Figure 2Thioperamide treatment of tissue culture cells . BHK , CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C . Low magnification views are shown after staining with anti - LBPA antibodies ( green ) and DAPI ( blue ) . n = 3 independent experiments with > 200 images acquired and analysed automatically . Cholesterol quantification by mass spectrometry . A431 cells were treated with DMSO alone , pitolisant 10µM ( Pito ) , thioperamide 10µM ( thio ) or U18666A 10µM ( U18 ) for 18h at 37 ° C . After extraction , free cholesterol , cholesteryl esters and total cholesterol were quantified by mass spectrometry , and normalized to the DMSO controls . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * * * * = p < 0 . 0001Screen data plot of the cholesterol content of LBPA - endosomes . Automated unbiased quantification of the filipin integrated fluorescence signal ( cholesterol content ) in LBPA - containing endosomes , after treatment with each compound of the Prestwick library . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The fluorescence signal of LBPA - endosomes was used to segment the imaged and generate a mask , which was then applied on the micrographs to quantify the integrated intensity of filipin staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated .

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Figure 2Thioperamide treatment of tissue culture cells. BHK, CHO and HeLa cells were treated with thioperamide and processed as in Fig 1C. Low magnification views are shown after staining with anti-LBPA antibodies (green) and DAPI (blue). n=3 independent experiments with > 200 images acquired and analysed automatically.Cholesterol quantification by mass spectrometry. A431 cells were treated with DMSO alone, pitolisant 10µM (Pito), thioperamide 10µM (thio) or U18666A 10µM (U18) for 18h at 37°C. After extraction, free cholesterol, cholesteryl esters and total cholesterol were quantified by mass spectrometry, and normalized to the DMSO controls. (n=3 independent experiments, error bars = SD, one-way ANOVA, ****=p<0.0001Screen data plot of the cholesterol content of LBPA-endosomes. Automated unbiased quantification of the filipin integrated fluorescence signal (cholesterol content) in LBPA-containing endosomes, after treatment with each compound of the Prestwick library. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The fluorescence signal of LBPA-endosomes was used to segment the imaged and generate a mask, which was then applied on the micrographs to quantify the integrated intensity of filipin staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated.


Figure 3Immunogold labelling with anti - LBPA antibodies . HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy . Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A - gold . Multivesicular endosomes are pseudocoloured ( see Fig EV1 for uncoloured images ) . Bars , 200nm . The data in ( A ) were quantified in a double - blind analysis of two sets of 16 micrographs for each condition ; the number of gold particles per endosome is shown in a scatter plot ( B ) for each endosome identified without any bias in each micrograph of the two control ( DMSO ) and thioperamide - treated ( THIO ) samples . Distribution of LBPA - containing endosomes in the perinuclear region . Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells , after treatment with each compound of the Prestwick library as in Fig 2C . As in Fig 1D , each dot is the average of replicates in the duplicate plates , and ≥ 600 cells were analysed per compound - only compounds that showed less than 20 % toxicity are plotted . The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell , calculated from the nuclei DAPI - staining . The samples treated with thioperamide ( pink ) , trimeprazine ( light blue ) , DMSO ( red ) and U18666A ( green ) are indicated . Number of perinuclear LBPA endosomes . The number of individual LBPA - positive structures was measured in the perinuclear region as in ( C ) . The z - factors are shown to evaluate the distribution of endosomes containing LBPA ; color code as in ( C ) .

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Figure 3Immunogold labelling with anti-LBPA antibodies. HeLa MZ cells were treated or not with thioperamide for 18h and processed for electron microscopy. Cryosections were labelled with antibodies against the late endosomal lipid LBPA followed by 5nm protein A-gold. Multivesicular endosomes are pseudocoloured (see Fig EV1 for uncoloured images). Bars, 200nm. The data in (A) were quantified in a double-blind analysis of two sets of 16 micrographs for each condition; the number of gold particles per endosome is shown in a scatter plot (B) for each endosome identified without any bias in each micrograph of the two control (DMSO) and thioperamide-treated (THIO) samples.Distribution of LBPA-containing endosomes in the perinuclear region. Automated unbiased quantification of LBPA fluorescence in the perinuclear region of the cells, after treatment with each compound of the Prestwick library as in Fig 2C. As in Fig 1D, each dot is the average of replicates in the duplicate plates, and ≥600 cells were analysed per compound - only compounds that showed less than 20% toxicity are plotted. The integrated intensity of the LBPA fluorescence signal was measured within a mask of the perinuclear region in each cell, calculated from the nuclei DAPI-staining. The samples treated with thioperamide (pink), trimeprazine (light blue), DMSO (red) and U18666A (green) are indicated. Number of perinuclear LBPA endosomes. The number of individual LBPA-positive structures was measured in the perinuclear region as in (C). The z-factors are shown to evaluate the distribution of endosomes containing LBPA; color code as in (C).


Figure 4Effect of Pitolisant on LBPA . HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37 ° C were labeled with DAPI and anti - LBPA antibodies and analysed by fluorescence microscopy ( quantification in fig EV3A ) . LBPA intensity in a mixed population of cells expressing HRH3 - GFP . After transfection with HRH3 - GFP , uncloned stably expressing cells ( C ) were labelled with anti - LBPA antibodies and analysed by automated microscopy ( C ) . Unbiased quantification ( D ) shows the inverse correlation between HRH3 - GFP expression ( high expressing cells in green ) and the endosome integrated intensity of LBPA staining ( high LBPA labelling in red ) . ( n = 2 independent experiments with 500 , 000 cells analysed automatically , error bars = SD ) . LBPA intensity in cells expressing or not HRH3 - GFP . HeLa MZ cells stably expressing HRH3 - GFP grown in 96 - well plates were separately treated , with 4 different siRNAs that target HRH3 ( siHRH3 # 1 ; siHRH3 # 2 ; siHRH3 # 3 ; siHRH3 # 4 ) or with control non - target ( siNT ) siRNA ( E ) . HRH3 - GFP was analysed by fluorescence microscopy in 4 rows of cells per condition ; micrographs are stitched together in the montage . Panel F shows an example of cells treated with siHRH3 # 2 or siNT , and then labelled with DAPI and antibodies against LBPA . Cells treated as in ( E ) and labelled with DAPI and antibodies against LBPA as in ( F ) , were analysed by automated microscopy and the integrated intensities of LBPA ( left panel ) and HRH3 - GFP ( right panel ) signals are compared ( G ) . ( n = 2 independent experiments with 192 images acquired and analysed automatically , error bars = SD ) .

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Figure 4Effect of Pitolisant on LBPA. HeLa MZ cells treated or not with pitolisant 10µM for 18h at 37°C were labeled with DAPI and anti-LBPA antibodies and analysed by fluorescence microscopy (quantification in fig EV3A).LBPA intensity in a mixed population of cells expressing HRH3-GFP. After transfection with HRH3-GFP, uncloned stably expressing cells (C) were labelled with anti-LBPA antibodies and analysed by automated microscopy (C). Unbiased quantification (D) shows the inverse correlation between HRH3-GFP expression (high expressing cells in green) and the endosome integrated intensity of LBPA staining (high LBPA labelling in red). (n=2 independent experiments with 500,000 cells analysed automatically, error bars = SD).LBPA intensity in cells expressing or not HRH3-GFP. HeLa MZ cells stably expressing HRH3-GFP grown in 96-well plates were separately treated, with 4 different siRNAs that target HRH3 (siHRH3#1; siHRH3#2; siHRH3#3; siHRH3#4) or with control non-target (siNT) siRNA (E). HRH3-GFP was analysed by fluorescence microscopy in 4 rows of cells per condition; micrographs are stitched together in the montage. Panel F shows an example of cells treated with siHRH3#2 or siNT, and then labelled with DAPI and antibodies against LBPA. Cells treated as in (E) and labelled with DAPI and antibodies against LBPA as in (F), were analysed by automated microscopy and the integrated intensities of LBPA (left panel) and HRH3-GFP (right panel) signals are compared (G). (n=2 independent experiments with 192 images acquired and analysed automatically, error bars = SD).


Figure 5Treatment of NPC fibroblasts and NPC null mice with thioperamide . Fibroblast lines obtained from patients with well - established heterozygote mutations in the NPC1 ( GM17912 line : NPC1 P1007A / T1036M ; GM17911 line : NPC1 I1061T / T1036M ) or with a homozygote mutation in the NPC2 gene ( GM17910 line : C93F / C93F ) were treated or not for 72h with thioperamide 10µM , stained with filipin ( cholesterol ) and analysed by fluorescence automated microscopy . Quantification of cholesterol and LBPA staining in NPC fibroblasts . Cells as in ( A ) were treated or not with thioperamide for 48h or 72h , labelled with filipin ( cholesterol ) and anti - LBPA antibodies , and analysed by fluorescence automated microscopy . The panels show the integrated intensity of the filipin ( left ) and LBPA ( right ) signals were quantified after 48 ( top ) and 72h ( bottom ) . The colour code of each fibroblast cell line is as in ( A ) . ( n = 3 independent experiments with 144 images acquired and analysed automatically , > 2000 cells per experiment , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ; * * * = p < 0 . 001 ) . Quantification of cholesterol in NPC fibroblasts by mass spectrometry . NPC cell lines ( A ) were treated or not with thioperamide or 0 . 1 % ( 2 - Hydroxypropyl ) - ß - cyclodextrin for 72h . After extraction , lipids were analysed and quantified by mass spectrometry . ( n = 3 ; error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ; * * * = p < 0 . 001 ) Quantification of cell counts in the 3 NPC cell lines treated with thioperamide . The experiment was as in ( A - C ) except that nuclei were labelled with DAPI and quantified by automated microscopy . Data are normalized to DMSO control ( n = 2 independent experiments , 75 images per experiments )

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 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 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 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

Figure 5Treatment of NPC fibroblasts and NPC null mice with thioperamide. Fibroblast lines obtained from patients with well-established heterozygote mutations in the NPC1 (GM17912 line: NPC1 P1007A / T1036M; GM17911 line: NPC1 I1061T / T1036M) or with a homozygote mutation in the NPC2 gene (GM17910 line: C93F / C93F) were treated or not for 72h with thioperamide 10µM, stained with filipin (cholesterol) and analysed by fluorescence automated microscopy.Quantification of cholesterol and LBPA staining in NPC fibroblasts. Cells as in (A) were treated or not with thioperamide for 48h or 72h, labelled with filipin (cholesterol) and anti-LBPA antibodies, and analysed by fluorescence automated microscopy. The panels show the integrated intensity of the filipin (left) and LBPA (right) signals were quantified after 48 (top) and 72h (bottom). The colour code of each fibroblast cell line is as in (A). (n=3 independent experiments with 144 images acquired and analysed automatically, >2000 cells per experiment, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001).Quantification of cholesterol in NPC fibroblasts by mass spectrometry. NPC cell lines (A) were treated or not with thioperamide or 0.1% (2-Hydroxypropyl)-ß-cyclodextrin for 72h. After extraction, lipids were analysed and quantified by mass spectrometry. (n=3; error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005;***=p<0.001)Quantification of cell counts in the 3 NPC cell lines treated with thioperamide. The experiment was as in (A-C) except that nuclei were labelled with DAPI and quantified by automated microscopy. Data are normalized to DMSO control (n=2 independent experiments, 75 images per experiments)


Figure 6Effects of thioperamide on cholesterol - dependent transcriptional regulation . The parental HeLa MZ cells , NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h . Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT - PCR . ( n = 3 independent experiments , error bars = SD , one - way ANOVA , * = p < 0 . 05 ; * * = p < 0 . 005 ) Quantification of cholesterol in NPC null mice by enzymatic analysis . WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed . The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue . The total content of LBPA in the same liver extracts was quantified by mass spectrometry ( see Appendix Figure S1 ) , and is therefore expressed as a percentage of total phospholipids . The total LBPA content of WT mice was only marginally increased by the drug , either because the doses were low , or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level ( despite much effort , we were unable to visualize LBPA in liver samples by immunocytochemistry ) . The relative ratio of LBPA vs . cholesterol values is shown .

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Figure 6Effects of thioperamide on cholesterol-dependent transcriptional regulation. The parental HeLa MZ cells, NPC1 KO cells or NPC2 KO cells were treated or not with thioperamide 10 μM for 18h. Total mRNA was extracted and both LDLR mRNA and HMG CoA reductase mRNA were quantified by RT-PCR. (n=3 independent experiments, error bars = SD, one-way ANOVA, *=p<0.05; **=p<0.005)Quantification of cholesterol in NPC null mice by enzymatic analysis. WT or NPC1 null mice were treated or not with thioperamide for 6 weeks after weaning and sacrificed. The total content of unesterified cholesterol in the corresponding liver extracts was then quantified using an enzymatic assay and is expressed in nmol per mg tissue. The total content of LBPA in the same liver extracts was quantified by mass spectrometry (see Appendix Figure S1), and is therefore expressed as a percentage of total phospholipids. The total LBPA content of WT mice was only marginally increased by the drug, either because the doses were low, or because the contribution from tissue material unaffected by the drug obscured selective changes at the cellular level (despite much effort, we were unable to visualize LBPA in liver samples by immunocytochemistry). The relative ratio of LBPA vs. cholesterol values is shown.


Figure 1Montage of Atoh7 + progenitor division generating an RGC ( magenta dot ) and a PRpr ( cyan dot ) . Dashed line indicates the apical side , arrowhead points to RGC axon . atoh7 : GFP - CAAX ( Atoh7 , grey ) . Scale bar 10 µm . Montage of Atoh7 + progenitor division generating an HC ( orange dot ) and a PRpr ( cyan dot ) . Dashed line indicates the apical side . atoh7 : GFP - CAAX ( Atoh7 , grey ) . Scale bar 10 µm . Montage of Atoh7 + progenitor division generating an AC ( yellow dot ) and a PRpr ( cyan dot ) . Dashed line indicates the apical side , arrowhead points to basal dendrites . atoh7 : GFP - CAAX ( Atoh7 , grey ) . Scale bar 10 µm . Event plot of all divisions analysed in ( H ) .

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Figure 1Montage of Atoh7+ progenitor division generating an RGC (magenta dot) and a PRpr (cyan dot). Dashed line indicates the apical side, arrowhead points to RGC axon. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.Montage of Atoh7+ progenitor division generating an HC (orange dot) and a PRpr (cyan dot). Dashed line indicates the apical side. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm. Montage of Atoh7+ progenitor division generating an AC (yellow dot) and a PRpr (cyan dot). Dashed line indicates the apical side, arrowhead points to basal dendrites. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.Event plot of all divisions analysed in (H).


Figure 2Two examples of retinas at 48 hpf in ( left ) control and ( right ) Prdm1a morphant ( MO ) . Atoh7 + cells ( magenta ) , inhibitory neurons ( yellow ) and photoreceptors ( cyan ) are labelled . Scale bar 50 µm . A ' ) Close up of Crx ( cyan ) signal ( upper panel ) and DAPI ( grey , lower panel ) from Fig A , for controls ( left ) and Prdm1a morphant ( right ) . Scale bar 20 µm . Staining for the photoreceptor cell marker zpr - 1 at 72 hpf in ( left ) control and ( right ) Prdm1a knockdown . Atoh7 + cells ( magenta ) , inhibitory neurons ( yellow ) , photoreceptors ( cyan ) and zpr - 1 ( grey ) . Scale bar 50 µm . Arrowheads indicate zpr - 1 staining . B ' ) Close up of Atoh7 ( magenta ) and Crx ( cyan ) signal ( upper panel ) , together with Zpr - 1 ( grey , lower panel ) from Fig B , for controls ( left ) and Prdm1a morphant ( right ) . Scale bar 20 µm . Measurements of retinal diameter in control ( black dots ) and Prdm1a knockdown ( empty dots ) at 24 , 36 , 48 and 72 hpf . N and p values are found in Table 2 . * * * * for p < 0 . 0001 , Two - way Anova with Bonferroni correction . Mean and single values are indicated . Layer thickness analysis in control and Prdm1a knockdown embryos at 72 hpf . N = 4 embryos ( control ) , 6 embryos ( Prdm1a morphant ) . Total thickness comparison : p = 0 . 0055 ; GCL comparison , p < 0 . 0001 ; INL comparison , ns ; ONL comparison , p = 0 . 0369 . Two - way Anova with Bonferroni correction . Mean and SD are indicated , as well as single values .

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Figure 2Two examples of retinas at 48 hpf in (left) control and (right) Prdm1a morphant (MO). Atoh7+ cells (magenta), inhibitory neurons (yellow) and photoreceptors (cyan) are labelled. Scale bar 50 µm. A') Close up of Crx (cyan) signal (upper panel) and DAPI (grey, lower panel) from Fig A, for controls (left) and Prdm1a morphant (right). Scale bar 20 µm. Staining for the photoreceptor cell marker zpr-1 at 72 hpf in (left) control and (right) Prdm1a knockdown. Atoh7+ cells (magenta), inhibitory neurons (yellow), photoreceptors (cyan) and zpr-1 (grey). Scale bar 50 µm. Arrowheads indicate zpr-1 staining. B') Close up of Atoh7 (magenta) and Crx (cyan) signal (upper panel), together with Zpr-1 (grey, lower panel) from Fig B, for controls (left) and Prdm1a morphant (right). Scale bar 20 µm. Measurements of retinal diameter in control (black dots) and Prdm1a knockdown (empty dots) at 24, 36, 48 and 72 hpf. N and p values are found in Table 2.**** for p < 0.0001, Two-way Anova with Bonferroni correction. Mean and single values are indicated.Layer thickness analysis in control and Prdm1a knockdown embryos at 72 hpf. N = 4 embryos (control), 6 embryos (Prdm1a morphant). Total thickness comparison: p = 0.0055; GCL comparison, p < 0.0001; INL comparison, ns; ONL comparison, p = 0.0369. Two-way Anova with Bonferroni correction. Mean and SD are indicated, as well as single values.


Figure 3Retina at 72 hpf in ( top left ) control , ( top right ) Atoh7 knockdown , ( bottom left ) Ptf1a knockdown and ( bottom right ) Atoh7 and Ptf1a knockdown . Atoh7 + cells ( magenta ) , inhibitory neurons ( yellow ) and photoreceptors ( cyan ) . Scale bar 50 µm , 20 µm in close - up panels . Layer thickness analysis in control and morphant embryos measured at 72 hpf . N = 9 embryos ( control ) , 7 embryos ( Atoh7 morphant ) , 7 embryos ( Ptf1a morphant ) , 7 embryos ( Atoh7 + Ptf1a morphant ) . p values for thickness measurements are found in Table 3 . Mixed effects analysis with Bonferroni correction . Mean and SD are indicated , as well as single values . Number of PH3 + cells per retina in control and morphant conditions at 28 , 32 and 36 hpf . N = 4 to 10 embryos per condition , mixed effects analysis with Dunnet ' s correction . All comparisons are statistically non - significant . Mean and SD are indicated , as well as single values . Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown , generating two PRpr ( cyan dots ) . Dashed line indicates the apical side . atoh7 : GFP - CAAX ( Atoh7 , grey ) . Scale bar 10 µm . Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown , generating a BC ( blue dot ) and a PRpr ( cyan dot ) . Dashed line labels the apical side , yellow arrow points at BC apical process , white arrows point at BC basal process . atoh7 : GFP - CAAX ( Atoh7 , grey ) . Scale bar 10 µm .

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Figure 3Retina at 72 hpf in (top left) control, (top right) Atoh7 knockdown, (bottom left) Ptf1a knockdown and (bottom right) Atoh7 and Ptf1a knockdown. Atoh7+ cells (magenta), inhibitory neurons (yellow) and photoreceptors (cyan). Scale bar 50 µm, 20 µm in close-up panels.Layer thickness analysis in control and morphant embryos measured at 72 hpf. N = 9 embryos (control), 7 embryos (Atoh7 morphant), 7 embryos (Ptf1a morphant), 7 embryos (Atoh7+Ptf1a morphant). p values for thickness measurements are found in Table 3. Mixed effects analysis with Bonferroni correction. Mean and SD are indicated, as well as single values.Number of PH3+ cells per retina in control and morphant conditions at 28, 32 and 36 hpf. N = 4 to 10 embryos per condition, mixed effects analysis with Dunnet's correction. All comparisons are statistically non-significant. Mean and SD are indicated, as well as single values.Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown, generating two PRpr (cyan dots). Dashed line indicates the apical side. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm. Montage of neurogenic progenitor division upon Atoh7 and Ptf1a knockdown, generating a BC (blue dot) and a PRpr (cyan dot). Dashed line labels the apical side, yellow arrow points at BC apical process, white arrows point at BC basal process. atoh7:GFP-CAAX (Atoh7, grey). Scale bar 10 µm.


Figure 4Distribution of each Atoh7 + division in time ( Event plots ) from 28 hpf in Atoh7 morphants , Ptf1a morphants and double Atoh7 / Ptf1a morphants . Coloured violin plots indicate different fates , grey violin plots show all neurogenic divisions analysed . Time in hpf .

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Figure 4Distribution of each Atoh7+ division in time (Event plots) from 28 hpf in Atoh7 morphants, Ptf1a morphants and double Atoh7/Ptf1a morphants. Coloured violin plots indicate different fates, grey violin plots show all neurogenic divisions analysed. Time in hpf.


Figure 1 ( A ) WT , Irgm1 - / - , Irgm2 - / - and Irgm3 - / - mice ( n = 4 mice / genotype ) were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hours post injection ( hpi ) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel . Relative concentration ( fold change relative mean of WT ) is shown for the indicated cytokines ( absolute cytokine concentrations of same experiment are shown in Fig EV1 ) . ( B ) WT ( n = 9 ) , Irgm1 - / - ( n = 7 ) , Irgm2 - / - ( n = 9 ) and Casp1 - / - Casp11 - / - ( n = 7 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β , IL - 18 , and TNFα was measured via ELISA .

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 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

Figure 1(A) WT, Irgm1-/-, Irgm2-/- and Irgm3-/- mice (n = 4 mice/genotype) were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hours post injection (hpi) and concentration of various cytokines determined via a preconfigured Luminex multiplex panel. Relative concentration (fold change relative mean of WT) is shown for the indicated cytokines (absolute cytokine concentrations of same experiment are shown in Fig EV1).(B) WT (n = 9), Irgm1-/- (n = 7), Irgm2-/- (n = 9) and Casp1-/-Casp11-/- (n = 7) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, IL-18, and TNFα was measured via ELISA.


Figure 2 ( A ) qPCR measurement of IL - 1β , and TNF - α mRNA levels in WT and Irgm2 - / - BMMs following 8 - hour stimulation with LPS ( 1 µg / ml ) . ( B ) WT , Irgm2 - / - and Casp1 - / - Casp11 - / - BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα , IL - 1β , and IL - 18 was measured by ELISA . ( C ) IFNγ - primed WT and Irgm2 - / - BMMs were treated with LPS , Pam3CSK4 , poly ( I : C ) or a combination of Pam3CSK4 and poly ( I : C ) ( 1 µg / ml for all treatments ) for 24 hours and cell supernatant IL - 1β and IL - 18 concentrations were assessed by ELISA . ( D ) WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) and IL - 1β , IL - 18 , and LDH release were assessed at 24 hours post treatment ( hpt ) . ( E ) IFNγ - primed WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 5 µg / ml ) for 4 hours and subsequently stained with anti - ASC antibody and Hoechst stain ( DNA / nuclei ) . Representative images are shown with white arrows pointing at ASC specks . Number of ASC specks per nuclei was quantified . Scale bars : 20 µm . ( F ) IFNγ - primed WT , Irgm2 - / - , Casp1 - / - Casp11 - / - and Irgm2 - / - Casp1 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) for 24 h and cell lysates and supernatants collected . Protein levels in cell lysates ( caspase - 1 , and actin ) and supernatants ( caspase - 1 p20 ) were visualized via immunoblotting .

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 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 O O O O O O O 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 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

Figure 2(A) qPCR measurement of IL-1β, and TNF-α mRNA levels in WT and Irgm2-/- BMMs following 8-hour stimulation with LPS (1 µg/ml).(B) WT, Irgm2-/- and Casp1-/-Casp11-/- BMMs were treated for 24 hours with LPS at indicated doses and supernatant TNFα, IL-1β, and IL-18 was measured by ELISA.(C) IFNγ-primed WT and Irgm2-/- BMMs were treated with LPS, Pam3CSK4, poly(I:C) or a combination of Pam3CSK4 and poly(I:C) (1 µg/ml for all treatments) for 24 hours and cell supernatant IL-1β and IL-18 concentrations were assessed by ELISA.(D) WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and IL-1β, IL-18, and LDH release were assessed at 24 hours post treatment (hpt).(E) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 hours and subsequently stained with anti-ASC antibody and Hoechst stain (DNA/nuclei). Representative images are shown with white arrows pointing at ASC specks. Number of ASC specks per nuclei was quantified. Scale bars: 20 µm.(F) IFNγ-primed WT, Irgm2-/-, Casp1-/-Casp11-/- and Irgm2-/-Casp1-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) for 24 h and cell lysates and supernatants collected. Protein levels in cell lysates (caspase-1, and actin) and supernatants (caspase-1 p20) were visualized via immunoblotting.


Figure 3 ( A ) IFNγ primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 5 µg / ml ) for 4 h . Following treatment , cells were stained with anti - ASC antibody and Hoechst ( DNA / nuclei ) . Representative images of ASC specks ( white arrows point at specks ) are shown and number of ASC specks per nuclei quantified . Scale bars : 20 µm . ( B ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) . IL - 1β , IL - 18 , and LDH release were assessed at 24 hours - post treatment ( hpt ) . ( C ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were infected with E . coli K - 12 at indicated MOIs and cell viability assessed via CellTiter - Glo . Cell death was calculated as a function of relative viability to uninfected cells . ( D ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were infected with E . coli K - 12 ( MOI 25 ) and 24 hpi supernatant IL - 1β and IL - 18 levels were measured by ELISA . ( E ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with OMVs at indicated concentrations for 24 h . Cell viability was assessed via CellTiter - Glo and cell death was calculated as a function of relative viability to untreated cells . ( F ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with OMVs ( 1 µg / ml ) and 24 hpt cell supernatant IL - 1β and IL - 18 levels were measured via ELISA .

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 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

Figure 3(A) IFNγ primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (5 µg/ml) for 4 h. Following treatment, cells were stained with anti-ASC antibody and Hoechst (DNA/nuclei). Representative images of ASC specks (white arrows point at specks) are shown and number of ASC specks per nuclei quantified. Scale bars: 20 µm.(B) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hours-post treatment (hpt). (C) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 at indicated MOIs and cell viability assessed via CellTiter-Glo. Cell death was calculated as a function of relative viability to uninfected cells.  (D) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were infected with E. coli K-12 (MOI 25) and 24 hpi supernatant IL-1β and IL-18 levels were measured by ELISA.  (E) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs at indicated concentrations for 24 h. Cell viability was assessed via CellTiter-Glo and cell death was calculated as a function of relative viability to untreated cells.  (F) WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were treated with OMVs (1 µg/ml) and 24 hpt cell supernatant IL-1β and IL-18 levels were measured via ELISA.


Figure 4 ( A ) WT , and Irgm2 - / - BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected . Lysates were assessed for Casp11 , Gbp2 , and actin protein levels via immunoblotting . ( B ) IFNγ - primed and unprimed WT , Irgm2 - / - , Gbpchr3 - / - and Aim2 - / - BMMs were infected with Francisella novicida ( MOI 10 ) and LDH release measured at 4 hpi . ( C ) WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) . IL - 1β , IL - 18 , and LDH release were assessed at 24 hpt . ( D ) IFNγ - primed WT , Irgm2 - / - , Gbpchr3 - / - and Irgm2 - / - Gbpchr3 - / - BMMs were treated with LPS ( 1 µg / ml ) for 24 hours and cell lysates and supernatants collected . Protein levels in cell lysates ( Caspase - 1 , and actin ) and supernatants ( Caspase - 1 p20 ) were visualized via immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT, and Irgm2-/- BMMs were stimulated overnight with IFNγ or left untreated and cell lysates were collected. Lysates were assessed for Casp11, Gbp2, and actin protein levels via immunoblotting.  (B) IFNγ-primed and unprimed WT, Irgm2-/-, Gbpchr3-/- and Aim2-/- BMMs were infected with Francisella novicida (MOI 10) and LDH release measured at 4 hpi.  (C) WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml). IL-1β, IL-18, and LDH release were assessed at 24 hpt.  (D) IFNγ-primed WT, Irgm2-/-, Gbpchr3-/- and Irgm2-/- Gbpchr3-/- BMMs were treated with LPS (1 µg/ml) for 24 hours and cell lysates and supernatants collected. Protein levels in cell lysates (Caspase-1, and actin) and supernatants (Caspase-1 p20) were visualized via immunoblotting.


Figure 5 ( A ) WT , Irgm2 - / - , and Nlrp3 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin for 1 h and IL - 1β / LDH release was measured . ( B ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 0 . 1 µg / ml ) for 3 h followed by nigericin and / or MCC950 for 1 h and IL - 1β / LDH release was measured . ( C ) WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) and / or MCC950 for 24 h and IL - 1β / LDH release was measured . ( D ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt . ( E ) IFNγ - primed WT , Irgm2 - / - , Casp11 - / - and Irgm2 - / - Casp11 - / - BMMs were co - treated with LPS ( indicated doses ) and Listeria monocytogenes ( MOI 10 ) and LDH release measured at 4 hpt .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) WT, Irgm2-/-, and Nlrp3-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin for 1 h and IL-1β/LDH release was measured.  (B) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (0.1 µg/ml) for 3 h followed by nigericin and/or MCC950 for 1 h and IL-1β/LDH release was measured.  (C) WT, Irgm2-/-, Casp11-/- and Irgm2-/-Casp11-/- BMMs were treated with LPS (1 µg/ml) and/or MCC950 for 24 h and IL-1β/LDH release was measured.  (D) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were transfected with LPS using lipofectamine LTX and LDH release was measured at 2 hpt.  (E) IFNγ-primed WT, Irgm2-/-, Casp11-/- and Irgm2-/- Casp11-/- BMMs were co-treated with LPS (indicated doses) and Listeria monocytogenes (MOI 10) and LDH release measured at 4 hpt.


Figure 6 ( A ) WT ( n = 9 ) , Irgm2 - / - ( n = 10 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 8 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Morbidity and mortality were observed for 48 h at 3 hour intervals . ( B ) WT ( n = 9 ) , Irgm2 - / - ( n = 8 ) , Casp11 - / - ( n = 7 ) and Irgm2 - / - Casp11 - / - ( n = 10 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β , and IL - 18 was measured by ELISA . Data shown are from 2 pooled experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 6 (A) WT (n = 9), Irgm2-/- (n = 10), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 8) mice were injected i.p. with LPS (8 mg/kg). Morbidity and mortality were observed for 48 h at 3 hour intervals.  (B) WT (n = 9), Irgm2-/- (n = 8), Casp11-/- (n = 7) and Irgm2-/-Casp11-/- (n = 10) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β, and IL-18 was measured by ELISA. Data shown are from 2 pooled experiments.


Figure 7 ( A ) WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 24 hpt ( n = 3 independent experiments ) . ( B ) IFNγ - primed WT , Irgm2 - / - , Irgm3 - / - , Irgm2 - / - Irgm3 - / - , panIrgm - / - , and Casp11 - / - BMMs were treated with LPS ( 1 µg / ml ) or infected with E . coli K - 12 ( MOI 25 ) and IL - 1β , and LDH release were assessed at 8 hpt / hpi ( n = 3 independent experiments ) . ( C ) WT ( n = 15 ) , Irgm2 - / - ( n = 12 ) , and panIrgm - / - ( n = 13 ) mice were injected i . p . with LPS ( 8 mg / kg ) . Serum was collected 4 hpi and concentration of IL - 1β and TNFα was measured via ELISA . Data shown are from 3 pooled experiments . ( D ) WT , Irgm2 - / - , panIrgm - / - and Casp11 - / - mice ( n = 8 mice / genotype ) were injected i . p . with LPS ( 8 mg / kg body weight ) . Morbidity and mortality were observed for 48 h at 3 - h intervals .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 (A) WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 24 hpt (n = 3 independent experiments). (B) IFNγ-primed WT, Irgm2-/-, Irgm3-/-, Irgm2-/-Irgm3-/-, panIrgm-/-, and Casp11-/- BMMs were treated with LPS (1 µg/ml) or infected with E. coli K-12 (MOI 25) and IL-1β, and LDH release were assessed at 8 hpt/ hpi (n = 3 independent experiments).  (C) WT (n = 15), Irgm2-/- (n = 12), and panIrgm-/- (n = 13) mice were injected i.p. with LPS (8 mg/kg). Serum was collected 4 hpi and concentration of IL-1β and TNFα was measured via ELISA. Data shown are from 3 pooled experiments.  (D) WT, Irgm2-/-, panIrgm-/- and Casp11-/- mice (n = 8 mice/genotype) were injected i.p. with LPS (8 mg/kg body weight). Morbidity and mortality were observed for 48 h at 3-h intervals.


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).


Figure 4 ( C ) Analysis of Flag - tagged Sox4 wildtype ( WT ) protein overexpressed in HEK293T ( 293T ) cells and detected with FLAG antibody . Lysates were treated with or without lambda - phosphatase ( λ - Phos ) . Antibody against phosphorylated β - catenin ( pβCAT ) was used to validate λ - Phos treatment . Purple arrowheads indicate Sox4 phosho - shift from 70kDa to 65kDa . ERK blot shows similar loading of samples . ( D ) Analysis of Flag - tagged Sox11 WT protein overexpressed in 293T cells and detected with FLAG antibody . Lysates treated with or without λ - Phos . Upper smear ( red arrowhead ) represents phosphorylated Sox11 . Antibody for pβCAT was used to validate λ - Phos treatment . ( G ) Co - transfection of myc - tagged GSK3β and Flag - Sox4 in 293T cells . BIO treatment ( 0 . 5 µM ) performed for 1 hour prior to harvest . Lower band ( 65kDa ) represents non - phosphorylated Sox4 . High and low exposure ( exp ) blots shown to better visualize non - phosphorylated Sox4 . Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs . controls . The sum of upper and lower bands was quantified ( n = 8 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 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

Figure 4(C) Analysis of Flag-tagged Sox4 wildtype (WT) protein overexpressed in HEK293T (293T) cells and detected with FLAG antibody. Lysates were treated with or without lambda-phosphatase (λ-Phos). Antibody against phosphorylated β-catenin (pβCAT) was used to validate λ-Phos treatment. Purple arrowheads indicate Sox4 phosho-shift from 70kDa to 65kDa. ERK blot shows similar loading of samples. (D) Analysis of Flag-tagged Sox11 WT protein overexpressed in 293T cells and detected with FLAG antibody. Lysates treated with or without λ-Phos. Upper smear (red arrowhead) represents phosphorylated Sox11. Antibody for pβCAT was used to validate λ-Phos treatment.(G) Co-transfection of myc-tagged GSK3β and Flag-Sox4 in 293T cells. BIO treatment (0.5 µM) performed for 1 hour prior to harvest. Lower band (65kDa) represents non-phosphorylated Sox4. High and low exposure (exp) blots shown to better visualize non-phosphorylated Sox4. Numbers above blot are quantification of Sox4 protein levels normalized to GFP and are expressed as fold change vs. controls. The sum of upper and lower bands was quantified (n=8 biological replicates).


Figure 5 ( B ) IF for Sox4 and pHH3 on neocortex sections of E13 . 5 control and DKO embryos . The level of Sox4 immunoreactivity is reduced in pHH3 + cells ( white arrowheads ) . ( C ) Quantification of Sox4 staining in SVZ mitotic cells from E13 . 5 control and DKO embryos . Mitotic Sox4 staining was defined as high if the pixel intensity was similar to , or higher than , non - mitotic cells , or low if the levels were below those observed in non - mitotic cells . Data are means ± SEM for ratios of cells with high vs . low Sox4 , and are displayed as fold change vs . controls ( n = 4 embryos , 3 litters ) . ( H ) IF for Sox11 and pHH3 in E13 . 5 control and DKO neocortices . Sox11 staining intensity is decreased in mitotic cells ( white arrowheads , insets ) . ( I ) Quantification of Sox11 protein levels in SVZ mitotic cells from E13 . 5 control and DKO embryos . Quantification performed as in ( C ) . Data are means ± SEM for ratios of high vs . low Sox11 and are displayed as fold change vs . controls ( n = 5 embryos , 3 litters ) . ( K ) Co - IF for Sox11 and Tbr2 in sections of neocortex of E13 . 5 control and DKO embryos . Sox11 immunoreactivity is reduced in some Tbr2 + cells ( asterisks , insets ) . ( L ) Quantification of percentage of Tbr2 + cells that show with high Sox11 staining intensity , in control and DKO E13 . 5 neocortices . High or low staining intensity was judged based on visual inspection , taking background staining levels into account . Data are means ± SEM and are shown as fold change vs . controls ( n = 5 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 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 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

Figure 5(B) IF for Sox4 and pHH3 on neocortex sections of E13.5 control and DKO embryos. The level of Sox4 immunoreactivity is reduced in pHH3+ cells (white arrowheads). (C) Quantification of Sox4 staining in SVZ mitotic cells from E13.5 control and DKO embryos. Mitotic Sox4 staining was defined as high if the pixel intensity was similar to, or higher than, non-mitotic cells, or low if the levels were below those observed in non-mitotic cells. Data are means ± SEM for ratios of cells with high vs. low Sox4, and are displayed as fold change vs. controls (n=4 embryos, 3 litters).(H) IF for Sox11 and pHH3 in E13.5 control and DKO neocortices. Sox11 staining intensity is decreased in mitotic cells (white arrowheads, insets). (I) Quantification of Sox11 protein levels in SVZ mitotic cells from E13.5 control and DKO embryos. Quantification performed as in (C). Data are means ± SEM for ratios of high vs. low Sox11 and are displayed as fold change vs. controls (n=5 embryos, 3 litters).(K) Co-IF for Sox11 and Tbr2 in sections of neocortex of E13.5 control and DKO embryos. Sox11 immunoreactivity is reduced in some Tbr2+ cells (asterisks, insets). (L) Quantification of percentage of Tbr2+ cells that show with high Sox11 staining intensity, in control and DKO E13.5 neocortices. High or low staining intensity was judged based on visual inspection, taking background staining levels into account. Data are means ± SEM and are shown as fold change vs. controls (n=5 embryos, 3 litters).


Figure 6 ( B ) IF for Tuj1 and Map2 ( not shown ) to identify newly formed neurons in NPCs after seven - day culture in differentiation medium . Empty CAG - IRES - EGFP vector was used as control in cells not transduced with Sox4 / 11 . Transduced cells were identified by IF for GFP ( Co and DKO ) or FLAG ( Co + Sox4 / 11 and DKO + Sox4 / 11 ) . Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension . Scale bar 20 μm . ( C ) Quantification of transduced Tuj1 + cells from ( B ) . Note the decrease in neuron number in DKO cultures compared to control , and the rescue in DKO cultures upon Sox4 / 11 expression ( experiment performed twice in triplicates , representative experiment shown ) . Data are means ± SEM . ( E ) IF for Tuj1 to identify newly formed neurons in NPCs after seven - day culture in differentiation medium . Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension . Scale bar 20 μm . ( F ) Quantification of Tuj1 + cells in control , control + CHIR ( 1 µM , 3 µM ) , DKO , and DKO + CHIR NPC cultures . Data are means ± SEM ( experiment performed twice in triplicates , representative experiment is shown ) . Numbers below asterisks show fold change .

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Figure 6(B) IF for Tuj1 and Map2 (not shown) to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Empty CAG-IRES-EGFP vector was used as control in cells not transduced with Sox4/11. Transduced cells were identified by IF for GFP (Co and DKO) or FLAG (Co + Sox4/11 and DKO + Sox4/11). Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm. (C) Quantification of transduced Tuj1+ cells from (B). Note the decrease in neuron number in DKO cultures compared to control, and the rescue in DKO cultures upon Sox4/11 expression (experiment performed twice in triplicates, representative experiment shown). Data are means ± SEM.(E) IF for Tuj1 to identify newly formed neurons in NPCs after seven-day culture in differentiation medium. Arrowheads indicate mature neurons and asterisks denote immature neurons lacking neurite extension. Scale bar 20 μm. (F) Quantification of Tuj1+ cells in control, control + CHIR (1 µM, 3 µM), DKO, and DKO + CHIR NPC cultures. Data are means ± SEM (experiment performed twice in triplicates, representative experiment is shown). Numbers below asterisks show fold change.


Figure 1 ( A , B and C ) Polyphenols TA , PGG and EGCG activate rhpR - lux in wild type but not rhpS deletion Psph strains . Data information : , * indicate significant differences between mock group and strains under the different test conditions . ( Student ' s t test . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) . ( D , E and F ) Polyphenols repress T3SS induction in P . syringae in MM . Transcripts of the indicated genes in the presence of 100 - μM TA ( D ) , 100 - µM PGG ( E ) or 2 - mM EGCG ( F ) were determined by RT - qPCR 6 hours after culturing in MM . Data information : , * indicate significant differences between mock group and strains under the different test conditions . ( Student ' s t test . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

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Figure 1(A, B and C) Polyphenols TA , PGG and EGCG activate rhpR-lux in wild type but not rhpS deletion Psph strains. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).(D, E and F) Polyphenols repress T3SS induction in P. syringae in MM. Transcripts of the indicated genes in the presence of 100-μM TA (D), 100-µM PGG (E) or 2-mM EGCG (F) were determined by RT-qPCR 6 hours after culturing in MM. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


Figure 2 ( D , E and F ) Polyphenols inhibit the phosphatase activity of RhpS . The GST - RhpR was phosphorylated by adding 5 - μM PSPPH _ 3550c protein as well as 10 - μCi [ γ - 32P ] - ATP for 10 minutes . The RhpS - FL protein was treated in the presence of different concentrations of TA ( D ) , PGG ( E ) , or EGCG ( F ) for 10 min . Then , the full - length RhpS protein was added into the mixture phosphorylated GST - RhpR for 1 hour before stopping the reaction . The phosphorylated proteins were separated by 12 % SDS - PAGE . After electrophoresis , the SDS - PAGE gels were dried with gel - dryer and then exposed to a medical X - ray film overnight . This experiment was repeated twice , yielding similar results . Data information : the reaction was stopped by adding 5 × loading buffer before SDS - PAGE . Abbreviation is as follows : CBB , coomassie bright blue stain ; CFU , colony - forming unit . ( I , J and K ) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants . Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants . Disease symptoms were recorded 5 days after inoculation . Leaf disks ( 1 cm2 ) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs . The experiment was repeated three times . Data information : , * indicate significant differences between mock group and strains under the different test conditions as indicated . ( Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

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Figure 2(D, E and F) Polyphenols inhibit the phosphatase activity of RhpS. The GST-RhpR was phosphorylated by adding 5-μM PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. Data information: the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit.(I, J and K) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


Figure 3 ( B , C and D ) Polyphenols bind to the RhpS - FL protein . The concentration of RhpS - FL protein was consistently measured as 0 . 6 - μM . The binding curves of TA ( B ) , PGG ( C ) and EGCG ( D ) are shown . The individual data points were shown ( n = 2 biological replicates ) . ( E ) Alanine - scanning mutagenesis of the RhpS sensor . RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector . LUX activity was estimated in the absence ( black frame ) and presence TA ( 50 - μM ) , PGG ( 100 - μM ) or EGCG ( 2 - mM ) . Error bars represent standard deviations ( n = 3 biological replicates ) . Data information : , * indicate significant differences between mock group and strains under the different test conditions as indicated . ( Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) .

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Figure 3(B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).(E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence TA (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates). Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).


Figure 4 ( C and D ) The rhpS / PSPPH _ 3550 / PSPPH _ 3736 / PSPPH _ 5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain . The wild type , rhpS deletion and quadruple deletion strains were cultured in MM ( C ) for 6 hours or KB ( D ) to OD600 = 0 . 6 . Transcripts of the indicated genes were determined by RT - qPCR . Data information : , * indicate significant differences between rhpS mutant and quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 . ( F ) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain . Bacterial virulence levels were estimated by inoculating wild type , rhpS deletion and quadruple deletion strains onto host bean plants . Disease symptoms were recorded 5 days after inoculation . The experiment was repeated three times . ( G ) Leaf disks ( 1 cm2 ) from ( G ) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs . The experiment was repeated three times . Data information : , * indicate significant differences between rhpS mutant and quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 .

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Figure 4(C and D) The rhpS/PSPPH_3550/PSPPH_3736/PSPPH_5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain. The wild type, rhpS deletion and quadruple deletion strains were cultured in MM (C) for 6 hours or KB (D) to OD600=0.6. Transcripts of the indicated genes were determined by RT-qPCR. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.(F) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain. Bacterial virulence levels were estimated by inoculating wild type, rhpS deletion and quadruple deletion strains onto host bean plants. Disease symptoms were recorded 5 days after inoculation. The experiment was repeated three times.(G) Leaf disks (1 cm2) from (G) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.


Figure 1 ( A ) The relative fold change of carbon metabolites with or without HU treatment for 60 min . Error bars represent standard deviations ( SD ) from six independent experiments . ( D , E ) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT , snf1Δ or snf1Δmig1Δ cells ( D ) . Protein extracts were probed with an anti - GFP antibody . Mcm2 was applied as a loading control . Three independent experiments were carried out and the relative fold changes were measured by Image J ( E ) . Error bars represent standard deviations ( SD ) from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 0 . 05 ; * * 0 . 001 < P - values < 0 . 01 ) .

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Figure 1(A) The relative fold change of carbon metabolites with or without HU treatment for 60 min. Error bars represent standard deviations (SD) from six independent experiments.(D, E) The relative levels of endogenous Zwf1 protein with a GFP tag were analyzed by immunoblots in WT, snf1Δ or snf1Δmig1Δ cells (D). Protein extracts were probed with an anti-GFP antibody. Mcm2 was applied as a loading control. Three independent experiments were carried out and the relative fold changes were measured by Image J (E). Error bars represent standard deviations (SD) from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01< P-values <0.05; **0.001< P-values <0.01).


Figure 2 ( C ) Rad53 phosphorylation in various snf1 alleles in response to HU . The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ . Rad53 phosphorylation was detected as above . ( D ) The snf1 kinase - defective mutants are sensitive to HU . Yeast spot assays were performed as in Fig 2A . The phospho - mimetic snf1T210D ( a constitutive active mutant ) was applied as a control for nonphosphrylable snf1T210A . ( F , G ) HU induces a rapid Snf1 T210 phosphorylation . Exponential cells were treated by 200 mM HU for the indicated time . Protein extracts were prepared as described in the Methods and Materials . Three independent experiments were done . Quantitation of Snf1 T210 and Snf1 signals was performed by Image J . The relative ratio of Snf1 T210 / Snf1 was shown in ( H ) . Error bars represent standard deviations ( SD ) from three biological repeats .

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Figure 2(C) Rad53 phosphorylation in various snf1 alleles in response to HU. The plasmids expressing the indicated snf1 alleles were transformed into snf1Δ. Rad53 phosphorylation was detected as above.(D) The snf1 kinase-defective mutants are sensitive to HU. Yeast spot assays were performed as in Fig 2A. The phospho-mimetic snf1T210D (a constitutive active mutant) was applied as a control for nonphosphrylable snf1T210A.(F, G) HU induces a rapid Snf1 T210 phosphorylation. Exponential cells were treated by 200 mM HU for the indicated time. Protein extracts were prepared as described in the Methods and Materials. Three independent experiments were done. Quantitation of Snf1 T210 and Snf1 signals was performed by Image J. The relative ratio of Snf1 T210/Snf1 was shown in (H). Error bars represent standard deviations (SD) from three biological repeats.


Figure 3 ( C ) Snap shot of Rfa1 ChIP - Seq at Chromosome VI . Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min . The sequencing reads were mapped to the yeast reference genome . The red dashed box indicated the relative read intensity of ARS607 . ( E , F ) SNF1 is required for efficient recruitment of Rfa1 , but not Psf2 , to HU - stalled replication forks . Cells were grown and synchronized in G1 by α - factor before release into the fresh medium supplemented with 200 mM HU for the indicated time . Cell extracts were prepared and subjected to MYC - ChIP of Rfa1 - 13MYC ( E ) or Psf2 - 13MYC ( F ) . The amounts of DNA in the precipitates were quantified by qPCR . Error bars represent SD from three biological repeats .

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Figure 3(C) Snap shot of Rfa1 ChIP-Seq at Chromosome VI. Cells were synchronized in G1 and released into the fresh medium containing 200 mM HU for 30 min. The sequencing reads were mapped to the yeast reference genome. The red dashed box indicated the relative read intensity of ARS607.(E, F) SNF1 is required for efficient recruitment of Rfa1, but not Psf2, to HU-stalled replication forks. Cells were grown and synchronized in G1 by α-factor before release into the fresh medium supplemented with 200 mM HU for the indicated time. Cell extracts were prepared and subjected to MYC-ChIP of Rfa1-13MYC (E) or Psf2-13MYC (F). The amounts of DNA in the precipitates were quantified by qPCR. Error bars represent SD from three biological repeats.


Figure 4mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU . The experiments were done as in Fig 2 . ( E , F ) snf1Δ shows less RPA on chromatin , which can be restored by mig1Δ . Cells were collected and fractionated into chromatin - bound ( Chromatin ) and non - chromatin - bound fractions ( non - Chromatin ) followed by immunoblotting . Tubulin and Orc2 were applied as controls for non - chromatin and chromatin fractions , respectively . The signals were quantified by Image J and quantifications were shown in ( F ) . Error bars represent SD from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 0 . 05 ) .

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Figure 4mig1Δ restores Rad53 phosphorylation of the snf1 mutants in the presence of HU. The experiments were done as in Fig 2.(E, F) snf1Δ shows less RPA on chromatin, which can be restored by mig1Δ. Cells were collected and fractionated into chromatin-bound (Chromatin) and non-chromatin-bound fractions (non-Chromatin) followed by immunoblotting. Tubulin and Orc2 were applied as controls for non-chromatin and chromatin fractions, respectively. The signals were quantified by Image J and quantifications were shown in (F). Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05).


Figure 5Phospho - mimetic mig1 - 2D bypasses the role of SNF1 in HU resistance ( C ) Phospho - mimetic mig1 - 2D bypasses the role of SNF1 in RPA recruitment ( D ) . ( E ) The diagram of the strategy to reinforce the Mig1 - Ssn6 interaction via a GFP and GBP pair ( left panel ) . The persistent Mig1 - Ssn6 association abolishes the phospho - mimicking effect of mig1 - 2D ( right panel ) .

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Figure 5Phospho-mimetic mig1-2D bypasses the role of SNF1 in HU resistance (C)Phospho-mimetic mig1-2D bypasses the role of SNF1 in RPA recruitment (D). (E) The diagram of the strategy to reinforce the Mig1-Ssn6 interaction via a GFP and GBP pair (left panel). The persistent Mig1-Ssn6 association abolishes the phospho-mimicking effect of mig1-2D (right panel).


Figure 6 ( C ) Rfa1 - 13MYC ChIP assays after HU treatment . Strains were synchronized in G1 phase by α - factor prior to release into fresh media containing 200 mM HU for the indicated time points . Cell extracts were prepared as in Fig 3 . Error bars represent SD from three biological repeats . ( D ) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ . The experiments were done as in Fig 1 . Error bars represent SD from three biological repeats . Statistical significance was evaluated based on Student ' s t - test ( * 0 . 01 < P - values < 0 . 05 ; * * 0 . 001 < P - values < 0 . 01 ) .

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Figure 6(C) Rfa1-13MYC ChIP assays after HU treatment. Strains were synchronized in G1 phase by α-factor prior to release into fresh media containing 200 mM HU for the indicated time points. Cell extracts were prepared as in Fig 3. Error bars represent SD from three biological repeats.(D) Phosphorylation of Mig1 T489 S495 completely restores the ZWF1 mRNA levels in snf1Δ. The experiments were done as in Fig 1. Error bars represent SD from three biological repeats. Statistical significance was evaluated based on Student's t-test (*0.01 < P-values < 0.05; **0.001 < P-values < 0.01).


Figure 7 ( B ) The effects of α - KG on the HU sensitivity of snf1Δ and other checkpoint mutants . Yeast spot assays were performed as in Fig 2A . ( D ) α - KG restores Rad53 phosphorylation in snf1Δ . CBB indicates coomassie bright blue ( CBB ) staining of the membrane as the loading control . ( E ) The effects of α - KG on the HU sensitivity of Rfa1 zinc finger mutants ( C486S , C491S , C505S , C508S ) . Yeast spot assays were performed as in Fig 2A .

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 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

Figure 7(B) The effects of α-KG on the HU sensitivity of snf1Δ and other checkpoint mutants. Yeast spot assays were performed as in Fig 2A.(D) α-KG restores Rad53 phosphorylation in snf1Δ. CBB indicates coomassie bright blue (CBB) staining of the membrane as the loading control.(E) The effects of α-KG on the HU sensitivity of Rfa1 zinc finger mutants (C486S, C491S, C505S, C508S). Yeast spot assays were performed as in Fig 2A.


Figure 1A Representative siliques from wild - type ( WT ; Col - 0 ) , herk1 , anj and herk1 anj plants prior to dehiscence . Siliques were placed on double - sided sticky tape and carpel walls separated from the replum to expose the developing seeds . Scale bar = 5 mm . B Developing seeds per silique in wild - type , herk1 , anj and herk1 anj plants . Fully expanded siliques were dissected and photographed under a stereomicroscope . n = 15 ( four independent experiments with at least three plants per line and five siliques per plant ) . Data presented are means ± SEM . * * * p < 0 . 001 ( Student ' s t - test ) . C Percentage of pollen tubes with normal reception at the female gametophyte ( black bars ; representative image middle centre of figure ) and with overgrowth ( grey bars ; representative image lower centre ) as assessed by aniline blue staining . 15 self - pollinated stage 16 flowers from wild - type , herk1 , anj and herk1 anj were analysed . Legend scale bars = 50 µm . * * * p < 0 . 001 ( χ - square tests ) . D Aniline blue staining of pollen tube reception in reciprocal crosses between wild - type and herk1 anj plants with at least two siliques per cross . Legend as per ( C ) . * * * p < 0 . 001 ( χ - square tests ) .

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 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 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 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

Figure 1A Representative siliques from wild-type (WT; Col-0), herk1, anj and herk1 anj plants prior to dehiscence. Siliques were placed on double-sided sticky tape and carpel walls separated from the replum to expose the developing seeds. Scale bar = 5 mm.B Developing seeds per silique in wild-type, herk1, anj and herk1 anj plants. Fully expanded siliques were dissected and photographed under a stereomicroscope. n = 15 (four independent experiments with at least three plants per line and five siliques per plant). Data presented are means ± SEM. *** p<0.001 (Student's t-test).C Percentage of pollen tubes with normal reception at the female gametophyte (black bars; representative image middle centre of figure) and with overgrowth (grey bars; representative image lower centre) as assessed by aniline blue staining. 15 self-pollinated stage 16 flowers from wild-type, herk1, anj and herk1 anj were analysed. Legend scale bars = 50 µm. *** p<0.001 (χ-square tests).D Aniline blue staining of pollen tube reception in reciprocal crosses between wild-type and herk1 anj plants with at least two siliques per cross. Legend as per (C). *** p<0.001 (χ-square tests).