Figure 3ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ intensities from five independent experiments . ST2 wild type or TBK1 / IKKε DKO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from five independent experiments is shown , statistical significance was determined by two ‐ tailed Mann - Whitney test . C . ST2 wild type or TBK1 / IKKε DKO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) for 15 minutes or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1 / IKKε DKO cells . D . Cells were pretreated with TAK1 inhibitor 7 - oxozeanol ( 2 µM ) and / or TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting . E . ST2 wild type or HOIP - deficient cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated , solubilized , and analyzed by immunoblotting . * indicates unspecific band . F . ST2 cells pretreated or not with TBK1 / IKKε inhibitor MRT67307 were stimulated with SF - IL - 17 for 15 minutes or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated and analyzed via immunoblotting .

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Figure 3ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ intensities from five independent experiments.ST2 wild type or TBK1/IKKε DKO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from five independent experiments is shown, statistical significance was determined by two‐tailed Mann-Whitney test.C. ST2 wild type or TBK1/IKKε DKO cells were stimulated with SF-IL-17 (500 ng/ml) for 15 minutes or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting. ACT1 phosphorylation detected as upper band in WT cells is absent in TBK1/IKKε DKO cells.D. Cells were pretreated with TAK1 inhibitor 7-oxozeanol (2 µM) and/or TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.E. ST2 wild type or HOIP-deficient cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM), stimulated with IL-17 (500 ng/ml) as indicated, solubilized, and analyzed by immunoblotting. * indicates unspecific band.F. ST2 cells pretreated or not with TBK1/IKKε inhibitor MRT67307 were stimulated with SF-IL-17 for 15 minutes or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated and analyzed via immunoblotting.


Figure 4A . ST2 wild type and ACT1 - deficient cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . As a control , cells were first solubilized and SF - IL - 17 was added only post lysis . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown . B . ST2 wild type , ACT1 KO or TRAF6 KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC and samples were analyzed by immunoblotting . C . ST2 cells deficient in TRAF6 reconstituted with TRAF6 ( WT ) or enzymatically inactive TRAF6 ( C70A ) were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . Number of identified peptides ( unique + razor ) and iBAQ intensities for each protein in two independent experiments are shown . D . TRAF6 deficient cells reconstituted with TRAF6 ( WT ) , TRAF6 ( C70A ) or empty vector were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated from lysates by anti - Flag immunoprecipitation and analyzed by immunoblotting . Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples , respectively . TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . ( E ) The activation of signaling pathways analyzed by immunoblotting . TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) and stimulated with IL - 17 ( 500 ng / ml ) for indicated time . The induction of mRNA for selected genes analyzed by real time PCR . Mean + SEM from four independent experiments is shown , statistical significance was determined using unpaired two ‐ tailed Student ' s t ‐ test . G . ST2 cells wild type or NEMO KO were stimulated with IL - 17 ( 500 ng / ml ) for indicated time points , solubilized and analyzed by immunoblotting . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC . Samples were analyzed by immunoblotting ( H ) . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 or were left unstimulated and IL - 17 was added post lysis . results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown ( I ) .

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Figure 4A. ST2 wild type and ACT1-deficient cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. As a control, cells were first solubilized and SF-IL-17 was added only post lysis. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.B. ST2 wild type, ACT1 KO or TRAF6 KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC and samples were analyzed by immunoblotting.C. ST2 cells deficient in TRAF6 reconstituted with TRAF6(WT) or enzymatically inactive TRAF6(C70A) were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. Number of identified peptides (unique + razor) and iBAQ intensities for each protein in two independent experiments are shown.D. TRAF6 deficient cells reconstituted with TRAF6(WT), TRAF6(C70A) or empty vector were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated from lysates by anti-Flag immunoprecipitation and analyzed by immunoblotting. Short and long exposure of the same membrane stained for Act1 are shown for lysate and IP samples, respectively.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. (E) The activation of signaling pathways analyzed by immunoblotting.TRAF6 deficient cells reconstituted with the indicated constructs were pretreated or not with TBK1/IKKε inhibitor MRT67307 (2 µM) and stimulated with IL-17 (500 ng/ml) for indicated time. The induction of mRNA for selected genes analyzed by real time PCR. Mean + SEM from four independent experiments is shown, statistical significance was determined using unpaired two‐tailed Student's t‐test.G. ST2 cells wild type or NEMO KO were stimulated with IL-17 (500 ng/ml) for indicated time points, solubilized and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC. Samples were analyzed by immunoblotting (H).ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 or were left unstimulated and IL-17 was added post lysis. results from four independent experiments were quantified by densitometry and mean + SEM from four independent experiments is shown (I).


Figure 5ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( A ) The heatmap shows the row - normalized Z - Score calculated from log2 transformed iBAQ values from two independent experiments . ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF - IL - 17 ( 500 ng / ml ) , solubilized and IL - 17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS . ( B ) The ratio between iBAQ intensities of selected IL - 17RSC components to iBAQ intensity of IL - 17RC . Mean + SEM from two independent experiments is shown . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were subjected to anti - Flag immunoprecipitation to isolate IL - 17RSC ( C ) and analyzed by immunoblotting . ST2 wild type or NEMO KO cells were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . Lysates were tested for activation of signaling pathways ( D ) and analyzed by immunoblotting . E . ST2 cells were pretreated with TBK1 / IKKε inhibitor MRT67307 ( 2 µM ) or IKKα / IKKβ inhibitor TPCA1 ( 10 µM ) , stimulated with IL - 17 ( 500 ng / ml ) as indicated and analyzed by immunoblotting . F . ST2 wild type or NEMO KO cells were stimulated with TNF ( 50 ng / ml ) for indicated time points and lysates were analyzed by immunoblotting .

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Figure 5ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (A) The heatmap shows the row-normalized Z-Score calculated from log2 transformed iBAQ values from two independent experiments.ST2 wild type or NEMO KO cells were stimulated for 15 minutes with SF-IL-17 (500 ng/ml), solubilized and IL-17RSC was isolated via consecutive Flag and Strep immunoprecipitation and analyzed by MS. (B) The ratio between iBAQ intensities of selected IL-17RSC components to iBAQ intensity of IL-17RC. Mean + SEM from two independent experiments is shown.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were subjected to anti-Flag immunoprecipitation to isolate IL-17RSC (C) and analyzed by immunoblotting.ST2 wild type or NEMO KO cells were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. Lysates were tested for activation of signaling pathways (D) and analyzed by immunoblotting.E. ST2 cells were pretreated with TBK1/IKKε inhibitor MRT67307 (2 µM) or IKKα/IKKβ inhibitor TPCA1 (10 µM), stimulated with IL-17 (500 ng/ml) as indicated and analyzed by immunoblotting.F. ST2 wild type or NEMO KO cells were stimulated with TNF (50 ng/ml) for indicated time points and lysates were analyzed by immunoblotting.


Figure 6B . Analysis of ACT1 structure using three different algorithms predicting disordered regions . Detected phosphorylation sites are all in the disordered region of the protein . ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho - sites mutated to alanines ( 9ST mut ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . E . ACT1 KO cells expressing either ACT1 ( WT ) or ACT1 ( 9ST mut ) were stimulated with SF - IL - 17 ( 500 ng / ml ) as indicated or were left unstimulated and IL - 17 was added post lysis . IL - 17RSC was isolated via anti - Flag immunoprecipitation and analyzed by immunoblotting . F . ACT1 KO cells were reconstituted with either ACT1 ( WT ) or ACT1 ( Δ20 - 380 ) . Cells were stimulated with IL - 17 ( 500 ng / ml ) as indicated and lysates were analyzed by immunoblotting . * indicates nonspecific band . G . Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL - 17RC . Data are composite of all 14 MS analyses of IL - 17RSC from wild type ST2 cells performed in this study . Mean + SEM is shown .

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Figure 6B. Analysis of ACT1 structure using three different algorithms predicting disordered regions. Detected phosphorylation sites are all in the disordered region of the protein.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.ACT1 KO cells were reconstituted with either wild type ACT1 or indicated mutants or ACT1 with all nine identified phospho-sites mutated to alanines (9ST mut). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.E. ACT1 KO cells expressing either ACT1(WT) or ACT1(9ST mut) were stimulated with SF-IL-17 (500 ng/ml) as indicated or were left unstimulated and IL-17 was added post lysis. IL-17RSC was isolated via anti-Flag immunoprecipitation and analyzed by immunoblotting.F. ACT1 KO cells were reconstituted with either ACT1(WT) or ACT1(Δ20-380). Cells were stimulated with IL-17 (500 ng/ml) as indicated and lysates were analyzed by immunoblotting. * indicates nonspecific band.G. Analysis of iBAQ intensity of ACT1 normalized to iBAQ intensity of IL-17RC. Data are composite of all 14 MS analyses of IL-17RSC from wild type ST2 cells performed in this study. Mean + SEM is shown.


Figure 1 ( B ) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX . Cut - off for what is regarded as expressed was set to 1 . 0 NX .

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Figure 1(B) ACE2 gene expression summary in human tissues in 61 different tissues and cells in NX. Cut-off for what is regarded as expressed was set to 1.0 NX.


Figure 4Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein ( brown ) , and counterstained with hematoxylin ( blue ) . Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules , in membranes of gallbladder epithelium , epididymis epithelium , testicular Sertoli cells and Leydig cells , a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes , with HPA000288 also staining the cardiac muscle fibers , while MAB933 only showed staining in a few cells . Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye , interlobular pancreatic ducts , as well as in placental villi , both in cytotrophoblasts , syncytiotrophoblasts , and also in extravillous trophoblasts , while placenta decidua was negative . ACE2 staining could be observed at the base of ciliated fallopian tube epithelium , however only for one of the antibodies . Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer . ACE2 also stained positive in endothelial cells and pericytes in several tissues , see Fallopian , thyroid , parathyroid , adrenal gland , pancreas and heart . Scale bar = 50µm . Scale bar in dashed squares = 10 µm . ( Brunner = Brunner ' s gland , EVT = extravillous trophoblasts , endo = endothelial cells ) .

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Figure 4Representative images of 20 tissue types and histological structures stained on consecutive sections with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Most intense antibody staining was observed in microvilli of the intestinal tract and renal proximal tubules, in membranes of gallbladder epithelium, epididymis epithelium, testicular Sertoli cells and Leydig cells, a subset of glandular cells in seminal vesicle and cytoplasm of cardiomyocytes, with HPA000288 also staining the cardiac muscle fibers, while MAB933 only showed staining in a few cells. Distinct ACE2 staining for both antibodies was also present in cornea and conjunctiva of the eye, interlobular pancreatic ducts, as well as in placental villi, both in cytotrophoblasts, syncytiotrophoblasts, and also in extravillous trophoblasts, while placenta decidua was negative. ACE2 staining could be observed at the base of ciliated fallopian tube epithelium, however only for one of the antibodies. Note that ACE2 protein expression was less prominent in the crypts of the mucosal intestinal layer. ACE2 also stained positive in endothelial cells and pericytes in several tissues, see Fallopian, thyroid, parathyroid, adrenal gland, pancreas and heart. Scale bar = 50µm. Scale bar in dashed squares = 10 µm. (Brunner = Brunner's gland, EVT = extravillous trophoblasts, endo=endothelial cells).


Figure 5Representative images of human respiratory tissues , all stained on consecutive sections , with immunohistochemistry using two antibodies targeting human ACE2 protein ( brown ) , and counterstained with hematoxylin ( blue ) . Respiratory tissues were composed of different structures in nasal mucosa , bronchus , smaller bronchioles and lung tissue . ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium ( arrowheads ) . Rare ACE2 staining was present in a few alveolar cells ( arrows ) . No staining was observed in nasal squamous epithelium , bronchioles , or submucosal glands in either tissue . Gender and age are shown for all individuals . Red and green colored squares mark the positions in the TMA cores shown as magnifications . Scale bar = 50µm . Scale bar for images in dashed squares = 10 µm . ( re = respiratory ; sq = squamous ) .

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Figure 5Representative images of human respiratory tissues, all stained on consecutive sections, with immunohistochemistry using two antibodies targeting human ACE2 protein (brown), and counterstained with hematoxylin (blue). Respiratory tissues were composed of different structures in nasal mucosa, bronchus, smaller bronchioles and lung tissue. ACE2 staining could be observed at the base of ciliated cells in both nasal mucosa and bronchial epithelium (arrowheads). Rare ACE2 staining was present in a few alveolar cells (arrows). No staining was observed in nasal squamous epithelium, bronchioles, or submucosal glands in either tissue. Gender and age are shown for all individuals. Red and green colored squares mark the positions in the TMA cores shown as magnifications. Scale bar = 50µm. Scale bar for images in dashed squares = 10 µm. (re=respiratory; sq=squamous).


Figure 6 ( B ) ACE2 protein abundance in different human tissues from ProteomicsDB , with the median expression presented in log10 ( iBAQ ) . ( C ) Western blot of ACE2 using five different human tissue lysates . For lung , two different lysates were used : one male ( left ) and one female ( right ) .

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Figure 6(B) ACE2 protein abundance in different human tissues from ProteomicsDB, with the median expression presented in log10(iBAQ).(C) Western blot of ACE2 using five different human tissue lysates. For lung, two different lysates were used: one male (left) and one female (right).


Figure 1 ( A ) Schematic overview of FMR1 , FXR1 , FXR2 , FMR2 and position of the 8 selected single nucleotide polymorphisms ( SNPs ) . Line represents introns , grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons . Gene structure plots generated using FancyGene ( Rambaldi Ciccarelli , 2009 ) ( B ) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls . ( C ) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls .

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Figure 1(A) Schematic overview of FMR1, FXR1, FXR2, FMR2 and position of the 8 selected single nucleotide polymorphisms (SNPs). Line represents introns, grey box at the beginning and end of each gene stands for UTR region and red boxes represent exons. Gene structure plots generated using FancyGene (Rambaldi Ciccarelli, 2009)(B) Distribution of repeat polymorphism lengths in FMR1 of male GRAS schizophrenia patients and healthy controls. (C) Distribution of repeat polymorphism lengths in FMR2 of male GRAS schizophrenia patients and healthy controls.


Figure 2Figure 2 : SNP overview and unbiased selection according to the standard operating procedure ( SOP ) developed for phenotype - based genetic association study ( PGAS ) approaches . ( A ) Genes from the ' broader fragile X family ' and their chromosomal position . ( B ) SNP numbers available through direct genotyping in the here used semi - custom genotyping array ( Affymetrix , Santa Clara , CA , USA ) . ( C ) SNPs fulfilling some of the first round of selection criteria ( ' functional ' = SNPs , i . e . located in promoter region , 3 ' UTR or coding sequence ; MAF = MAF ≥ 0 . 2 ; LD = SNPs that ' survive ' after linkage disequilibrium pruning : r² < 0 . 8 ) . Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS ( selection requirements : fulfilled 2 of the above criteria or were functional ) . Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene . ( D ) SNPs with a tendency in PGAS ( see Fig 3A ) at single SNP basis went into the final accumulation model .

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Figure 2Figure 2: SNP overview and unbiased selection according to the standard operating procedure (SOP) developed for phenotype-based genetic association study (PGAS) approaches. (A) Genes from the 'broader fragile X family' and their chromosomal position. (B) SNP numbers available through direct genotyping in the here used semi-custom genotyping array (Affymetrix, Santa Clara, CA, USA). (C) SNPs fulfilling some of the first round of selection criteria ('functional'=SNPs, i.e. located in promoter region, 3'UTR or coding sequence; MAF=MAF≥0.2; LD=SNPs that 'survive' after linkage disequilibrium pruning: r²<0.8). Underlined are the 13 SNPs selected for the PGAS approach using the PAUSS (selection requirements: fulfilled 2 of the above criteria or were functional). Not more than 3 SNPs per gene are selected to avoid overrepresentation of one gene. (D) SNPs with a tendency in PGAS (see Fig 3A) at single SNP basis went into the final accumulation model.


Figure 3 ( A - B ) A total of 13 SNPs pre - selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here : ( A ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which are eligible for the accumulation model . The genotype associated with the highest average PAUSS ( in this example CC ) is the ' proautistic genotype ' ( indicated by the black arrow ) and is assigned a score of 1 . The heterozygous genotype is assigned a score of 0 . 5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0 . Please note that the difference between genotypes does not have to be statistically significant . ( B ) PAUSS association pattern of an exemplary fictitious autosomal ( upper panel ) and a sex - chromosomal ( lower panel ) SNP which would not be selected for the accumulation model because of unclear phenotypical / biological relevance . ( C - D ) The specificity of the association of the 8SNP accumulation model with an autistic phenotype ( as determined using PAUSS ; compare Fig 4B ) is controlled by applying an unrelated ( or ' non - sense ' ) phenotype , e . g . delusional - depression : ( C ) Intercorrelation pattern of single items included in the delusional - depression composite score , used here as example control phenotype . Cronbach ' s alpha is presented as measure of internal consistency . ( D ) The delusional - depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample .

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

Figure 3(A-B) A total of 13 SNPs pre-selected according to the SOP presented in Fig 2 underwent PGAS screening as exemplified here: (A) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which are eligible for the accumulation model. The genotype associated with the highest average PAUSS (in this example CC) is the 'proautistic genotype' (indicated by the black arrow) and is assigned a score of 1. The heterozygous genotype is assigned a score of 0.5 and the homozygous genotype associated with the lowest PAUSS receives a score of 0. Please note that the difference between genotypes does not have to be statistically significant. (B) PAUSS association pattern of an exemplary fictitious autosomal (upper panel) and a sex-chromosomal (lower panel) SNP which would not be selected for the accumulation model because of unclear phenotypical/biological relevance.(C-D) The specificity of the association of the 8SNP accumulation model with an autistic phenotype (as determined using PAUSS; compare Fig 4B) is controlled by applying an unrelated (or 'non-sense') phenotype, e.g. delusional-depression: (C) Intercorrelation pattern of single items included in the delusional-depression composite score, used here as example control phenotype. Cronbach's alpha is presented as measure of internal consistency. (D) The delusional-depression composite score is not associated with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.


Figure 4 ( A ) PAUSS ( PANSS autism severity score ) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals ( discovery sample ) . Cronbach ' s alpha is presented as measure of internal consistency and also provided for the male replication samples I and II . ( B ) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample . ( C ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample ; binary logistic regression analysis with non - z - standardized PAUSS as dependent variable ; mean ± SEM . ( D ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients ; binary logistic regression analysis ; mean ± SEM . ( E ) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients ; Mann - Whitney U test ; mean ± SEM . F ) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype ; mean ± SEM . ( G ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample ; binary logistic regression analysis ; mean ± SEM . ( H ) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population ; binary logistic regression analysis ; mean ± SEM . For all replications p - values for one - sided tests are shown .

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

Figure 4(A) PAUSS (PANSS autism severity score) composition and item intercorrelation pattern in the GRAS sample of male schizophrenic individuals (discovery sample). Cronbach's alpha is presented as measure of internal consistency and also provided for the male replication samples I and II.(B) Association of PAUSS with the number of proautistic genotypes of the 8SNP risk model in the discovery sample.(C) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in the discovery sample; binary logistic regression analysis with non-z-standardized PAUSS as dependent variable; mean±SEM.(D) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample I of male schizophrenia patients; binary logistic regression analysis; mean±SEM.(E) PAUSS comparison of extreme groups with high and low numbers of accumulated proautistic genotypes in replication sample II of male disease control patients; Mann-Whitney U test; mean±SEM.F) The highly significant correlation of PAUSS and social support underlines the validity of social support as an autism proxy phenotype; mean±SEM.(G) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the discovery sample; binary logistic regression analysis; mean±SEM.(H) Comparison of the extreme groups with high and low numbers of accumulated proautistic genotypes for the autism proxy phenotype social support in the male replication sample III from general population; binary logistic regression analysis; mean±SEM. For all replications p-values for one-sided tests are shown.


Figure 5 ( A ) FXR1 expression in PBMC of individuals carrying the rs2601 GG ( low risk ; N = 16 ) versus AA genotype ( high risk ; N = 27 ) . ( B ) PBMC microRNA expression was normalized and data is plotted from all 4 miR - 181 - 5p members ( miR - 181a , b , c , d - 5p ) . ( C ) Sequence homology of all 4 human mature miR - 181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR - 181 family ( seed matches identified using Target Scan Human and SFOLD ) . The red letters specify seed sequences and seed matches , respectively . Chromosome positions for each seed sequence and seed match are denoted ( human genome assembly GRCh38 / hg38 ) . ( D ) Denoted are ΔΔG values for binding of each of the miR - 181 family members to different 3 ' UTR positions . Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values . ΔΔG is an energetic score , and the more negative its value , the stronger is the expected binding of the microRNA to the given

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

Figure 5(A) FXR1 expression in PBMC of individuals carrying the rs2601 GG (low risk; N=16) versus AA genotype (high risk; N=27).(B) PBMC microRNA expression was normalized and data is plotted from all 4 miR-181-5p members (miR-181a, b, c, d-5p).(C) Sequence homology of all 4 human mature miR-181 species shown together with sequences in the broader fragile X gene family containing binding sites for the miR-181 family (seed matches identified using Target Scan Human and SFOLD). The red letters specify seed sequences and seed matches, respectively. Chromosome positions for each seed sequence and seed match are denoted (human genome assembly GRCh38/hg38).(D) Denoted are ΔΔG values for binding of each of the miR-181 family members to different 3'UTR positions. Positions were identified using Target Scan Human and SFOLD and then processed using PITA algorithm to yield the denoted ΔΔG values. ΔΔG is an energetic score, and the more negative its value, the stronger is the expected binding of the microRNA to the given site


Figure 1 ( D ) The indicated amounts of recombinant wild - type and mutant PPM1H or PPM1J ( with a His - Sumo N - terminal tag , expressed in E . coli ) were incubated in vitro with 2 . 5 µg pThr72 phosphorylated Rab8a ( left ) or pThr73 phosphorylated Rab10 ( right ) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7 . 5 buffer . Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos - tag gel electrophoresis that separates phosphorylated ( slow migrating ) and dephosphorylated Rabs . The gel was stained with Instant Blue Coomassie . D288A is a substrate - trapping ( inactive ) variant of PPM1H and was used as a control .

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Figure 1(D) The indicated amounts of recombinant wild-type and mutant PPM1H or PPM1J (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pThr72 phosphorylated Rab8a (left) or pThr73 phosphorylated Rab10 (right) for 20 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.5 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated (slow migrating) and dephosphorylated Rabs. The gel was stained with Instant Blue Coomassie. D288A is a substrate-trapping (inactive) variant of PPM1H and was used as a control.


Figure 2 ( D ) Kinetics of phosphate hydrolysis . 25 nM recombinant wild - type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a ( GTPγS or GDP ) as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of released phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 phosphorylated Rab8a [ GTP bound conformation ] ( blue ) and pThr72 phosphorylated Rab8a [ GDP bound conformation ] ( red ) . The experiments were repeated twice , and both data points are shown in curves . Line fittings for the left and middle panel were performed using the mean of the two values . Kinetic constants ( Kcat , Vmax , Km ) were obtained using GraphPad software , and their uncertainties ( ± ) correspond to the SE of mean . ( E ) Kinetic analysis of PPM1H as in ( D ) using 50 nM PPM1H and phosphopeptide substrates , as described in Materials and Methods . Initial velocity ( V0 ) was calculated by dividing the concentration of phosphatase ( μM ) by time ( min ) and plotted against substrate concentration for pThr72 Rab8a phospho - peptide ( blue ) and Rab10 pThr73 phospho - peptide ( red ) . Each experiment was performed twice ( individual data points shown ) . ( F ) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis . 50 nM recombinant wild - type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein ( blue ) or Rab8a pThr72 phosphorylated peptide ( red ) for indicated times and analysed as described in Materials and Methods . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates .

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

Figure 2(D) Kinetics of phosphate hydrolysis. 25 nM recombinant wild-type PPM1H was incubated with increasing concentrations of pThr72 phosphorylated Rab8a (GTPγS or GDP) as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of released phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 phosphorylated Rab8a[GTP bound conformation] (blue) and pThr72 phosphorylated Rab8a[GDP bound conformation] (red). The experiments were repeated twice, and both data points are shown in curves. Line fittings for the left and middle panel were performed using the mean of the two values. Kinetic constants (Kcat, Vmax, Km) were obtained using GraphPad software, and their uncertainties (±) correspond to the SE of mean.(E) Kinetic analysis of PPM1H as in (D) using 50 nM PPM1H and phosphopeptide substrates, as described in Materials and Methods. Initial velocity (V0) was calculated by dividing the concentration of phosphatase (μM) by time (min) and plotted against substrate concentration for pThr72 Rab8a phospho-peptide (blue) and Rab10 pThr73 phospho-peptide (red). Each experiment was performed twice (individual data points shown).(F) Side by side comparison of the catalytic activity against protein and peptide by in vitro malachite green time course analysis. 50 nM recombinant wild-type PPM1H was incubated with 16 μM Rab8a GTPγS pThr72 phosphorylated protein (blue) or Rab8a pThr72 phosphorylated peptide (red) for indicated times and analysed as described in Materials and Methods. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates.


Figure 3 ( C ) His6 - SUMO - tagged full - length variants of PPM1H were used for catalytic assays ( left panel ) . 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie . Right , mass photometry histogram for 40nM His6 - SUMO - PPM1H WT ( blue ) and His6 - SUMO - PPM1H 2Glu ( brown ) , where WT is 130kDa ( ± 15 . 8 kDa , with 1004 single molecules counted ) and 2Glu is 83kDa ( ± 13 . 9 kDa , with 4117 single molecules counted ) . The calculated molecular weight of the fusion protein is approximately 68 . 5 kDa . ( D ) In vitro malachite green assay time course of recombinant His6 - SUMO - tagged PPM1H wild - type ( blue ) , 2Glu ( red ) , or D288A ( grey ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates .

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

Figure 3(C) His6-SUMO-tagged full-length variants of PPM1H were used for catalytic assays (left panel). 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie. Right, mass photometry histogram for 40nM His6-SUMO-PPM1H WT (blue) and His6-SUMO-PPM1H 2Glu (brown), where WT is 130kDa (±15.8 kDa, with 1004 single molecules counted) and 2Glu is 83kDa (±13.9 kDa, with 4117 single molecules counted). The calculated molecular weight of the fusion protein is approximately 68.5 kDa.(D) In vitro malachite green assay time course of recombinant His6-SUMO-tagged PPM1H wild-type (blue), 2Glu (red), or D288A (grey) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.


Figure 4 ( C ) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins ( 25 nM for the protein substrate , 50 nM for the peptide substrate ) against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . ( D ) In vitro malachite green assay against 32 μM pThr72 - Rab8a peptide ( left ) and pThr73 - Rab10 peptide mimic ( right ) . The experiments were repeated 3 times . The error bars represent standard error ( SE ) of mean of the technical replicates . ( E , F ) ( E ) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi - 2 for 90 min and then lysed . 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg / ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system . Left , each lane represents cell extract obtained from a different dish of cells ( two biological replicates per condition without MLi - 2 treatment , one biological replicate per condition with MLi - 2 treatment ) . Right , the ratio of phospho - Rab8a / total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild - type expressing cells . ( F ) As in ( E ) assessing phospho - Rab10 levels . For quantitation the two replicates from the presented blots were used . The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates .

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

Figure 4(C) In vitro malachite green assay time course to determine enzyme activity of recombinant PPM proteins (25 nM for the protein substrate, 50 nM for the peptide substrate) against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS). The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. (D) In vitro malachite green assay against 32 μM pThr72-Rab8a peptide (left) and pThr73-Rab10 peptide mimic (right). The experiments were repeated 3 times. The error bars represent standard error (SE) of mean of the technical replicates.(E, F) (E) HEK293 cells overexpressing indicated constructs were treated with ± 200 nM MLi-2 for 90 min and then lysed. 10 μg whole cell lysate was subjected to immunoblot analysis with the indicated antibodies at 1 μg/ml final concentration and membranes were analysed using the OdysseyClx Western Blot imaging system. Left, each lane represents cell extract obtained from a different dish of cells (two biological replicates per condition without MLi-2 treatment, one biological replicate per condition with MLi-2 treatment). Right, the ratio of phospho-Rab8a/total Rab8a was quantified using Image Studio software and data presented relative to the phosphorylation ratio observed in PPM1H wild-type expressing cells. (F) As in (E) assessing phospho-Rab10 levels. For quantitation the two replicates from the presented blots were used. The data points are shown on the graphs with the bar levels representing the mean value of the biological replicates.


Figure 5 ( B ) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS , left ) , 32 μM pThr72 phosphorylated Rab8a peptide ( middle ) , or 32 μM pThr73 phosphorylated Rab10 peptide ( right ) . The experiments were repeated 4 times . The error bars represent SE of mean of the technical replicates . ( C ) 3 μg of recombinant wild - type or indicated mutant PPM1H proteins were resolved on 4 - 12 % Bis - Tris gradient gel and stained with Instant Blue Coomassie . ( E ) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein ( GTPγS ) using the malachite green assay time course . The experiments were repeated 3 times . The error bars represent SE of mean of the technical replicates . Quality of purified proteins ( right panel ) is shown using the protocol in ( C ) . ( F ) HEK293 cells overexpressing indicated constructs were treated and analysed as described in ( D ) , with pRab10 analysed in the upper panel , and pRab8a in the lower panel .

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

Figure 5(B) In vitro malachite green assay time course of recombinant PPM1H mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS, left), 32 μM pThr72 phosphorylated Rab8a peptide (middle), or 32 μM pThr73 phosphorylated Rab10 peptide (right). The experiments were repeated 4 times. The error bars represent SE of mean of the technical replicates.(C) 3 μg of recombinant wild-type or indicated mutant PPM1H proteins were resolved on 4-12% Bis-Tris gradient gel and stained with Instant Blue Coomassie.(E) Activity of 25 nM PPM1H flap domain mutants against 16 μM pThr72 phosphorylated Rab8a protein (GTPγS) using the malachite green assay time course. The experiments were repeated 3 times. The error bars represent SE of mean of the technical replicates. Quality of purified proteins (right panel) is shown using the protocol in (C).(F) HEK293 cells overexpressing indicated constructs were treated and analysed as described in (D), with pRab10 analysed in the upper panel, and pRab8a in the lower panel.


Figure 6 ( B ) Incremental deletions of the N - terminus from residues 1 - 37 to 1 - 44 , one residue at a time . Upon deletion of R43 ( 44 - end ) , a reduced level of soluble PPM1H expression has no significant catalytic activity .

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Figure 6(B) Incremental deletions of the N-terminus from residues 1-37 to 1-44, one residue at a time. Upon deletion of R43 (44-end), a reduced level of soluble PPM1H expression has no significant catalytic activity.


Figure 1 ( B ) HEK293T cells were transfected with SFB control , SFB - tagged PR130 and SFB - B56δ , and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot ( WB ) . ( C ) Glutathione Sepharose beads bound with bacterially expressed recombinant GST , GST - B56δ or GST - PPP2R1A proteins were incubated with bacterially purified recombinant MBP - PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody . Interaction of B56δ - PPP2R1A was shown similarly using MBP - B56δ and GST - PPP2R1A proteins . Recombinant protein expression is shown by Coomassie staining . ( D ) HEK293T cells were transfected with SFB control , SFB - PPP2CA , SFB - PPP2CB , SFB - PPP2R1A , SFB - PPP2R1B and SFB - B56δ , and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody . ( E ) Cell lysates from HEK293T cells expressing SFB vector , SFB - B56α , SFB - B56β , SFB - B56γ , SFB - B56δ and SFB - B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody . ( G ) Cells expressing B56δ full length ( FL ) , Δ91 - 102 , L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting . ( H ) Various mutants of B56δ were transfected along with myc - PPP2R1A and pulled down using streptavidin beads and interaction with myc - PPP2R1A was detected by immunoblotting . ( I ) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio - layer interferometry . 6xHis - tagged PPM1G and PPP2R1A were immobilized on Ni - NTA biosensors and incubated with the GST - B56δ wild type ( WT ) or Δ91 - 102 mutant at various concentrations ( 100 - 400 nm ) . Curves represent experimental trace obtained from the BLI experiments . Binding affinities ( Kd ) ( + SD ; n = 3 independent experiments ) for B56δ - PPM1G and B56δ - PPP2R1A are shown .

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

Figure 1(B) HEK293T cells were transfected with SFB control, SFB-tagged PR130 and SFB-B56δ, and the cell lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot (WB).(C) Glutathione Sepharose beads bound with bacterially expressed recombinant GST, GST-B56δ or GST-PPP2R1A proteins were incubated with bacterially purified recombinant MBP-PPM1G and interaction of B56δ with PPM1G was detected by immunoblotting with MBP antibody. Interaction of B56δ-PPP2R1A was shown similarly using MBP- B56δ and GST-PPP2R1A proteins. Recombinant protein expression is shown by Coomassie staining.(D) HEK293T cells were transfected with SFB control, SFB- PPP2CA, SFB-PPP2CB, SFB-PPP2R1A, SFB-PPP2R1B and SFB- B56δ, and the lysates were pulled down using streptavidin beads and the interaction of endogenous PPM1G was detected by western blot using PPM1G antibody.(E) Cell lysates from HEK293T cells expressing SFB vector, SFB-B56α, SFB-B56β, SFB-B56γ, SFB-B56δ and SFB-B56ε were pulled down and interaction with PPM1G was detected by western blotting using PPM1G antibody.(G) Cells expressing B56δ full length (FL), Δ91-102, L183A and H282A mutants were pulled down with streptavidin beads and interaction with PPM1G was detected by western blotting.(H) Various mutants of B56δ were transfected along with myc-PPP2R1A and pulled down using streptavidin beads and interaction with myc-PPP2R1A was detected by immunoblotting.(I) Binding affinities of the B56δ with PPM1G and PPP2R1A subunit were determined by Bio-layer interferometry. 6xHis-tagged PPM1G and PPP2R1A were immobilized on Ni-NTA biosensors and incubated with the GST-B56δ wild type (WT) or Δ91-102 mutant at various concentrations (100-400 nm). Curves represent experimental trace obtained from the BLI experiments. Binding affinities (Kd) (+SD; n=3 independent experiments) for B56δ-PPM1G and B56δ-PPP2R1A are shown.


Figure 2 ( A ) MCF7 and MDA - MB - 231 cells transfected with MYC - B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization . Representative images from three independent experiments are shown , Scale bars 5 µm . ( B ) Quantification of cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell was plotted ( n = 35 for MCF - 7 cells ; n = 25 for MDA - MB - 231 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C ) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies . ( D ) Cells expressing either vector or SFB - B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively . ( E ) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting . Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively . ( F ) Localization of PPM1G in cells expressing either empty vector ( control ) , full length B56δ ( FL ) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies . Representative images from two independent experiments are shown , Scale bars 10 µm . ( G ) Cytoplasmic to nuclear ( Cyto / Nuc ) ratio of PPM1G intensity per each cell expressing either vector , B56δ full length ( FL ) or mutant versions was quantified and plotted ( n = 10 ) . Error bars represents SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

Figure 2(A) MCF7 and MDA-MB-231 cells transfected with MYC-B56δ were fixed and imaged using a confocal microscope after staining with antibodies against MYC tag and PPM1G to determine PPM1G localization. Representative images from three independent experiments are shown, Scale bars 5 µm. (B) Quantification of cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell was plotted (n=35 for MCF-7 cells; n=25 for MDA-MB-231 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C) Western blot showing expression levels of SFB tagged B56δ along with endogenous B56δ using in dicated antibodies.(D) Cells expressing either vector or SFB-B56δ were fractionated into nuclear and cytoplasmic fractions and levels of indicated proteins were determined using western blotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.(E) Cells treated with control or B56δ shRNA were fractionated into cytoplasmic and nuclear fractions and levels of indicated proteins were determined using immunoblotting. Tubulin and Histone H3 were used to mark cytoplasmic and nuclear fractions respectively.(F) Localization of PPM1G in cells expressing either empty vector (control), full length B56δ (FL) or various indicated mutants was determined by confocal imaging after staining with Flag and Myc antibodies. Representative images from two independent experiments are shown, Scale bars 10 µm. (G) Cytoplasmic to nuclear (Cyto/Nuc) ratio of PPM1G intensity per each cell expressing either vector, B56δ full length (FL) or mutant versions was quantified and plotted (n=10). Error bars represents SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 3 ( A ) Endogenous association of PPM1G with α - Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α - Catenin antibody . ( B ) SFB - PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads . Interaction with α - catenin was determined by western blotting . ( C ) In vitro phosphorylated MBP - tagged α - Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant ( D496A ) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent ( A620 nm ) . Data represents mean absorbance from three independent experiments . Error bars indicate SD , * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( D ) In vitro phosphate release assay was performed using wild type α - Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted , n = 3 , Error bars indicate SD ; * * p < 0 . 01 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( E ) MDA - MB - 231 breast cancer cells were transfected with vector , wild type PPM1G or catalytically inactive PPM1G D496A ( D / A ) mutant . Levels of phosphorylated α - catenin were detected by using phospho - specific antibodies . ( F ) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies . ( G ) Cells transfected with vector , SFB - PR130 and SFB - B56δ and levels of pS641 α - catenin was detected using western blotting . ( H ) 293T Cells transfected with either empty vector , full length B56δ or indicated mutants were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody . ( I ) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α - catenin was determined using pS641 α - catenin specific antibody . Cells were transduced with shRNAs as indicated and the effect of ( J ) B56δ expression on α - Catenin phosphorylation was assessed . , K ) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α - Catenin phosphorylation was assessed . ( L ) SFB - PPM1G and SFB - PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells . Lysates were pulled down using streptavidin beads and interaction with α - catenin was detected by western blotting using α - catenin antibody . ( M ) Control shRNA and B56δ shRNA cells were transfected with vector , MYC - PPM1G and MYC - PPM1G ΔNLS in indicated combination and pS641 α - catenin levels were determined using western blotting .

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

Figure 3(A) Endogenous association of PPM1G with α-Catenin was detected by immunoblotting with PPM1G antibody after performing immunoprecipitation with either IgG or α-Catenin antibody.(B) SFB-PPM1G was transfected in both Control shRNA and B56δ shRNA cells and pulled down using streptavidin beads. Interaction with α-catenin was determined by western blotting.(C) In vitro phosphorylated MBP-tagged α-Catenin was incubated with equal amounts of bacterially purified recombinant wild type and catalytically inactive mutant (D496A) of PPM1G and the amount of released phosphate was assayed colorimetrically using the malachite green reagent (A620 nm). Data represents mean absorbance from three independent experiments. Error bars indicate SD, **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(D) In vitro phosphate release assay was performed using wild type α-Catenin and S641A mutant as substrates and PPM1G activity on these proteins is plotted, n=3, Error bars indicate SD; **p<0.01 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(E) MDA-MB-231 breast cancer cells were transfected with vector, wild type PPM1G or catalytically inactive PPM1G D496A (D/A) mutant. Levels of phosphorylated α-catenin were detected by using phospho-specific antibodies.(F) Cells treated with control shRNA and PPM1G specific shRNAs were lysed and blotted against specific antibodies.(G) Cells transfected with vector, SFB-PR130 and SFB-B56δ and levels of pS641 α-catenin was detected using western blotting.(H) 293T Cells transfected with either empty vector, full length B56δ or indicated mutants were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.(I) Cells treated with control shRNA and B56δ specific shRNAs were lysed and the levels of phosphorylated α-catenin was determined using pS641 α-catenin specific antibody.Cells were transduced with shRNAs as indicated and the effect of (J) B56δ expression on α-Catenin phosphorylation was assessed., K) Cells were transduced with shRNAs as indicated and the effect of PPM1G expression on α-Catenin phosphorylation was assessed.(L) SFB-PPM1G and SFB-PPM1G ΔNLS were transfected in both control shRNA and B56δ shRNA transduced cells. Lysates were pulled down using streptavidin beads and interaction with α-catenin was detected by western blotting using α-catenin antibody.(M) Control shRNA and B56δ shRNA cells were transfected with vector, MYC-PPM1G and MYC-PPM1G ΔNLS in indicated combination and pS641 α-catenin levels were determined using western blotting.


Figure 4 ( A ) Cells transduced with the indicated shRNAs were fixed and localization of α - catenin and β - catenin was determined by imaging with a confocal microscope after staining with antibodies against α - catenin and β - catenin . Representative images from three independent experiments are shown . Scale bars , 10 μm . ( B ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4A was plotted ( n = 40 cells ) . Error bars indicate SD . * * * p < 0 . 001 , ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C ) Control , PPM1G and B56δ shRNA transduced cells were transfected with SFB - α - catenin . Cell lysates were pulled down using streptavidin beads and interaction with β - catenin was determined by western blotting . ( D ) F - Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope . Scale bars , 20 μm . E ) Localization of α - catenin and β - catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB - PPM1G and SFB - PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies . Representative images are shown , Scale bars , 10 μm . ( F ) Quantification of plasma membrane ( PM ) to cytoplasmic ratio of α - catenin intensity per each cell from figure 4F was plotted ( n = 30 cells ) . Error bars indicate SD . * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

Figure 4(A) Cells transduced with the indicated shRNAs were fixed and localization of α-catenin and β-catenin was determined by imaging with a confocal microscope after staining with antibodies against α-catenin and β-catenin. Representative images from three independent experiments are shown. Scale bars, 10 μm. (B) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4A was plotted (n=40 cells). Error bars indicate SD. ***p < 0.001, (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C) Control, PPM1G and B56δ shRNA transduced cells were transfected with SFB-α-catenin. Cell lysates were pulled down using streptavidin beads and interaction with β-catenin was determined by western blotting.(D) F-Actin in control and PPM1G depleted cells was stained using rhodamine labelled Phalloidin and the cytoskeleton arrangement was detected by using confocal microscope. Scale bars, 20 μm.E) Localization of α-catenin and β-catenin in control shRNA cells or B56δ depleted cells with or without expression of SFB-PPM1G and SFB-PPM1G ΔNLS was detected by imaging with confocal microscope after staining with specific antibodies. Representative images are shown, Scale bars, 10 μm. (F) Quantification of plasma membrane (PM) to cytoplasmic ratio of α-catenin intensity per each cell from figure 4F was plotted (n=30 cells). Error bars indicate SD. ***p < 0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 5 ( A , B ) MDA - MB - 231 cells were transfected with vector control , PPM1G wild type , catalytically inactive PPM1G D496A mutant or α - Catenin and transwell migration assay was performed . Phase contrast microscopic images ( A ) were taken after staining the migrated cells with crystal violet and ( B ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( C , D ) MCF - 7 cells were transfected with SFB - vector , SFB - B56δ or indicated mutants and transwell migration assay was performed . Phase contrast microscopic images ( C ) were taken after staining the migrated cells with crystal violet and ( D ) Quantification data for migrated cells per field ( derived from average of 3 different fields ) from three independent experiments is shown . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( E , F ) SFB - B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells . Representative images from transwell migration assay ( E ) are shown and ( F ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( G , H ) SFB - PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells . Representative images from transwell migration assay ( G ) are shown and ( H ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( I , J ) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC - PPM1G and MYC - PPM1G ΔNLS . Representative images from transwell migration assay ( I ) performed with these cells are shown and ( J ) quantified data for migrated cells from three independent experiments per field was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) . ( K , L ) SFB - tagged α - catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown . Representative images from transwell migration assay ( K ) performed with these cells are shown and ( L ) quantified data for migrated cells per field from three independent experiments was plotted . Error bars indicate standard deviation ( n = 3 ) , * * * p < 0 . 001 ( one - way ANOVA , post hoc test : Bonferroni ' s multiple comparison test ) .

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

Figure 5(A, B) MDA-MB-231 cells were transfected with vector control, PPM1G wild type, catalytically inactive PPM1G D496A mutant or α-Catenin and transwell migration assay was performed. Phase contrast microscopic images (A) were taken after staining the migrated cells with crystal violet and (B) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(C, D) MCF-7 cells were transfected with SFB-vector, SFB-B56δ or indicated mutants and transwell migration assay was performed. Phase contrast microscopic images (C) were taken after staining the migrated cells with crystal violet and (D) Quantification data for migrated cells per field (derived from average of 3 different fields) from three independent experiments is shown. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(E, F) SFB-B56δ was expressed in control shRNA and PPM1G shRNA transduced MCF7 cells. Representative images from transwell migration assay (E) are shown and (F) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(G, H) SFB-PPM1G was expressed in control shRNA and B56δ shRNA transduced MCF7 cells. Representative images from transwell migration assay (G) are shown and (H) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(I, J) Control shRNA and B56δ shRNA transduced MCF7 cells were transfected with MYC-PPM1G and MYC-PPM1G ΔNLS. Representative images from transwell migration assay (I) performed with these cells are shown and (J) quantified data for migrated cells from three independent experiments per field was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).(K, L) SFB-tagged α-catenin S641A and S641D mutants were expressed in PPM1G shRNA and B56δ shRNA transduced MCF7 cells as shown. Representative images from transwell migration assay (K) performed with these cells are shown and (L) quantified data for migrated cells per field from three independent experiments was plotted. Error bars indicate standard deviation (n=3), ***p<0.001 (one-way ANOVA, post hoc test: Bonferroni's multiple comparison test).


Figure 1 ( B ) Hoechst ( DNA ) staining and IF for Tbr2 to calculate the thickness ( white bars ) of the VZ , SVZ and intermediate zone + cortical plate ( IZ + CP ) in control and DKO E13 . 5 neocortex . Scale bar 20 μm . ( C ) Quantification of VZ , SVZ and IZ + CP thickness in control and DKO E13 . 5 neocortex . Each zone is represented as percentage of total thickness ( measured in µm ) of the neocortex . Columns are means ± SEM ( n = 6 embryos , 4 litters ) . ( ( D ) IF for Pax6 and Tbr2 ( not shown ) in control and DKO E13 . 5 neocortex . White bars indicate thickness of Pax6 + zone ( i . e . VZ ) . Scale bar 20 μm . ( E ) Quantification of the number of APs ( Pax6 + Tbr2 - ) from ( D ) . Pax6 + Tbr2 + cells were excluded from final counts . AP numbers are expressed as percentage of total cell number ( sum of D , F and H ) . Columns are means ± SEM ( n = 4 embryos , 3 litters ) . ( F ) IF for Tbr2 in control and DKO E13 . 5 neocortex . White bars indicate thickness of Tbr2 + zone ( i . e . SVZ ) . Scale bar 20 μm . ( G ) Quantification of the number of BPs from ( F ) . Columns are means ± SEM ( n = 6 embryos , 3 litters ) . ( H ) IF for Tbr1 in control and DKO E13 . 5 neocortex . White bars indicate thickness of Tbr1 + zone ( i . e . mostly CP ) . Scale bar 15 μm . ( I ) Quantification the number of deep - layer post - mitotic neurons from ( H ) . Columns are means ± SEM ( n = 3 embryos , 3 litters ) . ( J ) IF for Ccny ( a ) , Ccnyl1 ( b ) and LRP6 ( c ) ( low - density lipoprotein receptor - related protein 6 ) phosphorylated at T1479 ( pLRP6 ) in the neocortex of E13 . 5 control embryos . White arrowheads in ( a , b ) indicate apical membrane / cortex , shown at higher magnification in the insets . High levels of LRP6 phosphorylation are detected in mitotic APs ( c , white arrowheads ) . Scale bar 50 μm . ( K ) IF for Ccny ( a ) , Ccnyl1 ( b ) and pLRP6 ( c ) , each together with IF for Tbr2 , in the neocortex of E12 . 5 control embryos . White arrowheads in the insets of ( a , b ) depict Ccny / l1 puncta in Tbr2 + BPs . High levels of LRP6 phosphorylation are detected in mitotic BPs ( c , inset , white arrowhead ) . DNA staining for mitotic pLRP6 + BP shown in bottom right inset ( c , green arrowhead ) . Scale bar 20 μm . ( L ) IF for pLRP6 ( T1479 ) in the neocortex of E13 . 5 control and DKO embryos . White arrowheads depict mitotic APs and purple arrowheads depict mitotic BPs . Scale bar 20 μm . ( M ) Quantification of relative pLRP6 staining intensity in mitotic APs and BPs from ( L ) . Data are expressed as fold change vs . controls and are means ± SEM ( n = 3 embryos , 3 litters ) . ( O ) Expression analysis of the β - catenin target gene Axin2 by RNAScope in situ hybridization on neocortex sections from E13 . 5 control and DKO embryos . Scale bar 20 μm . ( P ) Quantification of ( O ) . Axin2 + area was calculated and normalized to total neocortex area . Data are expressed as fold change vs . controls and are means ± SEM ( n = 4 embryos , 3 litters ) .

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

Figure 1(B) Hoechst (DNA) staining and IF for Tbr2 to calculate the thickness (white bars) of the VZ, SVZ and intermediate zone + cortical plate (IZ+CP) in control and DKO E13.5 neocortex. Scale bar 20 μm. (C) Quantification of VZ, SVZ and IZ+CP thickness in control and DKO E13.5 neocortex. Each zone is represented as percentage of total thickness (measured in µm) of the neocortex. Columns are means ± SEM (n=6 embryos, 4 litters). ( (D) IF for Pax6 and Tbr2 (not shown) in control and DKO E13.5 neocortex. White bars indicate thickness of Pax6+ zone (i.e. VZ). Scale bar 20 μm. (E) Quantification of the number of APs (Pax6+Tbr2-) from (D). Pax6+Tbr2+ cells were excluded from final counts. AP numbers are expressed as percentage of total cell number (sum of D, F and H). Columns are means ± SEM (n=4 embryos, 3 litters). (F) IF for Tbr2 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr2+ zone (i.e. SVZ). Scale bar 20 μm. (G) Quantification of the number of BPs from (F). Columns are means ± SEM (n=6 embryos, 3 litters). (H) IF for Tbr1 in control and DKO E13.5 neocortex. White bars indicate thickness of Tbr1+ zone (i.e. mostly CP). Scale bar 15 μm. (I) Quantification the number of deep-layer post-mitotic neurons from (H). Columns are means ± SEM (n=3 embryos, 3 litters).(J) IF for Ccny (a), Ccnyl1 (b) and LRP6 (c) (low-density lipoprotein receptor-related protein 6) phosphorylated at T1479 (pLRP6) in the neocortex of E13.5 control embryos. White arrowheads in (a,b) indicate apical membrane / cortex, shown at higher magnification in the insets. High levels of LRP6 phosphorylation are detected in mitotic APs (c, white arrowheads). Scale bar 50 μm. (K) IF for Ccny(a), Ccnyl1(b) and pLRP6(c), each together with IF for Tbr2, in the neocortex of E12.5 control embryos. White arrowheads in the insets of (a, b) depict Ccny/l1 puncta in Tbr2+ BPs. High levels of LRP6 phosphorylation are detected in mitotic BPs (c, inset, white arrowhead). DNA staining for mitotic pLRP6+ BP shown in bottom right inset (c, green arrowhead). Scale bar 20 μm.(L) IF for pLRP6 (T1479) in the neocortex of E13.5 control and DKO embryos. White arrowheads depict mitotic APs and purple arrowheads depict mitotic BPs. Scale bar 20 μm. (M) Quantification of relative pLRP6 staining intensity in mitotic APs and BPs from (L). Data are expressed as fold change vs. controls and are means ± SEM (n=3 embryos, 3 litters).(O) Expression analysis of the β-catenin target gene Axin2 by RNAScope in situ hybridization on neocortex sections from E13.5 control and DKO embryos. Scale bar 20 μm. (P) Quantification of (O). Axin2+ area was calculated and normalized to total neocortex area. Data are expressed as fold change vs. controls and are means ± SEM (n=4 embryos, 3 litters).


Figure 2 ( A ) Triple - IF for BrdU , Pax6 and Tbr2 ( not shown ) in E12 . 5 control and DKO neocortices . BrdU was injected into pregnant dams one hour before sacrifice . ( B ) Quantification of the proportion of Pax6 + Tbr2 - cells that are BrdU + , in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are expressed as fold change vs . controls , with the control values set to 1 . 0 , and are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 5 embryos , 5 litters and E13 . 5 n = 5 embryos , 4 litters ) . ( C ) Average percentage of APs ( Pax6 + Tbr2 - ) ( from B ) that are BrdU + , in E13 . 5 control and DKO neocortices . Data are means ± SEM . ( D ) Co - IF for Tbr2 and BrdU ( double positive cells depicted by white arrowheads ) in E12 . 5 control and DKO neocortices . BrdU was injected into pregnant dams one hour before sacrifice . ( E ) Quantification of the proportion of Tbr2 + cells that are BrdU + , in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are expressed as fold change vs controls , with the control values set to 1 . 0 , and are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 5 embryos , 5 litters and E13 . 5 n = 5 embryos , 4 litters ) . ( F ) Average percentage of BPs ( Tbr2 + ) ( from E ) that are BrdU + , in E13 . 5 control and DKO neocortices . Data are means ± SEM . ( K ) Co - IF for the proliferation marker Ki67 , the mitotic marker phospho - histone H3 ( pHH3 ) , and Tbr2 to identify mitotic APs ( division at apical surface ) and BPs ( division in basal VZ or SVZ ) in control and DKO E13 . 5 neocortices . Arrowheads depict pHH3 + Tbr2 + cells . ( L ) Average percentage of Ki67 + APs and BPs that are pHH3 + in control and DKO E13 . 5 neocortices . Data are means ± SEM ( n = 4 embryos , 3 litters ) . ( N ) IF for cleaved caspase 3 ( Casp3 ) to identify apoptotic cells in E13 . 5 control and DKO neocortices . ( O ) Quantification of apoptotic cells in control and DKO neocortices at E11 . 5 , E12 . 5 and E13 . 5 . Data are represented as number of Casp3 + cells per 5000 cells . Data are means ± SEM ( E11 . 5 n = 4 embryos , 3 litters ; E12 . 5 n = 3 embryos , 3 litters , and E13 . 5 n = 4 embryos , 3 litters ) .

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

Figure 2(A) Triple-IF for BrdU, Pax6 and Tbr2 (not shown) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (B) Quantification of the proportion of Pax6+Tbr2- cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs. controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (C) Average percentage of APs (Pax6+Tbr2-) (from B) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM. (D) Co-IF for Tbr2 and BrdU (double positive cells depicted by white arrowheads) in E12.5 control and DKO neocortices. BrdU was injected into pregnant dams one hour before sacrifice. (E) Quantification of the proportion of Tbr2+ cells that are BrdU+, in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are expressed as fold change vs controls, with the control values set to 1.0, and are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=5 embryos, 5 litters and E13.5 n=5 embryos, 4 litters). (F) Average percentage of BPs (Tbr2+) (from E) that are BrdU+, in E13.5 control and DKO neocortices. Data are means ± SEM.(K) Co-IF for the proliferation marker Ki67, the mitotic marker phospho-histone H3 (pHH3), and Tbr2 to identify mitotic APs (division at apical surface) and BPs (division in basal VZ or SVZ) in control and DKO E13.5 neocortices. Arrowheads depict pHH3+Tbr2+ cells. (L) Average percentage of Ki67+ APs and BPs that are pHH3+ in control and DKO E13.5 neocortices. Data are means ± SEM (n=4 embryos, 3 litters).(N) IF for cleaved caspase 3 (Casp3) to identify apoptotic cells in E13.5 control and DKO neocortices. (O) Quantification of apoptotic cells in control and DKO neocortices at E11.5, E12.5 and E13.5. Data are represented as number of Casp3+ cells per 5000 cells. Data are means ± SEM (E11.5 n=4 embryos, 3 litters; E12.5 n=3 embryos, 3 litters, and E13.5 n=4 embryos, 3 litters).


Figure 3 ( C ) IF for alpha - tubulin to visualize mitotic spindle and astral microtubules in E13 . 5 control and DKO neocortices . Central and apical - basal astral microtubules denoted by white and purple arrows , respectively . Scale bar 2 μm . ( D ) Quantification of apical - basal , central and total ( sum ) number of astral microtubules in mitotic APs of control and DKO E13 . 5 neocortices . Data are means of microtubules per cell ± SEM ( at least 10 cells counted per embryo ) ( n = 3 embryos , 3 litters ) . ( E , F ) In utero electroporation ( IUE ) of plasmids expressing GFP plus shControl ( Co ) or shCcny / l1 into the lateral ventricles of E13 . 5 wildtype embryos , followed by analysis at E15 . 5 or E17 . 5 . ( E ) IF for GFP and Tbr2 in the E15 . 5 neocortex . ( F ) IF for GFP and the deep - layer neuron marker CTIP2 in the E17 . 5 neocortex . ( E , F ) White arrowheads denote double - positive cells . Insets of magnified cells ( indicated by blue arrowheads ) depict representative GFP + Tbr2 ( E ) or GFP + Ctip2 ( F ) co - staining . Scale bars 20 μm . ( G ) Quantification of the percentage of GFP + cells that are Tbr2 + , in control and shCcny / l1 neocortices at E15 . 5 upon IUE at E13 . 5 . Data are means ± SEM ( n = 3 embryos ) . ( H ) Quantification of the percentage of GFP + cells that are Ctip2 + , in control and shCcny / l1 neocortices at E17 . 5 upon IUE at E13 . 5 . Data are means ± SEM ( n = 4 embryos ) . ( I ) IF for βIII - tubulin ( Tuj1 ) of control ( Ccny + / - ; Ccnyl1 - / - ; shRNA Co ) and mutant ( Ccny + / - ; Ccnyl1 - / - ; shRNA Ccny ) NPC monolayer cultures grown in differentiation medium for seven days . Insets depict neurons with representative neurite morphology for controls and mutants . Scale bar 20 μm . ( J ) Quantification of the percentage of Tuj1 + cells from ( I ) . Data are means ± SEM ( experiment performed twice in triplicates , representative experiment shown ) .

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

Figure 3(C) IF for alpha-tubulin to visualize mitotic spindle and astral microtubules in E13.5 control and DKO neocortices. Central and apical-basal astral microtubules denoted by white and purple arrows, respectively. Scale bar 2 μm. (D) Quantification of apical-basal, central and total (sum) number of astral microtubules in mitotic APs of control and DKO E13.5 neocortices. Data are means of microtubules per cell ± SEM (at least 10 cells counted per embryo)(n=3 embryos, 3 litters).(E, F) In utero electroporation (IUE) of plasmids expressing GFP plus shControl (Co) or shCcny/l1 into the lateral ventricles of E13.5 wildtype embryos, followed by analysis at E15.5 or E17.5. (E) IF for GFP and Tbr2 in the E15.5 neocortex. (F) IF for GFP and the deep- layer neuron marker CTIP2 in the E17.5 neocortex. (E, F) White arrowheads denote double-positive cells. Insets of magnified cells (indicated by blue arrowheads) depict representative GFP + Tbr2 (E) or GFP + Ctip2 (F) co-staining. Scale bars 20 μm. (G) Quantification of the percentage of GFP+ cells that are Tbr2+, in control and shCcny/l1 neocortices at E15.5 upon IUE at E13.5. Data are means ± SEM (n=3 embryos). (H) Quantification of the percentage of GFP+ cells that are Ctip2+, in control and shCcny/l1 neocortices at E17.5 upon IUE at E13.5. Data are means ± SEM (n=4 embryos).(I) IF for βIII-tubulin (Tuj1) of control (Ccny+/-; Ccnyl1-/-; shRNA Co) and mutant (Ccny+/-; Ccnyl1-/-; shRNA Ccny) NPC monolayer cultures grown in differentiation medium for seven days. Insets depict neurons with representative neurite morphology for controls and mutants. Scale bar 20 μm. (J) Quantification of the percentage of Tuj1+ cells from (I). Data are means ± SEM (experiment performed twice in triplicates, representative experiment shown).