 independent experiments ; t - test ; NS , not significant ; * * p < 0 . 01 ) .

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(E) Flow cytometry analysis confirmed that 4BPPNit treatment (20 µM) significantly increased IIH6-reactive glycan in mouse H2K 2B4 cells, compared with untreated cells. There was no significant difference in the percentage of IIH6-positive cells. Values indicate mean ± s.e.m. (n= 6, triplicates of 2 independent experiments; t-test; NS, not significant; ** p<0.01).