B . miRNA mimic screen for miRNAs that regulate mitosis genes . miRNA - mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A . Individual points correspond to the mean z - score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA - mimic .

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B. miRNA mimic screen for miRNAs that regulate mitosis genes. miRNA-mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in A. Individual points correspond to the mean z-score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA-mimic.


C . Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics . Nuclear envelope breakdown is at t = 0 min . ( n ≥ 71 in all conditions ) .

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C. Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics. Nuclear envelope breakdown is at t = 0 min. (n ≥ 71 in all conditions).


D . Confocal time lapse microscopy images of HeLa cells stably expressing H2B - mCherry ( red ) and a microtubule marker ( α - Tub - eGFP ; green ) 48 h after transfection of miR - 449a mimic , or non - targeting control siRNA . Scale bar , 10 μm . E . Quantification of spindle rotation and duration from prometaphase to anaphase onset in time - lapse movies as in D . miR - 449a mimic promotes spindle rotation . Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset . Spindle rotation during metaphase was measured as described in methods . Individual data points correspond to single cells ( n ≥ 72 in all conditions ) . Normality was tested with Kolmogorov - Smirnov test . Variance between samples was tested using F test . Significance was tested by Welch ' s t test : p - value = 2 . 598e - 06 comparing spindle rotation of negative control versus miR - 449a mimic - transfected cells ( all data points are compared ) . p - value = 0 . 0005621 ( for cells with prometaphase - anaphase onset duration lower than 60 min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Confocal time lapse microscopy images of HeLa cells stably expressing H2B-mCherry (red) and a microtubule marker (α-Tub-eGFP; green) 48 h after transfection of miR-449a mimic, or non-targeting control siRNA. Scale bar, 10 μm.E. Quantification of spindle rotation and duration from prometaphase to anaphase onset in time-lapse movies as in D. miR-449a mimic promotes spindle rotation. Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset. Spindle rotation during metaphase was measured as described in methods. Individual data points correspond to single cells (n ≥ 72 in all conditions). Normality was tested with Kolmogorov-Smirnov test. Variance between samples was tested using F test. Significance was tested by Welch's t test: p-value = 2.598e-06 comparing spindle rotation of negative control versus miR-449a mimic-transfected cells (all data points are compared). p-value = 0.0005621 (for cells with prometaphase-anaphase onset duration lower than 60 min.


A . The expression levels of endogenous miR - 34 / 449 family members were measured by RT - qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14 . The levels of the different miR - 34 / 449 family members and miR - 7a - 1 , a highly expressed miRNA relevant in cortical progenitor biology , were determined . All concentrations were normalized ( norm . ) using miR - 7a - 1 concentration ( n = 8 cortices , 2 different litters ) . Error bars indicate standard error .

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A. The expression levels of endogenous miR-34/449 family members were measured by RT-qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14. The levels of the different miR-34/449 family members and miR-7a-1, a highly expressed miRNA relevant in cortical progenitor biology, were determined. All concentrations were normalized (norm.) using miR-7a-1 concentration (n = 8 cortices, 2 different litters). Error bars indicate standard error.


B , C . Expression analysis of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b and miR - 34c are preferentially expressed in the subventricular ( SVZ ) and ventricular ( VZ ) zones of the neocortex . Scale bar , 50 μm ( A ) , 10 μm ( B ) .

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B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (A), 10 μm (B).


B , C . Expression analysis of miR - 34 / 449 by in situ hybridization using locked nucleic acid ( LNA ) probes in wild type cortices at E14 . Mature miR - 449 , miR - 34b and miR - 34c are preferentially expressed in the subventricular ( SVZ ) and ventricular ( VZ ) zones of the neocortex . Scale bar , 50 μm ( B ) , 10 μm ( C ) .

B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild type cortices at E14. Mature miR-449, miR-34b and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (B), 10 μm (C).


D , E . Brains of adult mice ( P23 ) , and quantification of brain weight . Dots indicate individual brains ; red line indicates median . Mice lacking miR - 449abc and miR - 34bc ( DKO ) or miR - 449abc , miR - 34bc , and miR - 34a ( TKO ) have significantly smaller brains compared to littermate controls ( Het ) . Significance was tested by Pairwaise t - test with Bonferroni correction ; Het ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00215 ; Het ( n = 6 brains , 2 different litters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00054 . Subfigure G was statistically tested as in E . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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D, E. Brains of adult mice (P23), and quantification of brain weight. Dots indicate individual brains; red line indicates median. Mice lacking miR-449abc and miR-34bc (DKO) or miR-449abc, miR-34bc, and miR-34a (TKO) have significantly smaller brains compared to littermate controls (Het). Significance was tested by Pairwaise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00215; Het (n = 6 brains, 2 different litters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00054. Subfigure G was statistically tested as in E. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


F , G . Confocal images of coronal brain sections ( P23 ) and quantification of cortex width from miR - 34 / 449 DKO and TKO mice and littermate controls ( Het ) . Sections were stained with DAPI . DKO or TKO mice have significantly thinner cortices compared to littermate controls ( Het ) in adult mice ( P23 ) . ( n = 6 brains , 2 different litters ) vs . DKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00084 , Het ( n = 6 brains , 2 different liters ) vs . TKO ( n = 4 brains , 2 different litters ) p - value = 0 . 00211 . Scale bar , 500 μm .

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F, G. Confocal images of coronal brain sections (P23) and quantification of cortex width from miR-34/449 DKO and TKO mice and littermate controls (Het). Sections were stained with DAPI. DKO or TKO mice have significantly thinner cortices compared to littermate controls (Het) in adult mice (P23). (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters) p-value = 0.00084, Het (n = 6 brains, 2 different liters) vs. TKO (n = 4 brains, 2 different litters) p-value = 0.00211. Scale bar, 500 μm.


A . Confocal images of coronal sections from E16 brains of miR - 34 / 449 KO mice and littermate controls ( Het ) , stained with anti - Satb2 - antibody to label neurons of layers II - IV ( green ) and DAPI to label all cell nuclei ( blue ) . Scale bars : 50 μm . B . Quantification of the number of Satb2 - positive cells per 100 μm ventricular zone surface . Bars indicate mean ± SEM , as for all quantifications shown in this figure ; n = 3 brains per genotype group , from two independent litters . Data was normalized ( norm . ) to the ventricular zone surface analyzed ( 100 µm ) and relativized to the heterozygous control average value . Significance was tested by Welch ' s t test : p - value = 0 . 01146 ( Het vs . KO ) ( n = 4 cortices per genotype group , 2 independent litters ) . All subfigures were statistically tested as in B . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 .

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A. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Satb2-antibody to label neurons of layers II-IV (green) and DAPI to label all cell nuclei (blue). Scale bars: 50 μm.B. Quantification of the number of Satb2-positive cells per 100 μm ventricular zone surface. Bars indicate mean ± SEM, as for all quantifications shown in this figure; n = 3 brains per genotype group, from two independent litters. Data was normalized (norm.) to the ventricular zone surface analyzed (100 µm) and relativized to the heterozygous control average value. Significance was tested by Welch's t test: p-value = 0.01146 (Het vs. KO) (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in B. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : C , D . E16 brains stained with anti - Ctip2 antibody to label layer Vneurons ( n = 4 brains per genotype group , 2 independent litters ) . Scale bars : 50 μm . Images in C were taken from same brain slices as shown in A . p - value = 0 . 03224 ( Het vs . KO ) ( immuno co - staining of Ctip2 ( red , C ) ) .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:C, D. E16 brains stained with anti-Ctip2 antibody to label layer Vneurons (n = 4 brains per genotype group, 2 independent litters). Scale bars: 50 μm. Images in C were taken from same brain slices as shown in A. p-value = 0.03224 (Het vs. KO) (immuno co-staining of Ctip2(red, C)).


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : E , F . E14 brains stained with anti - Tbr1 antibody to label layer IVneurons p - value = 0 . 01392 ( Het vs . KO ) ( n = 3 and 4 brains per genotype group from 2 independent litters ) . Scale bars : 50 μm .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:E, F. E14 brains stained with anti-Tbr1 antibody to label layer IVneurons p-value = 0.01392 (Het vs. KO) (n = 3 and 4 brains per genotype group from 2 independent litters). Scale bars: 50 μm.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : G , H . E16 brains stained with anti - Tbr2 antibody to label intermediate progenitors . p - value = 0 . 01647 ( Het vs . KO ) ( n = 7 brains for each genotype group , 5 independent litters ) . Scale bars : 50 μm .

C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:G, H. E16 brains stained with anti-Tbr2 antibody to label intermediate progenitors. p-value = 0.01647 (Het vs. KO) (n = 7 brains for each genotype group, 5 independent litters). Scale bars: 50 μm.


C - J . Confocal images and cell density quantifications as in A using molecular markers for different cell types : I , J . E16 brains stained with anti - Pax6 antibody to label radial glia progenitors . p - value = 0 . 002768 ( n = 5 brains per genotype group , 3 independent litters ) . Scale bars : 50 μm .

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C-J. Confocal images and cell density quantifications as in A using molecular markers for different cell types:I, J. E16 brains stained with anti-Pax6 antibody to label radial glia progenitors. p-value = 0.002768 (n = 5 brains per genotype group, 3 independent litters). Scale bars: 50 μm.


K . 3D reconstruction of a dividing radial glial progenitor at early anaphase . A coronal section of an E14 brain of a heterozygous control mouse was stained with anti - phospho - Vimentin antibody ( red ) , anti - γ - tubulin antibody ( green / yellow ) , phalloidin ( magenta ) , and DAPI ( blue ) and imaged by 3D confocal microscopy . " s " indicates spindle axis , " α " indicates angle relative to ventricular surface plane , which was determined by a vector path as indicated by thin white lines located on the left side of the image . Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell . Scale bars : 5 μm . L . Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR - 34 / 449 KO and littermate controls ( Het ) . Each dot represents a single dividing cell ; p - value = 3 . 166e - 05 ( Het vs . KO ) ( n = 131 vs . 107 cells , n = 4 brains per genotype group , 2 independent litters ) .

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K. 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti-phospho-Vimentin antibody (red), anti-γ-tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. "s" indicates spindle axis, "α" indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. Scale bars: 5 μm.L. Quantification of spindle orientation in radial glial cells as in K for E14 brains of miR-34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; p-value = 3.166e-05 (Het vs. KO) (n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters).


A . Quantification of mRNA expression by RT - qPCR of candidate miR - 34 / 449 targets that might regulate spindle orientation in E14 cortex samples from miR - 34 / 449 KO mice compared with littermate controls ( Het ) . Expression was normlaized ( norm . ) to Phosphoglycerate kinase ( PGK ) mRNA levels . Significance was tested by Welch ' s t test : p = 0 . 009756 ( JAM - A ( Het ) vs . JAM - A ( KO ) ) , all the rest Het vs . KO comparisons p = n . s . ( n = 4 cortices per genotype group , 2 independent litters ) . All subfigures were statistically tested as in A . * indicates p ≤ 0 . 05 , * * indicates p ≤ 0 . 01 , * * * indicates p ≤ 0 . 001 . Red bar indicates median .

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A. Quantification of mRNA expression by RT-qPCR of candidate miR-34/449 targets that might regulate spindle orientation in E14 cortex samples from miR-34/449 KO mice compared with littermate controls (Het). Expression was normlaized (norm.) to Phosphoglycerate kinase (PGK) mRNA levels. Significance was tested by Welch's t test: p = 0.009756 (JAM-A (Het) vs. JAM-A (KO)), all the rest Het vs. KO comparisons p=n.s. (n = 4 cortices per genotype group, 2 independent litters). All subfigures were statistically tested as in A. * indicates p ≤ 0.05, ** indicates p ≤ 0.01, *** indicates p ≤ 0.001. Red bar indicates median.


B . RT - qPCR quantification of JAM - A mRNA expression in HeLa cells 48 h after transfection of miR - 449a mimic compared to cells transfected with non - targeting negative control siRNA . JAM - A mRNA Expression was normalized ( norm . ) to Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) mRNA levels . p = 0 . 0001218 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 measurements from 3 independent replicates ) .

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B. RT-qPCR quantification of JAM-A mRNA expression in HeLa cells 48 h after transfection of miR-449a mimic compared to cells transfected with non-targeting negative control siRNA. JAM-A mRNA Expression was normalized (norm.) to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. p = 0.0001218 (Neg. ctrol. vs. miR-449a mimic) (n = 9 measurements from 3 independent replicates).


C , D . mouse J110 cells were transfected with either miR - 449a mimic or a negative control mimic . Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM - A and ACTIN . ACTIN was used to normalize JAM - A expression ( norm . ) as loading control . p = 0 . 04454 ( Neg . ctrol . vs . miR - 449a mimic ) ( n = 4 independent wells , 2 independent transfections ) .

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C, D. mouse J110 cells were transfected with either miR-449a mimic or a negative control mimic. Whole cell extracts were subjected to immunoblot analysis to detect endogenous JAM-A and ACTIN. ACTIN was used to normalize JAM-A expression (norm.) as loading control. p = 0.04454 (Neg. ctrol. vs. miR-449a mimic) (n = 4 independent wells, 2 independent transfections).


E , F . HeLa cells were cotransfected with either miR - 449a mimic or a negative control mimic , together with JAM - A - GFP fusion protein and GFP . Whole cell extracts were subjected to immunoblot analysis to detect GFP . GFP was used to normalize ( norm . ) by transfection efficiency as loading control . p = 0 . 001176 ( JAM - A WT Neg . ctrol . vs . miR - 449a mimic ) ( n = 9 independent wells , 4 independent transfections ) .

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E, F. HeLa cells were cotransfected with either miR-449a mimic or a negative control mimic, together with JAM-A-GFP fusion protein and GFP. Whole cell extracts were subjected to immunoblot analysis to detect GFP. GFP was used to normalize (norm.) by transfection efficiency as loading control. p = 0.001176 (JAM-A WT Neg. ctrol. vs. miR-449a mimic) (n = 9 independent wells, 4 independent transfections).


G , H . Confocal images of coronal sections from E14 brains of wild type mice stained with anti - JAM - A antibody ( green ) , phalloidin to label actin ( gray ) and DAPI to label cell nuclei ( blue ) . Scale bars : 10 μm ( G ) , 5 μm ( H ) . I . Quantification of JAM - A protein expression in the ventricular zone of E14 brain coronal sections from miR - 34 / 449 KO mice compared with littermate controls ( Het ) by immunofluorescence . ACTIN ( phalloidin signal ) was used to normalize ( norm . ) the expresion levels of JAM - A . p = 0 . 04842 ( n = 5 ( KO ) or 7 ( het ) cortices per genotype group , 3 independent litters ) .

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G, H. Confocal images of coronal sections from E14 brains of wild type mice stained with anti-JAM-A antibody (green), phalloidin to label actin (gray) and DAPI to label cell nuclei (blue). Scale bars: 10 μm (G), 5 μm (H).I. Quantification of JAM-A protein expression in the ventricular zone of E14 brain coronal sections from miR-34/449 KO mice compared with littermate controls (Het) by immunofluorescence. ACTIN (phalloidin signal) was used to normalize (norm.) the expresion levels of JAM-A. p = 0.04842 (n = 5 (KO) or 7 (het) cortices per genotype group, 3 independent litters).


A , D . Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker ( Centrin2 - GFP ; green ) , the microtubule marker ( α - tubulin - mRFP ; red ) and no extra protein ( A ) or mouse JAM - A ( D ) , 48 h after transfection of miR - 449a mimic , JAM - A siRNA , or non - targeting negative control siRNA . Scale bar , 10 μm . B , E . Quantification of spindle rotation for cells as shown in A and D . Each dot represents a single cell . Red bar indicates median . Significance was tested by Pairwise t - test with Bonferroni correction . p - value = 2e - 11 ( Neg . control versus miR449 mimic ) , p - value = 0 . 01928 ( Neg . control versus JAM - A siRNA ) , p - value = 0 . 17492 ( Neg . control versus miR449 mimic , under JAM - A overexpression ) . * indicates p ≤ 0 . 05 ; * * p ≤ 0 . 01 ; p * * * p ≤ 0 . 001 for all panels . C , F . Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset , measured for cells as shown in A and D ; n ≥ 20 cells for all conditions , 2 independent replicates .

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A, D. Confocal time lapse images of metaphase HeLa cells stably expressing a centriole marker (Centrin2-GFP; green), the microtubule marker (α-tubulin-mRFP; red) and no extra protein (A) or mouse JAM-A (D), 48 h after transfection of miR-449a mimic, JAM-A siRNA, or non-targeting negative control siRNA. Scale bar, 10 μm.B, E. Quantification of spindle rotation for cells as shown in A and D. Each dot represents a single cell. Red bar indicates median. Significance was tested by Pairwise t-test with Bonferroni correction. p-value = 2e-11 (Neg. control versus miR449 mimic), p-value = 0.01928 (Neg. control versus JAM-A siRNA), p-value = 0.17492 (Neg. control versus miR449 mimic, under JAM-A overexpression). * indicates p ≤ 0.05; ** p ≤ 0.01; p*** p ≤ 0.001 for all panels.C, F. Cumulative histograms of mitotic duration from nuclear envelope breakdown until anaphase onset, measured for cells as shown in A and D; n ≥ 20 cells for all conditions, 2 independent replicates.


A - Distribution of Casp3 + cells in rostral and ventral domains of an E11 . 5 Rx - Dicer mutant embryo . NCx , neocortex ; OB , olfactory bulb ; Spt , septum .

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A - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.


B , C - Marker analysis of Casp3 + cells in the rostral telencephalon of E12 . 5 Rx - Dicer mutant embryos . Most apoptotic cells are Pax6 + RGCs ( solid arrowheads ) and not Tbr1 + neurons ( open arrowheads ) . N = 3 replicates per marker .

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B,C - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker.


D , E - Distribution and abundance of apoptotic cells ( Casp3 + ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 replicates per genotype and age .

D,E - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3 replicates per genotype and age.


F , G - Distribution and abundance of mitoses ( PH3 + ) and neurons ( Tuj1 ) in the rostral telencephalic primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted line indicates basal surface , dashed line apical surface . N = 3 - 4 replicates per genotype and age . No significant differences were found between control and mutant embryos in apical nor basal mitoses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F,G - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.


A - DAPI stain of sagittal sections through the rostral telencephalon of E17 . 5 control and Rx - Dicer mutant littermates showing the neocortex ( NCx ) and olfactory bulb ( OB ; dashed line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).


B - Expression pattern of Grm1 mRNA in OB ( dotted line ) of control and Rx - Dicer mutants . C - Quantification of OB perimeter ( mean ± SEM ; symbols indicate values for individual embryos ) ; t - test , * * * P < 0 . 001 . N = 14 replicates per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 - Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants. C - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype.


D - Immunostains of E17 . 5 control and Rx - Dicer mutant brains showing the distribution of progenitor cells ( Ki67 , red ) and neurons ( Tuj1 , green ) . Arrowheads indicate rosettes . Sp , septum .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D - Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.


E - Detail of a rosette from an E17 . 5 Rx - Dicer mutant embryo displaying typical features : closed apical surface ( dotted line ) with PH3 + apical mitoses ( white arrowhead ) and basal mitoses , surrounded by Tuj1 + neurons ( green ) . Dashed line indicates the basal border of the rosette .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E - Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.


F - Rostral half of an Rx - Dicer mutant E17 . 5 brain immunostained for Pax6 , clarified and segmented to reveal rosettes ( yellow ) . C , caudal ; R , rostral .

F - Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.


G , H - BrdU incorporation and cell cycle re - entry analysis with the progenitor cell marker Ki67 at E17 . 5 , in the rostral cortex of control embryos compared to rostral rosettes of Rx - Dicer mutants . Dotted lines indicate apical surface , dashed lines indicate basal border of VZ . Arrowheads indicate double - positive cells . Data in histograms are mean ± SEM , symbols indicate values for individual embryos ; n ≥ 3 embryos ; X2 - test , * P < 0 . 05 , * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G,H - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; X2-test, *P < 0.05, ***P < 0.001.


I - Apical lumen of rosettes immunostained against apical complex proteins ( Par3 , β - Catenin ) , primary cilia ( Arl13b ) , apical mitoses ( PhVim ) and neurons ( Tuj1 ) .

I - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).


J - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo illustrating the high abundance and location of rosettes ( arrows ) , as revealed by the distribution of mitoses ( PH3 ) and neurons ( Tuj1 ) . K - S - Analysis of the regional identity of rosettes in E14 . 5 Rx - Dicer mutants . ( K ) Schema of normal transcription factor expression patterns defining telencephalic regional identity . Expression of Ngn2 , Tbr2 and Pax6 identifies rosettes in the rostro - dorsal telencephalon as having dorsal identity ( L - N , P ) ; Gsx2 , Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity ( O , Q ) ; Nkx2 . 1 identifies MGE rosettes as being normotopic ( R ) ; absence of Gsx2 in LGE rosette cells identifies them as ectopic ( S ) . Tuj1 labels neurons . In ( L , M ) , arrowheads indicate the border between dorsal and ventral territories , and dotted line indicates the outer border of the telencephalon . LGE , lateral ganglionic eminence ; MGE , medial ganglionic eminence ; P , pallium ; SP , subpallium .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.


A - Coronal section through the rostral telencephalon of an E14 . 5 Rx - Dicer mutant embryo displaying the three types of rosettes .

O O O O O O O O O O O O O O O O O O O O O O O O O

A - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.


B - High magnification of rosettes in Rx - Dicer mutants at E12 . 5 ( Type 1 ) , E14 . 5 ( Type 2 ) and E17 . 5 ( Type 3 ) , immunostained for PH3 and Tuj1 . Dashed line indicates ventricular surface ; arrowheads indicate basal mitoses ; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle .

B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.


C , D - Abundance of rosette types at the indicated ages ( C ) , and rostro - caudal distribution of total rosette abundance per age , independent of type ( D ) . n ≥ 2 brains per age .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.


F , G - Distribution of neurons ( Tuj1 + cells ) and apical adherens junction protein Par3 in the OB primordium of control and Rx - Dicer mutant embryos at the indicated ages . Dotted lines indicate basal border , dashed lines indicate apical border . White arrowheads indicate ectopic neurons and / or absence of Par3 . Arrow in ( G ) indicates accumulation of Par3 at the lumen of a nascent rosette .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.


A , B - Changes in expression levels of miRNAs between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ) ( B ) . Dashes lines indicate stringency limits in evaluation of results .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.


D , E - Changes in mRNA expression levels between control and Rx - Dicer mutants at E11 . 5 in the rostral telencephalon . Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic ( DESeq2 analysis ; Adj . P < 0 . 1 and FC > 1 . 25 ) are highlighted in red , and the three with the highest Adj . P value are identified by name . Blue dot indicates Irs2 : FC = 1 . 748 , Adj . P = 5 . 95e - 08 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.


G - Heatmaps of relative expression levels of DEGs related to apoptosis ( top ) and proliferation ( bottom ) . Rows correspond to independent biological replicates . Indicated are some example genes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.


H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis ( NES = 2 . 98 ; p = 0 ; Adj . P = 0 . 00045 ) , Regulation of cell proliferation ( NES = 2 . 40 ; p = 0 ; Adj . P = 0 . 0033 ) , Positive regulation of cell adhesion ( NES = 2 . 06 ; p = 0 . 0058 ; Adj . P = 0 . 013 ) , Regulation of response to stress ( NES = 2 . 50 ; p = 0 ; Adj . P = 0 . 0023 ) , Positive regulation of cell death ( NES = 1 . 84 ; p = 0 . 0179 ; Adj . P = 0 . 0326 ) , Apoptotic signaling pathway ( NES = 1 . 59 ; p = 0 . 047 ; Adj . P = 0 . 084 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).


I , J - ISH stains of Irs2 mRNA , and qPCR for Irs2 mRNA and let - 7 - 5p miRNA , in the rostral telencephalon of control and Rx - Dicer mutant embryos . Dashed line indicates basal border , dotted line indicates apical surface . Arrowheads indicate area with the greatest increase in Irs2 expression . BG , basal ganglia ; H , hippocampus ; NCx , neocortex ; OB , olfactory bulb ; Spt , septum . Histograms represent mean ± SEM ( logarithmic scale ) ; symbols in plots indicate values for individual embryos ; t - test , * P < 0 . 05 , * * P < 0 . 01 . N = 6 - 9 replicates per group . Scale bar , 100 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I,J - ISH stains of Irs2 mRNA, and qPCR for Irs2 mRNA and let-7-5p miRNA, in the rostral telencephalon of control and Rx-Dicer mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in Irs2 expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *P <0.05, **P <0.01. N = 6-9 replicates per group. Scale bar, 100 µm.


A - C - Immunostains for activated p53 ( red ) in the rostral telencephalon of control and Rx - Dicer mutant embryos at E12 . 5 , and quantification of positive cells ( C ) . Dashed lines indicate apical side , dotted lines indicate basal side . N = 4 replicates per genotype . Scale bar , 50µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A-C - Immunostains for activated p53 (red) in the rostral telencephalon of control and Rx-Dicer mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. N = 4 replicates per genotype. Scale bar, 50µm.


D - L - Rostral telencephalon of E12 . 5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells ( Casp3 ) , neurons ( Tuj1 ) and the apical complex protein Par3 , and quantification of cells positive for Casp3 ( H ) . N = 4 - 7 replicates per genotype . Scale bar , 50 µm .

D-L - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). N = 4-7 replicates per genotype. Scale bar, 50 µm.


M - O - Sagittal sections through the rostral telencephalon of E17 . 5 embryos of the indicated genotypes . Note the rosettes and general disorganization in Rx - Dicer single mutants , and complete rescue of this phenotype in Rx - Dicer - p53 double mutants . NCx , neocortex ; OB , olfactory bulb . Scale bar , 1 mm .

M-O - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in Rx-Dicer single mutants, and complete rescue of this phenotype in Rx-Dicer-p53 double mutants. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.


A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp - or Irs2 - encoding plasmids . Mean ± SEM ; t - test , * * * * P < 0 . 0001 . N = 6 replicates per group .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.


B - D - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitoses ( PH3 , white ) in the rostral telencephalon of wild - type E14 . 5 or E17 . 5 mouse embryos , as indicated , electroporated at E12 . 5 with the indicated plasmid combinations . Dashed line indicates the apical surface . Insets show the same field of view , with location and extent of electroporations . Note the overlap of rosettes ( arrows ) with ectopic neurons at the apical surface ( arrowheads ) and increased basal mitoses , upon overexpression of Irs2 ( C ) . Asterisk indicates absence of malformation in nearby cortex . At E17 . 5 ( D ) GFP + rosettes remain in deep cortical layers ( arrowheads ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of Irs2 (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).


E - H - Details of rosettes ( large arrows in E ; dashed lines in F - H ) from Irs2 + Gfp - overexpressing E14 . 5 embryos immunostained as indicated . Small arrows indicate apical mitoses , arrowheads indicate basal mitoses ( F , G ) and Par3 + apical surface at the centre of rosettes ( H ) , dotted lines indicate apical surface , dashed lines indicate basal side of rosettes . Inset in ( H ) is at the same scale .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H - Details of rosettes (large arrows in E; dashed lines in F-H) from Irs2+Gfp-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.


I - M - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitotic ( PH3 , white ) or apoptotic ( Casp3 , white ) cells in the rostral telencephalon of wild - type E13 . 5 embryos electroporated at E12 . 5 with the indicated plasmids , and quantification of Casp3 + cells ( M ) . Dashed line indicates apical surface , dotted lines indicate borders of the cortical plate ( CP ) . Nascent rosettes ( arrows ) are next to ectopic ventricular neurons ( arrowheads in K ' ) and basal mitoses , without significant apoptosis in VZ ( L ' , M ) . Mean ± SEM ; t - test ; ns , not significant , * P < 0 . 05 . N = 3 - 6 replicates per group . N - Distribution of neurons ( Tuj1 , red ) in the rostral telencephalon of wild - type E14 . 5 embryos electroporated at E12 . 5 with a combination of plasmids encoding Irs2 , let - 7a , let - 7b and let - 7c , and Gfp . Dashed line indicates apical surface . Formation of rosettes in Irs2 - expressing embryos is rescued by overexpression of let - 7 . Inset shows the same field of view , with location and extent of electroporation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I-M - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *P < 0.05. N = 3-6 replicates per group. N - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding Irs2, let-7a, let-7b and let-7c, and Gfp. Dashed line indicates apical surface. Formation of rosettes in Irs2-expressing embryos is rescued by overexpression of let-7. Inset shows the same field of view, with location and extent of electroporation.


A - Frequency of rosette formation in the experimental conditions indicated . n , number of embryos per condition .

A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition.


B - G - Distribution of neurons ( Tuj1 , red ) , GFP ( green ) and mitoses ( PH3 , white ) in the rostral telencephalon of wild - type E14 . 5 mouse embryos electroporated at E12 . 5 with the indicated plasmid combinations . Inset shows the same field of view , with location and extent of electroporations . In ( C ) and ( E ) , arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias . ( D ) and ( F ) are high magnification details of individual rosettes ( dashed lines ) , where arrowheads indicate apical and basal mitoses . Loss of endogenous let - 7 drives the formation of rosettes and ventricular neuronal ectopias ( C ) , which is rescued with the additional loss of Irs2 ( G ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).


B - G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations , to reveal the frequency of electroporated cells remaining as progenitors ( % GFP + Ki67 + / GFP + ) . Histograms represent mean ± SEM ; symbols in plots indicate values for individual organoids ; X2 - test , * P < 0 . 05 . N = 8 - 11 replicates per group . Dashed lines indicate ventricular surface , solid arrowheads indicate Ki67 + / GFP + cells , open arrowheads indicate Ki67 - / GFP + cells . Scale bars , 50 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-G - Stains and quantifications of human cerebral organoids electroporated with the indicated construct combinations, to reveal the frequency of electroporated cells remaining as progenitors (%GFP+Ki67+/GFP+). Histograms represent mean ± SEM; symbols in plots indicate values for individual organoids; X2-test, *P < 0.05. N = 8-11 replicates per group. Dashed lines indicate ventricular surface, solid arrowheads indicate Ki67+/GFP+ cells, open arrowheads indicate Ki67-/GFP+ cells. Scale bars, 50 µm.


B Negative stain image of 2D crystals containing the FERM - kinase region of FAK bound to a PI ( 4 , 5 ) P2 monolayer . Top right insert : Fourier transform of the 2D crystal image . Bottom right insert : 2 . 5x zoom of the main image .

O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O 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 Negative stain image of 2D crystals containing the FERM-kinase region of FAK bound to a PI(4,5)P2 monolayer. Top right insert: Fourier transform of the 2D crystal image. Bottom right insert: 2.5x zoom of the main image.


A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles ( PC ; phosphatidylcholine ) or PI ( 4 , 5 ) P2 containing vesicles . Plotted are mean values from two independent autophosphorylation reactions , each determined in duplicates ( n = 4 ) . Mutant nomenclature is as shown in Table 2 .

O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Autophosphorylation as measured in a ELISA assay is shown for WT and interaction mutants in presence of control vesicles (PC; phosphatidylcholine) or PI(4,5)P2 containing vesicles. Plotted are mean values from two independent autophosphorylation reactions, each determined in duplicates (n=4). Mutant nomenclature is as shown in Table 2.


B Catalytic turnover activity as measured in a coupled kinase assay is shown . Plotted are mean values from at least three independent 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

B Catalytic turnover activity as measured in a coupled kinase assay is shown. Plotted are mean values from at least three independent experiments.


C To determine whether autophosphorylation occurs in cis or trans , autophosphorylation was measured for WT or a kinase dead form of FAK ( KD , contains K454R mutation ) in presence or absence of a substrate deficient FAK mutant ( Y397F ) and / or PI ( 4 , 5 ) P2 vesicles . PI ( 4 , 5 ) P2 negative experiments contain control PC vesicles instead . Note that the scales of the two plots are different , i . e . autophosphorylation is increased in presence of PI ( 4 , 5 ) P2 . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) . Left : Schematic of possible cis - and trans - autophosphorylation ( red arrows ) in mixtures of FAK - WT / FAK - Y397F and FAK - KD / FAK - Y397F .

O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C To determine whether autophosphorylation occurs in cis or trans, autophosphorylation was measured for WT or a kinase dead form of FAK (KD, contains K454R mutation) in presence or absence of a substrate deficient FAK mutant (Y397F) and/or PI(4,5)P2 vesicles. PI(4,5)P2 negative experiments contain control PC vesicles instead. Note that the scales of the two plots are different, i.e. autophosphorylation is increased in presence of PI(4,5)P2. Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4). Left: Schematic of possible cis- and trans-autophosphorylation (red arrows) in mixtures of FAK-WT/FAK-Y397F and FAK-KD/FAK-Y397F.


D Src phosphorylation of Y577 in FAK in presence of PC or PI ( 4 , 5 ) P2 vesicles as determined by an ELISA method . The plot on the left is obtained in presence Src containing only the kinase domain ( Src254 - 536 ) and the plot on the right with Src containing additionally the SH3 and SH2 domains ( Src84 - 536 ) . Plotted are mean values from two independent phosphorylation reactions , each determined in duplicates ( n = 4 ) .

O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Src phosphorylation of Y577 in FAK in presence of PC or PI(4,5)P2 vesicles as determined by an ELISA method. The plot on the left is obtained in presence Src containing only the kinase domain (Src254-536) and the plot on the right with Src containing additionally the SH3 and SH2 domains (Src84-536). Plotted are mean values from two independent phosphorylation reactions, each determined in duplicates (n=4).


Interactions between WT or interface mutant FAK with a PI ( 4 , 5 ) P2 containing membrane was probed using a pulldown assay with membrane coated silica beads . Plotted are bound protein relative to the binding for WT at the highest PI ( 4 , 5 ) P2 concentration . Mutant naming is as in Table 2 . For WT the non - specific binding to a PC membrane is shown for comparison . Plotted are mean values from at least three independent experiments . Error bars : s . d .

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

Interactions between WT or interface mutant FAK with a PI(4,5)P2 containing membrane was probed using a pulldown assay with membrane coated silica beads. Plotted are bound protein relative to the binding for WT at the highest PI(4,5)P2 concentration. Mutant naming is as in Table 2. For WT the non-specific binding to a PC membrane is shown for comparison. Plotted are mean values from at least three independent experiments. Error bars: s.d.


A FAK phosphorylation of WT or interface mutant FAK in SCC cells . Whole cell lysates were subjected to western blot analysis with anti - pFAK Y397 , anti - pFAK Y576 / 577 , anti - pFAK Y861 , anti - pFAK Y925 and anti - FAK . Anti - GAPDH served as a loading control . The graph shows densitometric analysis of relative pFAK / FAK ratios ( mean ) from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

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A FAK phosphorylation of WT or interface mutant FAK in SCC cells. Whole cell lysates were subjected to western blot analysis with anti-pFAK Y397, anti-pFAK Y576/577, anti-pFAK Y861, anti-pFAK Y925 and anti-FAK. Anti-GAPDH served as a loading control. The graph shows densitometric analysis of relative pFAK/FAK ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


B Downstream signaling in SCC cells expressing WT or interface mutant FAK . Whole cell lysates are analyzed by western blot analysis with the indicated antibodies . The graph shows densitometric analysis of relative phospho / total protein ratios ( mean ) from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Downstream signaling in SCC cells expressing WT or interface mutant FAK. Whole cell lysates are analyzed by western blot analysis with the indicated antibodies. The graph shows densitometric analysis of relative phospho/total protein ratios (mean) from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


C Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK , Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti - pSrc Y416 , anti - FAK , anti - Src or non - specific IgG - agarose , followed by western blot analysis with anti - FAK , anti - pSrc Y416 and anti - Src . Anti - GAPDH served as a loading control . For full blots see source data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Src phosphorylation and interactions between FAK and Src or pSrc Y416 in SCC cells . FAK, Src or pSrc Y416 were immunoprecipitated from whole cell SCC lysates using anti-pSrc Y416, anti-FAK, anti-Src or non-specific IgG-agarose, followed by western blot analysis with anti-FAK, anti-pSrc Y416 and anti-Src. Anti-GAPDH served as a loading control. For full blots see source data.


FAK - expressing SCC cells were grown on glass coverslips , fixed and stained with anti - FAK anti - Paxillin and DAPI Representative immunofluorescence images are shown . Scale bars , 20 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-FAK anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm.


B FAK - expressing SCC cells were grown on glass coverslips , fixed and stained with anti - pSrc Y416 , anti - Paxillin and DAPI Representative immunofluorescence images are shown . Scale bars , 20 μm . The graph ( B ) shows the quantification of internalized active Src from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars , s . d . * = p < 0 . 01 .

B FAK-expressing SCC cells were grown on glass coverslips, fixed and stained with anti-pSrc Y416 , anti-Paxillin and DAPI Representative immunofluorescence images are shown. Scale bars, 20 μm. The graph (B) shows the quantification of internalized active Src from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.d. * = p < 0.01.


C Subcellular fractionation of SCC cells expressing FAK - WT or interface mutant FAK . Whole cell lysates ( WCL ) and subcellular fractions were subjected to western blot analysis with anti - FAK , anti - Tubulin ( marker for cytoplasmic fraction ; C ) , anti - RCAS ( marker for perinuclear fraction ; PN ) and anti - H4 ( marker for nuclear fraction ; N ) . The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars : s . d . * = p < 0 . 01 , # = p < 0 . 05 . For full blots see source data .

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C Subcellular fractionation of SCC cells expressing FAK-WT or interface mutant FAK. Whole cell lysates (WCL) and subcellular fractions were subjected to western blot analysis with anti-FAK, anti-Tubulin (marker for cytoplasmic fraction; C), anti-RCAS (marker for perinuclear fraction; PN) and anti-H4 (marker for nuclear fraction; N). The graph shows densitometric analysis of relative FAK amount of the FAK mutants in the different fractions from three independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars: s.d. * = p < 0.01, # = p < 0.05. For full blots see source data.


A FAK - WT , interface mutant FAK or FAK - / - SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation . Images were taken from 10 random fields after nine days ( representative images are shown ) and the relative colony size ( top graph ) as well as the number of colonies ( bottom graph ) were assessed from three independent experiments . Scale bars , 200 μm . Student ' s t - test was carried out to calculate the statistical significance . Error bars s . d . * = p < 0 . 01 , # = p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A FAK-WT, interface mutant FAK or FAK -/- SCC cells were resuspended in methylcellulose solution in growth medium on a layer of agarose and observed for 3D sphere formation. Images were taken from 10 random fields after nine days (representative images are shown) and the relative colony size (top graph) as well as the number of colonies (bottom graph) were assessed from three independent experiments. Scale bars, 200 μm. Student's t-test was carried out to calculate the statistical significance. Error bars s.d. * = p < 0.01, # = p < 0.05.


B FAK - WT , interface mutant FAK or FAK - / - SCC cells were seeded on growth factor - reduced Matrigel in serum - free media . After 72 h , invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel . Representative images of the invasion assay are shown . Scale bars , 200 μm . The graph shows relative invasion from five independent experiments . Student ' s t - test was carried out to calculate the statistical significance . Error bars , s . e . m . * = p < 0 . 01 , # = p < 0 . 05 .

B FAK-WT, interface mutant FAK or FAK -/- SCC cells were seeded on growth factor-reduced Matrigel in serum-free media. After 72 h, invasion towards a serum gradient was analyzed by staining the cells with calcein and taking images at 10 μm increments through the matrigel. Representative images of the invasion assay are shown. Scale bars, 200 μm. The graph shows relative invasion from five independent experiments. Student's t-test was carried out to calculate the statistical significance. Error bars, s.e.m. * = p < 0.01, # = p < 0.05.


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) and 2 mice were uninfected ( UI ) . Weights were compared over 15 days post infection ( pi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Weights were compared over 15 days post infection (pi).


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) and 2 mice were uninfected ( UI ) . Kaplan - Meier survival were compared over 15 days post infection ( pi ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type (WT) and TRIM21 knockout (KO) mice were infected with 10^5 FFU LCMV clone 13 (6 mice per group) and 2 mice were uninfected (UI). Kaplan-Meier survival were compared over 15 days post infection (pi).


C ) Sera from WT mice was analysed for N protein ( N ) and glycoprotein ( GP ) - specific antibodies by ELISA , each data point corresponds to one mouse from the same experiment .

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

C) Sera from WT mice was analysed for N protein (N) and glycoprotein (GP)-specific antibodies by ELISA, each data point corresponds to one mouse from the same experiment.


D ) N - specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice .

O O O O O O O O O O O O O O O O O O O O O

D) N-specific antibody ELISA was used to compare antibody responses in sera of WT and KO mice.


E - F ) In vitro neutralisation experiments were performed with sera collected day 8pi from WT , KO and UI mice . E ) Sera was pre - incubated with LCMV , then the virus - serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA . F ) Sera was electroporated into MEFs , then cells were plated in triplicate and LCMV was added 4 hours later . LCMV infection titre after 16 hours was measured by FFA ( three replicates ) .

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E-F) In vitro neutralisation experiments were performed with sera collected day 8pi from WT, KO and UI mice. E) Sera was pre-incubated with LCMV, then the virus-serum mix was added to MEF cells and LCMV infection titre after 16 hours was measured by FFA. F) Sera was electroporated into MEFs, then cells were plated in triplicate and LCMV was added 4 hours later. LCMV infection titre after 16 hours was measured by FFA (three replicates).


G ) Anti - N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O

G) Anti-N mAb KL53 was electroporated into WT and KO MEFs and subsequent LCMV infection titres were measured by FFA.


H ) Anti - N mAb KL53 was co - electroporated with recombinant N protein into WT and KO MEFs , and immunoblotting for N was performed after 3 hours . Electroporation of recombinant KL53 expressing the TRIM21 non - binding mutation H433A was unable to mediate N protein degradation .

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O

H) Anti-N mAb KL53 was co-electroporated with recombinant N protein into WT and KO MEFs, and immunoblotting for N was performed after 3 hours. Electroporation of recombinant KL53 expressing the TRIM21 non-binding mutation H433A was unable to mediate N protein degradation.


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 0 . 5 x 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) , and either received anti - N mAb KL53 ( + ) or control ( - ) intraperitoneally on days 1 and 3pi . Two mice were uninfected ( UI ) . A ) Weights were monitored throughout infection , with final day 8 weights of individual mice presented separately .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). A) Weights were monitored throughout infection, with final day 8 weights of individual mice presented separately.


Wild type ( WT ) and TRIM21 knockout ( KO ) mice were infected with 0 . 5 x 10 ^ 5 FFU LCMV clone 13 ( 6 mice per group ) , and either received anti - N mAb KL53 ( + ) or control ( - ) intraperitoneally on days 1 and 3pi . Two mice were uninfected ( UI ) . Viral titres in the spleen , liver , lung and kidney of all mice were determined by FFA day 8pi . Each data point represents one mouse , with results from two repeat experiments combined .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Wild type (WT) and TRIM21 knockout (KO) mice were infected with 0.5 x 10^5 FFU LCMV clone 13 (6 mice per group), and either received anti-N mAb KL53 (+) or control (-) intraperitoneally on days 1 and 3pi. Two mice were uninfected (UI). Viral titres in the spleen, liver, lung and kidney of all mice were determined by FFA day 8pi. Each data point represents one mouse, with results from two repeat experiments combined.


A ) CD8 T cells in WT mice were depleted by administration of anti - CD8 mAb 1 day prior to infection with 10 ^ 5 FFU LCMV . Anti - N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi . Viral titres in the spleen , lung , liver and kidney of all mice were determined by FFA day 8pi .

O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) CD8 T cells in WT mice were depleted by administration of anti-CD8 mAb 1 day prior to infection with 10^5 FFU LCMV. Anti-N mAb KL53 or PBS control was passively transferred IP on days 1 and 3pi. Viral titres in the spleen, lung, liver and kidney of all mice were determined by FFA day 8pi.


B ) Spleens from WT and KO mice + / - mAb KL53 day 8pi with 0 . 5 x 10 ^ 5 FFU LCMV were analysed for the presence of LCMV N - specific CTLs by staining with the class I N396 - 404 tetramer .

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B) Spleens from WT and KO mice +/- mAb KL53 day 8pi with 0.5 x 10^5 FFU LCMV were analysed for the presence of LCMV N-specific CTLs by staining with the class I N396-404 tetramer.


C ) Spleens from naïve WT and KO mice day 10pi with 0 . 5 x 10 ^ 5 FFU LCMV were stained with N tetramer .

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C) Spleens from naïve WT and KO mice day 10pi with 0.5 x 10^5 FFU LCMV were stained with N tetramer.


D ) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0 . 5 x 10 ^ 5 FFU LCMV . KL53 was administered on days 1 and 3pi , and N396 - specific CTLs in the spleen were measured day 8pi by tetramer staining .

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D) Macrophages were depleted in WT mice by administration of clodronate liposomes 1 day prior to infection with 0.5 x 10^5 FFU LCMV. KL53 was administered on days 1 and 3pi, and N396-specific CTLs in the spleen were measured day 8pi by tetramer staining.


A ) Splenocytes from uninfected CD45 . 1 mice , either pulsed with 3 concentrations of N peptide and cell trace violet ( CTV ) or unlabelled control cells , were transfused intravenously into WT and KO mice ( CD45 . 2 ) that had been infected with 0 . 5 x 10 ^ 5 FFU LCMV 8 days earlier . After 3 hours , spleens from recipient mice were harvested and the proportion of CTV - labelled CD45 . 1 cells was analysed by flow cytometry . Histograms from single representative uninfected ( UI ) , WT and KO mice are presented , showing the proportion of CD45 . 1 cells remaining for each of the labelled fractions normalised to mode . Summary data from all individual mice in the same experiment is presented in associated scatter plot , showing the mean ± standard error . B ) Labelled splenocytes as for ( A ) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier , and received mAb KL53 on days 1 and 3pi . Flow cytometry histograms from single representative mice of each genotype . Summary data from all mice in the experiment is presented , showing the mean ± standard error .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A) Splenocytes from uninfected CD45.1 mice, either pulsed with 3 concentrations of N peptide and cell trace violet (CTV) or unlabelled control cells, were transfused intravenously into WT and KO mice (CD45.2) that had been infected with 0.5 x 10^5 FFU LCMV 8 days earlier. After 3 hours, spleens from recipient mice were harvested and the proportion of CTV-labelled CD45.1 cells was analysed by flow cytometry. Histograms from single representative uninfected (UI), WT and KO mice are presented, showing the proportion of CD45.1 cells remaining for each of the labelled fractions normalised to mode. Summary data from all individual mice in the same experiment is presented in associated scatter plot, showing the mean ± standard error. B) Labelled splenocytes as for (A) were transfused into WT and KO mice that had been infected with LCMV 8 days earlier, and received mAb KL53 on days 1 and 3pi. Flow cytometry histograms from single representative mice of each genotype. Summary data from all mice in the experiment is presented, showing the mean ± standard error.


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four individual donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Infectivity in cell culture supernatants by plaque titration assay Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test comparing mock - and Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.


PBMCs from four individual donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Infectivity in cell culture supernatants by plaque titration assay Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Infectivity in cell culture supernatants by plaque titration assay Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four individual donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Viral RNA ( genome equivalents / ml ) concentrations in cell culture supernatants by Q - RT - PCR Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test comparing mock - and Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; Ruxo. = Ruxolitinib.


PBMCs from four individual donors were exposed to SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : Viral RNA ( genome equivalents / ml ) concentrations in cell culture supernatants by Q - RT - PCR Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test p - values > 0 . 05 were considered not significant and are not shown in the figure . n . d . = not detectable ; h . p . e . = hours post - exposure ; RMV =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O

PBMCs from four individual donors were exposed to SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: Viral RNA (genome equivalents/ml) concentrations in cell culture supernatants by Q-RT-PCR Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test p-values > 0.05 were considered not significant and are not shown in the figure. n.d. = not detectable; h.p.e. = hours post-exposure; RMV = Remdesivir


Untreated or Ruxolitinib ( 10 µM ) - treated PBMCs from four individual donors were exposed to SARS - CoV or SARS - CoV - 2 ( MOI 0 . 5 ) . PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum - free medium supplemented with 0 . 5 % gelatine were used as control condition ( Mock ) . Supernatants and individual cell fractions were collected at indicated time points post - inoculation and analyzed for : ( E ) Relative changes of cell - associated viral genomic RNA quantities by Q - RT - PCR and normalized to RNASEP levels . Data information : Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M . Statistical significance was tested using paired Student ' s t - test comparing mock - and Ruxolitinib - treated samples . p - values > 0 . 05 were considered not significant and are not shown in the figure . h . p . e . = hours post - exposure Ruxo . = Ruxolitinib .

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

Untreated or Ruxolitinib (10 µM)-treated PBMCs from four individual donors were exposed to SARS-CoV or SARS-CoV-2 (MOI 0.5). PBMCs inoculated with supernatant from Vero E6 cell cultures mixed with PBS and OptiPro serum-free medium supplemented with 0.5% gelatine were used as control condition (Mock). Supernatants and individual cell fractions were collected at indicated time points post-inoculation and analyzed for: (E) Relative changes of cell-associated viral genomic RNA quantities by Q-RT-PCR and normalized to RNASEP levels. Data information: Data were generated in four individual experiments using cells from at least four individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M. Statistical significance was tested using paired Student's t-test comparing mock- and Ruxolitinib-treated samples. p-values > 0.05 were considered not significant and are not shown in the figure. h.p.e. = hours post-exposure Ruxo. = Ruxolitinib.


RNA extracted from Ruxolitinib - treated or mock - treated , and SARS - CoV - , SARS - CoV - 2 - PBMCs was analyzed for ( A ) IFIT1 , ( B ) IFNA1 and IFNB1 mRNA expression by Q - RT - PCR at indicated time points . Suspension and adherent cell fractions were analyzed separately , except at the four hours time point . Values were normalized to cellular RNASEP expression and are shown as fold change over mock - inoculated conditions . The dotted line indicates the expression level of mock - inoculated cell cultures and is set to 1 . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M ( A - B Statistical significance between mock - and Ruxolitinib - treated samples was tested using paired Student ' s t - testing and comparing SARS - CoV to SARS - CoV - 2 - treated samples from the same donor and time points . P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) .

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

RNA extracted from Ruxolitinib-treated or mock-treated, and SARS-CoV-, SARS-CoV-2- PBMCs was analyzed for (A) IFIT1, (B) IFNA1 and IFNB1 mRNA expression by Q-RT-PCR at indicated time points. Suspension and adherent cell fractions were analyzed separately, except at the four hours time point. Values were normalized to cellular RNASEP expression and are shown as fold change over mock-inoculated conditions. The dotted line indicates the expression level of mock-inoculated cell cultures and is set to 1. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M (A-B Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor and time points. P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( C ) Supernatants from Ruxolitinib - or mock - treated and SARS - CoV - , SARS - CoV - 2 or mock - inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN - α2 , IP - 10 / CXCL10 , MCP - 1 / CCL2 and MCP - 3 / CCL7 were quantified using a Luminex - based immunoassay . PHA - or LPS - treated PBMCs were used as a positive control . Bars represent the results of a pool of four individual samples per condition . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , Statistical significance between mock - and Ruxolitinib - treated samples was tested using paired Student ' s t - testing and comparing SARS - CoV to SARS - CoV - 2 - treated samples from the same donor P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) . n . d . = not detectable .

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

(C) Supernatants from Ruxolitinib- or mock-treated and SARS-CoV-, SARS-CoV-2 or mock-inoculated PBMCs were collected 48 hours post exposure and cytokine expression of IFN-α2, IP-10/CXCL10, MCP-1/CCL2 and MCP-3/CCL7 were quantified using a Luminex-based immunoassay. PHA- or LPS-treated PBMCs were used as a positive control. Bars represent the results of a pool of four individual samples per condition. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, Statistical significance between mock- and Ruxolitinib-treated samples was tested using paired Student's t-testing and comparing SARS-CoV to SARS-CoV-2-treated samples from the same donor P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown). n.d. = not detectable.


( D ) Supernatants collected from SARS - CoV - , SARS - CoV - 2 - or mock - exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay . Data information : Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols , bars represent the mean , error bars indicate the S . E . M P - values < 0 . 05 were considered significant ( * ) , < 0 . 01 very significant ( * * ) or ≥ 0 . 05 not significant ( not shown ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Supernatants collected from SARS-CoV-, SARS-CoV-2- or mock-exposed PBMCs at indicated time points were analyzed for the release of bioactive IFN using the HL116 reporter cell assay. Data information: Data were generated in four individual experiments using PBMCs from four or more individual donors represented by different symbols, bars represent the mean, error bars indicate the S.E.M P-values < 0.05 were considered significant (*), < 0.01 very significant (**) or ≥ 0.05 not significant (not shown).


( A ) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock - , SARS - CoV - and SARS - CoV - 2 - challenged conditions for indicated cell types . Data information : Data shown in this figure are based on the analysis of two donors .

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(A) Pseudotime cell trajectory analysis and GSEA analysis using genes differentially regulated between mock-, SARS-CoV- and SARS-CoV-2-challenged conditions for indicated cell types. Data information: Data shown in this figure are based on the analysis of two donors.


( B ) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O 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) Representative UMAPs showing IFIT1 and ISG15 mRNA expression in the indicated conditions. Data information: Data shown in this figure are based on the analysis of two donors.


( C ) Volcano plot of all DEGs in SARS - CoV - 2 - exposed cells compared to mock - exposed cells in the indicated cell types . Known ISGs were colored in green based on their presence in the interferome database Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Volcano plot of all DEGs in SARS-CoV-2-exposed cells compared to mock-exposed cells in the indicated cell types. Known ISGs were colored in green based on their presence in the interferome database Data information: Data shown in this figure are based on the analysis of two donors.


( D ) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN - module gene set . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Cell trajectory maps of indicated cell types with cells colored by expression of the genes in an IFN-module gene set. Data information: Data shown in this figure are based on the analysis of two donors.


( E ) Dot plot depicting expression of selected ISGs and cytokines . Expression levels are color - coded , the percentage of cells expressing the respective gene is coded by symbol size . Data information : Data shown in this figure are based on the analysis of two donors .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Dot plot depicting expression of selected ISGs and cytokines. Expression levels are color-coded, the percentage of cells expressing the respective gene is coded by symbol size. Data information: Data shown in this figure are based on the analysis of two donors.