( D ) LDH and IL - 1β release from WT , Irgm2 - / - and Casp11 - / - BMDMs silenced for Gate16 and treated for 16 hours with 2 , 5µg / 2 . 105 cells of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O

(D) LDH and IL-1β release from WT, Irgm2-/- and Casp11-/- BMDMs silenced for Gate16 and treated for 16 hours with 2,5µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( E ) Western blot examination of caspase - 11 and processed caspase - 1 ( p20 ) and gasdermin - D ( p30 ) in supernatants and pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) , pro - caspase - 11 , Gate16 and GAPDH in cell lysates of Gate16 - silenced WT , Irgm2 - / - and GBPChr3 - / - BMDMs exposed to 2 , 5 . 105µg / 2 . 105 cells of OMVs for 16 hours . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(E) Western blot examination of caspase-11 and processed caspase-1 (p20)and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55), pro-caspase-11, Gate16 and GAPDH in cell lysates of Gate16-silenced WT, Irgm2-/- and GBPChr3-/- BMDMs exposed to 2,5.105µg/2.105 cells of OMVs for 16 hours. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( F ) Representative confocal fluorescence microscopy images and associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red , MOI 10 ) in IFNγ - primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection . Nucleus ( blue ) was stained with Hoechst ; scale bar 5µm . For quantifications , the percentage of bacteria positive for caspase - 11 - C254G - GFP was determined by combining the bacterial counts from n = 3 independent experiments and expressed as mean ± SEM . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 for the indicated comparisons using t - test with bonferroni correction . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) Representative confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red, MOI 10) in IFNγ-primed iWT BMDMs silenced for Gate16 after 4 and 8 hours of infection. Nucleus (blue) was stained with Hoechst; scale bar 5µm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n=3 independent experiments and expressed as mean ± SEM. **p ≤ 0.01, ***p ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( A ) LDH and IL - 1B release from siRNA - treated primary human Monocyte - Derived Macrophages ( hMDMs ) infected with S . Typhimurium orgA - ( MOI25 ) for 16 hours . When specified , the caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or the NLRP3 inhibitor MCC950 ( 10µM ) were added to the experiments .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O

(A) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs) infected with S. Typhimurium orgA- (MOI25) for 16 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( B ) LDH and IL - 1B release from siRNA - treated primary human Monocyte - Derived Macrophages ( hMDMs ) , primed with IFNγ ( 10UI / mL ) and PAM3CSK4 ( 100ng / mL ) and then stimulated with Nigericin ( 20µM ) for 4 hours . When specified , the caspase - 4 / 5 inhibitor Z - LEVD ( 25µM ) or the NLRP3 inhibitor MCC950 ( 10µM ) were added to the experiments .

O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-ORGANISM B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O

(B) LDH and IL-1B release from siRNA-treated primary human Monocyte-Derived Macrophages (hMDMs), primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then stimulated with Nigericin (20µM) for 4 hours. When specified, the caspase-4/5 inhibitor Z-LEVD (25µM) or the NLRP3 inhibitor MCC950 (10µM) were added to the experiments.


( C , D ) LDH and IL - 1B release from siRNA - treated WT or GBP1 / 2 / 5 - / - U937 Monocytic cell line , primed with IFNγ ( 10UI / mL ) and PAM3CSK4 ( 100ng / mL ) and then infected with ( C ) with S . Typhimurium orgA - ( MOI25 ) for 10 hours or ( D ) electroporated with 1µg of E . coli LPS for 4 hours . Φ indicates that no LPS electroporation was performed .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-CELL_LINE B-CELL_TYPE O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O

(C, D) LDH and IL-1B release from siRNA-treated WT or GBP1/2/5-/- U937 Monocytic cell line, primed with IFNγ (10UI/mL) and PAM3CSK4 (100ng/mL) and then infected with (C) with S. Typhimurium orgA- (MOI25) for 10 hours or (D) electroporated with 1µg of E. coli LPS for 4 hours. Φ indicates that no LPS electroporation was performed.


B Analysis of cell differentiation ( left ) and proliferation ( right ) in the intestine after CDK8 deletion as in ( A ) . Olfm4 , Lysozyme , PAS and Dclk1 staining was used to reveal , respectively , stem , Paneth , goblet and tuft cells . β - Catenin staining allows detection of cancer cells ( cytoplasmic vs nuclear localisation ) . Cell proliferation was assessed by PCNA , Ki - 67 and BrdU ( 2h pulse ) staining . Scatter plots represent the percentage of the area stained by each antibody ( relative to the area occupied by hematoxylin ) . For Paneth cells , BrdU , PCNA and Ki67 , only crypts were analysed . For goblet cells , crypts and villi were analysed . For Tuft cell quantification , Dclk1 - positive cells were counted in 50 villi . Colour code depicts small intestine ( green ) , proximal colon ( blue ) , and distant colon ( red ) . Mean ± SEM is shown . P - value of unpaired two - tailed t - test is indicated ( ns , not significant ; p > 0 . 05 ) . Scale bars , 25μm ( Olfm4 , Lysozyme , Dclk1 and β - Catenin ) and 50μm ( PAS , BrdU , Ki - 67 and PCNA ) . ( n = 9 biological replicates )

O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O B-TISSUE O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O B-CELL_TYPE O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD O O B-TISSUE O O O O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O B-TISSUE O O O O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O O O O O O O B-TISSUE O O O O B-TISSUE I-TISSUE O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O 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-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O

B Analysis of cell differentiation (left) and proliferation (right) in the intestine after CDK8 deletion as in (A). Olfm4, Lysozyme, PAS and Dclk1 staining was used to reveal, respectively, stem, Paneth, goblet and tuft cells. β-Catenin staining allows detection of cancer cells (cytoplasmic vs nuclear localisation). Cell proliferation was assessed by PCNA, Ki-67 and BrdU (2h pulse) staining. Scatter plots represent the percentage of the area stained by each antibody (relative to the area occupied by hematoxylin). For Paneth cells, BrdU, PCNA and Ki67, only crypts were analysed. For goblet cells, crypts and villi were analysed. For Tuft cell quantification, Dclk1-positive cells were counted in 50 villi. Colour code depicts small intestine (green), proximal colon (blue), and distant colon (red). Mean ± SEM is shown. P-value of unpaired two-tailed t-test is indicated (ns, not significant; p > 0.05). Scale bars, 25μm (Olfm4, Lysozyme, Dclk1 and β-Catenin) and 50μm (PAS, BrdU, Ki-67 and PCNA). (n=9 biological replicates)


C Analysis of mouse colon after AOM / DSS treatment . Quantification of the number of neoplastic lesions ( n = 10 for Cdk8 + / + , and n = 7 for Cdk8 - / - mice ) . P - value of unpaired t - test is indicated : ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown .

O O O B-ORGANISM B-TISSUE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O O O 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 Analysis of mouse colon after AOM/DSS treatment. Quantification of the number of neoplastic lesions (n = 10 for Cdk8+/+, and n = 7 for Cdk8 -/- mice). P-value of unpaired t-test is indicated: ns, not significant (p > 0.05). Mean ± SD is shown.


D Quantification of the percentage of the colon surface occupied by tumours . Intestine samples were stained for β - Catenin , and tumour regions with nuclear β - Catenin localisation were quantified using NDP . view software . Two - tailed p - value of unpaired t - test is indicated ; ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown . ( n = 6 biological replicates )

O O O O O O O B-TISSUE O O O O O B-TISSUE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Quantification of the percentage of the colon surface occupied by tumours. Intestine samples were stained for β-Catenin, and tumour regions with nuclear β-Catenin localisation were quantified using NDP.view software. Two-tailed p-value of unpaired t-test is indicated; ns, not significant (p > 0.05). Mean ± SD is shown. (n=6 biological replicates)


E Example of IHC with β - Catenin staining of tumour - free regions with membrane β - catenin localisation ( left ) and tumour regions with nuclear β - catenin localisation ( right ) . Scale bars , 50μm .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-EXP_ASSAY O O O O O O O O O

E Example of IHC with β-Catenin staining of tumour-free regions with membrane β-catenin localisation (left) and tumour regions with nuclear β-catenin localisation (right). Scale bars, 50μm.


A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 - / - and Cdk8 - / - / Cdk19 - / - organoids after 7 days of OH - tamoxifen treatment .

O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O

A Genotyping confirms the loss of Cdk8 exon 2 in Cdk8 -/- and Cdk8 -/-/Cdk19 -/- organoids after 7 days of OH-tamoxifen treatment.


B WB of organoid samples after 7 days of OH - tamoxifen treatment ; β - actin was used as loading control .

O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

B WB of organoid samples after 7 days of OH-tamoxifen treatment; β-actin was used as loading control.


C Phase contrast images of organoids before and after 6 days of OH - tamoxifen treatment . Scale bars , 150μm . D Quantification of organoid size at day 6 , shown in ( C ) ( mean ± SEM are shown ) . P - value , one - way ANOVA ( Bonferroni test ) : ( * ) p ≤ 0 . 05 , ( * * * ) p ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . ( n = 47 biological replicates )

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Phase contrast images of organoids before and after 6 days of OH-tamoxifen treatment. Scale bars, 150μm. D Quantification of organoid size at day 6, shown in (C) (mean ± SEM are shown). P-value, one-way ANOVA (Bonferroni test): (*) p ≤ 0.05, (***) p ≤ 0.001; ns, not significant (p > 0.05). (n=47 biological replicates)


E IHC staining of organoids ( day 7 of OH - tamoxifen treatment ) with Ki - 67 antibody . Scale bars , 100μm . F Quantification of Ki - 67 positive area ( % of the total area of the organoids ; mean ± SEM ) in the four different genotypes presented in ( E ) . Areas with positive Ki - 67 signal were detected and quantified using QuPath and ImageJ programs . Four technical replicates from 2 biological replicates of each genotype were analysed . Adjusted p - values of ordinary one - way ANOVA ( Bonferroni test ) are indicated : ( * * ) p - values ≤ 0 . 01 ; ( * ) p - values ≤ 0 . 05 ; ns , not significant ( p > 0 . 05 ) .

O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 IHC staining of organoids (day 7 of OH-tamoxifen treatment) with Ki-67 antibody. Scale bars, 100μm. F Quantification of Ki-67 positive area (% of the total area of the organoids; mean ± SEM) in the four different genotypes presented in (E). Areas with positive Ki-67 signal were detected and quantified using QuPath and ImageJ programs. Four technical replicates from 2 biological replicates of each genotype were analysed. Adjusted p-values of ordinary one-way ANOVA (Bonferroni test) are indicated: (**) p-values ≤ 0.01; (*) p-values ≤ 0.05; ns, not significant (p > 0.05).


C Left , IHC staining of WT and Cdk8 - / - / Cdk19 - / - organoids with Olfm4 ( stem cells ) , Lysozyme ( Paneth cells ) , Muc2 ( goblet cells ) , and Dclk1 ( tuft cells ) antibodies . Scale bars , 50μm ( Olfm4 , Muc2 , Dclk1 ) and 25μm ( Lysozyme ) . Right , quantification of Olfm4 , Lysozyme and Muc2 positive area ( % of the total organoid area as indicated by hematoxylin staining ; mean ± SEM ) , or the number of Dclk1 positive cells per organoid . P - value , unpaired two - tailed t - test : ( * * * ) p - value ≤ 0 . 001 ; ns , not significant ( p > 0 . 05 ) . 20 different organoids from 2 biological replicates of each genotype were used for the analysis .

O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Left, IHC staining of WT and Cdk8 -/-/Cdk19 -/- organoids with Olfm4 (stem cells), Lysozyme (Paneth cells), Muc2 (goblet cells), and Dclk1 (tuft cells) antibodies. Scale bars, 50μm (Olfm4, Muc2, Dclk1) and 25μm (Lysozyme). Right, quantification of Olfm4, Lysozyme and Muc2 positive area (% of the total organoid area as indicated by hematoxylin staining; mean ± SEM), or the number of Dclk1 positive cells per organoid. P-value, unpaired two-tailed t-test: (***) p-value ≤ 0.001; ns, not significant (p > 0.05). 20 different organoids from 2 biological replicates of each genotype were used for the analysis.


D WB of indicated proteins extracted from Wt and Cdk8 - / - / Cdk19 - / - organoids . Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation ( CFF ) . Amidoblack was used as loading control .

O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-DISEASE I-DISEASE O O O O O B-SMALL_MOLECULE O O O O O O

D WB of indicated proteins extracted from Wt and Cdk8 -/-/Cdk19 -/- organoids. Included are CFTR positive and negative control cell line samples obtained from the Cystic Fibrosis Foundation (CFF). Amidoblack was used as loading control.


A Histological PAS staining of organoids treated for 7 days with OH - tamoxifen . Scale bar , 50μm . B Quantification of PAS signal ( % of total organoid area ; mean ± SD are shown ) in the four different genotypes presented in ( A ) . Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) . Four technical replicates from 2 biological replicates of each genotype were analysed .

O B-EXP_ASSAY B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Histological PAS staining of organoids treated for 7 days with OH-tamoxifen. Scale bar, 50μm. B Quantification of PAS signal (% of total organoid area; mean ± SD are shown) in the four different genotypes presented in (A). Areas containing positive PAS staining were detected and quantified using QuPath and ImageJ programs. Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05). Four technical replicates from 2 biological replicates of each genotype were analysed.


C Representative phase contrast images of organoids at the indicated time points after 7 days of OH - tamoxifen treatment are shown . Scale bar , 100 μm . D Quantification of the time needed for mucus release ( observed as a dark staining in the center of the organoid ; mean ± SD are shown ) . Adjusted p - values of ordinary one - way Anova followed by Tukey ' s multiple comparison test are indicated : ( * * * ) p - value ≤ 0 . 001 ; ns : not significant ( p > 0 . 05 ) ; ( n = 17 for Wt , 11 for Cdk8 - / - , 18 for Cdk19 - / - , and 12 for Cdk8 - / - / Cdk19 - / - , all biological replicates ) .

O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O

C Representative phase contrast images of organoids at the indicated time points after 7 days of OH-tamoxifen treatment are shown. Scale bar, 100 μm. D Quantification of the time needed for mucus release (observed as a dark staining in the center of the organoid; mean ± SD are shown). Adjusted p-values of ordinary one-way Anova followed by Tukey's multiple comparison test are indicated: (***) p-value ≤ 0.001; ns: not significant (p > 0.05); (n= 17 for Wt, 11 for Cdk8 -/-, 18 for Cdk19 -/-, and 12 for Cdk8 -/-/ Cdk19 -/- , all biological replicates).


E Fluorescence confocal microscopy images of Calcein green - labeled WT and Cdk8 - / - / Cdk19 - / - organoids treated with forskolin . Scale bars , 100 μm . F Quantification of forskolin - induced swelling in WT organoids treated for 1hour or 24 hours with 0 . 1μM , 1μM or 10μM Senexin B ( SenB ) , as indicated , or double KO organoids ; DMSO vehicle was used as control . The surface area of individual organoids at different time points relative to the area at t = 0 ( 100 % ) was measured ( mean ± SD , n = 8 biological replicates ) . Linear regression lines are shown .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence confocal microscopy images of Calcein green-labeled WT and Cdk8-/-/Cdk19-/- organoids treated with forskolin. Scale bars, 100 μm. F Quantification of forskolin-induced swelling in WT organoids treated for 1hour or 24 hours with 0.1μM, 1μM or 10μM Senexin B (SenB), as indicated, or double KO organoids; DMSO vehicle was used as control. The surface area of individual organoids at different time points relative to the area at t = 0 (100%) was measured (mean ± SD, n=8 biological replicates). Linear regression lines are shown.


H WB of indicated proteins extracted from Senexin B - treated ( 10μM , 2h and 24h ) organoids ( left ) , and WT and Cdk8 - / - / Cdk19 - / - organoids ( right ) . Amidoblack was used as loading control .

O B-EXP_ASSAY O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O

H WB of indicated proteins extracted from Senexin B-treated (10μM, 2h and 24h) organoids (left), and WT and Cdk8 -/-/ Cdk19 -/- organoids (right). Amidoblack was used as loading control.


A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 ( Snx 631 ) for 4 or 11 days .

O B-EXP_ASSAY O O O O O B-TISSUE I-TISSUE O O B-ORGANISM O B-ORGANISM O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O

A WB of indicated proteins extracted from intestinal epithelium of control mice or mice treated with Senexin 631 (Snx 631) for 4 or 11 days.


B Left , immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 ( Snx 631 ) treatment ; scale bars 50μm . Right , quantification of PAS - and hematoxilin ( Hema ) - positive area ( mean ± SEM ) , using QuPath and ImageJ software . Adjusted p - values of unpaired t - test are shown : ( * ) p < 0 . 05 ; ns , not significant ( p > 0 . 05 ) . 8 mice ( 4 treated with Senexin 631 and and 4 control ) were used in this quantification .

O O O B-EXP_ASSAY B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE I-TISSUE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O

B Left, immunohistochemical PAS and hematoxylin staining of mouse small intestines collected at 4 and 11 days of Senexin 631 (Snx 631) treatment; scale bars 50μm. Right, quantification of PAS- and hematoxilin (Hema)-positive area (mean ± SEM), using QuPath and ImageJ software. Adjusted p-values of unpaired t-test are shown: (*) p < 0.05; ns, not significant (p > 0.05). 8 mice (4 treated with Senexin 631 and and 4 control) were used in this quantification.


Changes with time in amounts of SH - EP , α - amylase , and VmPE - 1 in cotyledons of V . mungo seedlings ( Attached cotyledons ) or embryo axis - removed cotyledons of V . mungo seeds ( Detached cotyledons ) . Attached or detached cotyledons were prepared as described in Materials and methods . 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris - Cl ( pH 7 . 4 ) , and the homogenates were centrifuged at 15 , 000 g for 10 min . Supernatants ( 10 μl each ) were analyzed by SDS - PAGE immunoblotting with anti - SH - EP , α - amylase , or VmPE - 1 antibody .

O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-TISSUE O O O O O O B-TISSUE O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-TISSUE O O O O O B-TISSUE O O O O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O

Changes with time in amounts of SH-EP, α-amylase, and VmPE-1 in cotyledons of V. mungo seedlings (Attached cotyledons) or embryo axis-removed cotyledons of V. mungo seeds (Detached cotyledons). Attached or detached cotyledons were prepared as described in Materials and methods. 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris-Cl (pH 7.4), and the homogenates were centrifuged at 15,000 g for 10 min. Supernatants (10 μl each) were analyzed by SDS-PAGE immunoblotting with anti-SH-EP, α-amylase, or VmPE-1 antibody.


Subcellular distribution of α - amylase in V . mungo cotyledon cells . Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods . Extracts from dry seeds and day 3 cotyledons , microsomes , and PSV fractions ( 20 μg protein each ) were separated by SDS - PAGE . Proteins in the gel were analyzed by Coomassie Brilliant blue staining ( A ) , or by immunoblotting with anti - SH - EP antibody ( B ) or anti - α - amylase antibody ( C ) . Bracket indicates the polypeptides derived from storage globulins . Arrowhead indicates α - amylase .

O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM I-ORGANISM I-ORGANISM B-TISSUE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-TISSUE O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD I-GENEPROD O

Subcellular distribution of α-amylase in V. mungo cotyledon cells. Microsome and PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials and methods. Extracts from dry seeds and day 3 cotyledons, microsomes, and PSV fractions (20 μg protein each) were separated by SDS-PAGE. Proteins in the gel were analyzed by Coomassie Brilliant blue staining (A), or by immunoblotting with anti-SH-EP antibody (B) or anti- α-amylase antibody (C). Bracket indicates the polypeptides derived from storage globulins. Arrowhead indicates α-amylase.


Electron micrographs showing the immunogold localization of α - amylase in attached cotyledons cells ( A - E ) , detached cotyledons cells ( F ) , or control for immunogold labeling ( G ) . ( A ) Anti - α - amylase antibody immunogold - stained PSV , but not SG . ( B and C ) Golgi complex and PSVs were both immunogold - labeled with anti - α - amylase antibody . ( D ) Immunogold localization of α - amylase ( 15 - nm particles ) and SH - EP ( 10 - nm particles ) . α - Amylase and SH - EP were localized in PSVs and KVs , respectively . ( E ) Gold particles from anti - α - amylase antibody were detected in LVs as well as PSVs . ( F ) PSVs in detached cotyledons were immunogold labeled with anti - α - amylase antibody . ( G ) Immunogold staining of detached cotyledon cells without first antibody ( anti - α - amylase antibody ) . No gold particles were observed in the cell . CW , cell wall ; G , Golgi complex ; KV , KDEL - tailed cysteine proteinase - accumulating vesicle ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O O B-TISSUE O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-EXP_ASSAY O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-SUBCELLULAR O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SUBCELLULAR O O O O O O O O O O O B-TISSUE O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O O O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

Electron micrographs showing the immunogold localization of α-amylase in attached cotyledons cells (A-E), detached cotyledons cells (F), or control for immunogold labeling (G). (A) Anti-α-amylase antibody immunogold-stained PSV, but not SG. (B and C) Golgi complex and PSVs were both immunogold-labeled with anti- α-amylase antibody. (D) Immunogold localization of α-amylase (15-nm particles) and SH-EP (10-nm particles). α-Amylase and SH-EP were localized in PSVs and KVs, respectively. (E) Gold particles from anti-α-amylase antibody were detected in LVs as well as PSVs. (F) PSVs in detached cotyledons were immunogold labeled with anti- α-amylase antibody. (G) Immunogold staining of detached cotyledon cells without first antibody (anti-α-amylase antibody). No gold particles were observed in the cell. CW, cell wall; G, Golgi complex; KV, KDEL-tailed cysteine proteinase-accumulating vesicle; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


Electron micrographs showing ultrastructures of cotyledon cells of germinatedV . mungo seeds ( A - G ) , immunogold localization of α - amylase in cotyledon cells ( H ) , and ultrastructure of cells of detached cotyledons ( I ) . Toluidine blue ( TB ) staining of sections from cotyledons of day 3 V . mungo seedlings . Conversion from TB - stained cells ( region I ) to TB - stainless cells ( region III ) was accompanied with that of the PSV to LV . ( B ) SGs and PSVs in TB - stained cells ( A , region I ) . The PSV was filled with storage proteins . Arrowheads indicate border between SGs and the cytoplasm . ( C ) SGs and PSVs in cotyledons at region II in A . Electron density of PSVs became low . SGs were surrounded with membranous structure ( arrows ) . LED areas were found between SGs and the cytoplasm . ( D ) SG and LV in TB - stainless cells ( region III in A ) . The PSV was converted to the LV in the cells . The areas around SG with LED were enlarged . ( E ) Ultrastructure of TB - stainless cells . SGs wrapped with a LED area contacted with LVs ( arrowheads ) . Vesicles with similar density to LVs were observed . ( F ) LED membranes around the SGs fused with the LV membranes ( arrow ) . ( G ) SGs were observed in LVs . The shape of SGs were largely different from those in B - E . ( H ) An Immunogold image representing degradation of SGs by α - amylase localized in LVs . Gold particles from anti - α - amylase antibody were densely detected in the peripheral region of SGs , which is inserted into the inside of the LV . ( I ) PSVs were not converted to LVs , and SGs were not taken up into PSVs in the cells of detached cotyledons . Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells . LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars : ( A ) 50 μm ; ( E and G ) 2 μm ; ( D , F , and I ) 1 μm ; ( B , C , and H ) 200 nm .

B-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-ORGANISM I-ORGANISM O O O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O B-TISSUE O O O O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O O B-ORGANISM I-ORGANISM I-ORGANISM B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR 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-TISSUE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Electron micrographs showing ultrastructures of cotyledon cells of germinatedV. mungo seeds (A-G), immunogold localization of α-amylase in cotyledon cells (H), and ultrastructure of cells of detached cotyledons (I). Toluidine blue (TB) staining of sections from cotyledons of day 3 V. mungo seedlings. Conversion from TB-stained cells (region I) to TB-stainless cells (region III) was accompanied with that of the PSV to LV. (B) SGs and PSVs in TB-stained cells (A, region I). The PSV was filled with storage proteins. Arrowheads indicate border between SGs and the cytoplasm. (C) SGs and PSVs in cotyledons at region II in A. Electron density of PSVs became low. SGs were surrounded with membranous structure (arrows). LED areas were found between SGs and the cytoplasm. (D) SG and LV in TB-stainless cells (region III in A). The PSV was converted to the LV in the cells. The areas around SG with LED were enlarged. (E) Ultrastructure of TB- stainless cells. SGs wrapped with a LED area contacted with LVs (arrowheads). Vesicles with similar density to LVs were observed. (F) LED membranes around the SGs fused with the LV membranes (arrow). (G) SGs were observed in LVs. The shape of SGs were largely different from those in B-E. (H) An Immunogold image representing degradation of SGs by α-amylase localized in LVs. Gold particles from anti-α-amylase antibody were densely detected in the peripheral region of SGs, which is inserted into the inside of the LV. (I) PSVs were not converted to LVs, and SGs were not taken up into PSVs in the cells of detached cotyledons. Neither low density areas around SGs nor vesicles with similar density to LV was observed in the cells. LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars: (A) 50 μm; (E and G) 2 μm; (D, F, and I) 1 μm; (B, C, and H) 200 nm.


LysoTracker red staining of partially broken cotyledon cells , SGs , or cotyledon sections . Cotyledon cells , SGs , and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods . ( A ) Differential interface - contrast image of a broken cell . ( B ) Fluorescent image of A . ( C ) Differential interface - contrast image of a broken cell . ( D ) Fluorescent image of C . ( E ) Differential interface - contrast image of cells of a section from attached cotyledons . ( F ) Fluorescent image of E . ( G ) Magnified image of E . ( H ) Magnified image of F . ( I ) Differential interface - contrast image of an SG . ( J ) Fluorescent image of I . ( K ) Differential interface - contrast image of an SG . ( L ) Fluorescent image of K . ( M ) Differential interface - contrast image of cells of a section from detached cotyledons . ( N ) Fluorescent image of M . ( O ) Magnified image of M . ( P ) Magnified image of O . Bars : ( A - D , G - L , O , and P ) 20 μm ; ( E , F , M , and N ) 50 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-TISSUE O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O O O O O O O B-TISSUE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

LysoTracker red staining of partially broken cotyledon cells, SGs, or cotyledon sections. Cotyledon cells, SGs, and sections were prepared from day 3 attached or detached cotyledons and stained with LysoTracker red as described in Materials and methods. (A) Differential interface-contrast image of a broken cell. (B) Fluorescent image of A. (C) Differential interface-contrast image of a broken cell. (D) Fluorescent image of C. (E) Differential interface-contrast image of cells of a section from attached cotyledons. (F) Fluorescent image of E. (G) Magnified image of E. (H) Magnified image of F. (I) Differential interface-contrast image of an SG. (J) Fluorescent image of I. (K) Differential interface-contrast image of an SG. (L) Fluorescent image of K. (M) Differential interface-contrast image of cells of a section from detached cotyledons. (N) Fluorescent image of M. (O) Magnified image of M. (P) Magnified image of O. Bars: (A-D, G-L, O, and P) 20 μm; (E, F, M, and N) 50 μm.


Electron micrographs showing autophagosome - mediated autophagy in attached ( A - C ) and detached ( D and E ) cotyledon cells . ( A ) Two autophagosomes containing cytoplasm were observed . Mitochondria and the cytoplasm appeared to be enclosed with an ER - like double membrane . ( B ) An autophagosome containing mitochondria and cytoplasm was fused with LV ( arrowheads ) . ( C ) Autophagic body containing mitochondria was found in LV . ( D ) An autophagosome containing mitochondria and cytoplasm was observed . The PSV was not converted into the LV . ( E ) Autophagic body was observed in the PSV of detached cotyledon cells . AB , autophagic body ; AP , autophagosome ; ER , endoplasmic reticulum ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , protein storage vacuole ; SG , starch granule . Bars , 200 nm .

B-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O B-TISSUE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O

Electron micrographs showing autophagosome-mediated autophagy in attached (A-C) and detached (D and E) cotyledon cells. (A) Two autophagosomes containing cytoplasm were observed. Mitochondria and the cytoplasm appeared to be enclosed with an ER-like double membrane. (B) An autophagosome containing mitochondria and cytoplasm was fused with LV (arrowheads). (C) Autophagic body containing mitochondria was found in LV. (D) An autophagosome containing mitochondria and cytoplasm was observed. The PSV was not converted into the LV. (E) Autophagic body was observed in the PSV of detached cotyledon cells. AB, autophagic body; AP, autophagosome; ER, endoplasmic reticulum; LV, lytic vacuole; Mt, mitochondrion; PSV, protein storage vacuole; SG, starch granule. Bars, 200 nm.


E The representative images of IF - IHC staining of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 200 μm . F Quantification of numbers of Iba - 1 + cells on the meningeal regions . Mock n = 5 ; ZIKV n = 7 embryos . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The representative images of IF-IHC staining of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 200 μm. F Quantification of numbers of Iba-1+ cells on the meningeal regions. Mock n=5; ZIKV n=7 embryos. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


G The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) and DAPI ( blue ) . Scale bar , 50 μm . H Quantification of total number of Iba - 1 + cells per ChP area . I Percentage of Iba - 1 + cells on the surface of ChP . J Percentage of round - shaped Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM . Mock n = 5 ; ZIKV n = 6 biological

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-TISSUE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-TISSUE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O

G The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green) and DAPI (blue). Scale bar, 50 μm. H Quantification of total number of Iba-1+ cells per ChP area. I Percentage of Iba-1+ cells on the surface of ChP. J Percentage of round-shaped Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM. Mock n=5; ZIKV n=6 biological replicates


K The representative images of IF - IHC staining of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) , CD68 ( red ) and DAPI ( blue ) . Scale bar , 50 μm . L Percentage of CD68 + ; Iba - 1 + cells out of total Iba - 1 + cells . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test . All data are presented as mean ± SEM .

O O O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE I-TISSUE O O O O B-TISSUE I-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K The representative images of IF-IHC staining of choroid plexus of E14.5 embryonic brains with antibodies against Iba-1 (green), CD68 (red) and DAPI (blue). Scale bar, 50 μm. L Percentage of CD68+; Iba-1+ cells out of total Iba-1+ cells. Data information: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, two-tailed unpaired t-test. All data are presented as mean ± SEM.


B Representative animal tracks in the sociability test . C Percentage of time the subject mice spent in each chamber in the sociability session of three - chamber test . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . empty ( E ) chamber ; ZIKV ( n = 16 ) stranger 1 chamber vs . empty chamber ; one - way ANOVA with Bonferroni post hoc test . D Quantification of time spent investigating the stranger mouse and empty cage during sociability session . Mock ( n = 11 ) sniffing time at stranger 1 mouse vs . empty cup , ZIKV ( n = 16 ) sniffing time at stranger 1 mouse vs . empty cup . E Preference indices in sociability . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . F Representative animal tracks in the social memory test . G Percentage of time the subject mice spent in each in the social memory session . Mock ( n = 11 ) stranger 1 ( S1 ) chamber vs . stranger 2 ( S2 ) chamber , p < 0 . 0001 ; ZIKV ( n = 16 ) stranger 1 chamber vs . stranger 2 chamber ; one - way ANOVA with Bonferroni post hoc test . H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session . Mock ( n = 11 ) sniffing time at stranger 1 vs . stranger 2 mouse ; ZIKV ( n = 16 ) sniffing time at stranger 1 vs . stranger 2 mouse . I Preference indices in the social novelty session of the three - chamber test . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - tailed unpaired t - test unless described otherwise ; n represent biological replicates .

O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O 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-ORGANISM O O O O O O O O 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-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 animal tracks in the sociability test. C Percentage of time the subject mice spent in each chamber in the sociability session of three-chamber test. Mock (n=11) stranger 1 (S1) chamber vs. empty (E) chamber; ZIKV (n=16) stranger 1 chamber vs. empty chamber; one-way ANOVA with Bonferroni post hoc test. D Quantification of time spent investigating the stranger mouse and empty cage during sociability session. Mock (n=11) sniffing time at stranger 1 mouse vs. empty cup, ZIKV (n=16) sniffing time at stranger 1 mouse vs. empty cup. E Preference indices in sociability. Mock (n=11) vs. ZIKV (n=16). F Representative animal tracks in the social memory test. G Percentage of time the subject mice spent in each in the social memory session. Mock (n=11) stranger 1 (S1) chamber vs. stranger 2 (S2) chamber, p< 0.0001; ZIKV (n=16) stranger 1 chamber vs. stranger 2 chamber; one-way ANOVA with Bonferroni post hoc test. H Quantification of time spent investigating the familiar mouse and novel stranger mouse during social novelty session. Mock (n=11) sniffing time at stranger 1 vs. stranger 2 mouse; ZIKV (n=16) sniffing time at stranger 1 vs. stranger 2 mouse. I Preference indices in the social novelty session of the three-chamber test. Mock (n=11) vs. ZIKV (n=16). Data information: All data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, two-tailed unpaired t-test unless described otherwise; n represent biological replicates.


B Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC . C Quantitation of sEPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 4 mice ; ZIKV , n = 15 neurons from 5 mice in B and C . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

O O O B-EXP_ASSAY I-EXP_ASSAY B-TISSUE O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O O B-ORGANISM O B-ORGANISM O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O 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 Quantitation of sEPSC frequency mPFC from pyramidal neurons in mPFC. C Quantitation of sEPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 4 mice; ZIKV, n= 15 neurons from 5 mice in B and C. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


E Quantifications of sIPSC frequency from pyramidal neurons in mPFC . F Quantifications of sIPSC amplitude of mPFC pyramidal neurons . Mock , n = 14 neurons from 5 mice ; ZIKV , n = 18 neurons from 6 mice in E and F . Data information : Data were collected from PL and IL regions of mPFC , and were presented as mean ± SEM . * p < 0 . 05 , * * p < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-CELL_TYPE I-CELL_TYPE O O O O O O B-CELL_TYPE O O B-ORGANISM O B-ORGANISM O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O B-TISSUE O B-TISSUE O O B-TISSUE O O O O O O O O O O O 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 Quantifications of sIPSC frequency from pyramidal neurons in mPFC. F Quantifications of sIPSC amplitude of mPFC pyramidal neurons. Mock, n=14 neurons from 5 mice; ZIKV, n= 18 neurons from 6 mice in E and F. Data information: Data were collected from PL and IL regions of mPFC, and were presented as mean ± SEM. *p<0.05, **p<0.01. Two-tailed unpaired t-test unless described otherwise.


A Representative images of PV + , SST + and CAL + cells in the prelimbic ( PL , upper panel ) and infralimbic ( IL , lower panel ) cortex . Scale bars : 200 μm .

O O O O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-TISSUE O B-TISSUE O O O O O B-TISSUE O B-TISSUE O O O O B-TISSUE O O O O O O O

A Representative images of PV+, SST+ and CAL+ cells in the prelimbic (PL, upper panel) and infralimbic (IL, lower panel) cortex. Scale bars: 200 μm.


H IHC staining of mPFC stained with antibodies against vGLUT1 . Scale bars , 5 μm . I Quantification of vGLUT1 + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O 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 IHC staining of mPFC stained with antibodies against vGLUT1. Scale bars, 5 μm. I Quantification of vGLUT1+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


J IHC staining of mPFC stained with antibodies against vGAT . Scale bars , 5 μm . K Quantification of vGAT + puncta numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 . Two - tailed unpaired t - test unless described otherwise .

J IHC staining of mPFC stained with antibodies against vGAT. Scale bars, 5 μm. K Quantification of vGAT+ puncta numbers out of 100 μm2 region. Mock, n=4 mice; ZIKV, n=4 mice. Data information: All data are presented as mean ± SEM. *P<0.05, **P<0.01. Two-tailed unpaired t-test unless described otherwise.


B Representative confocal images of the vHIP stained with mCherry ( red ) and c - Fos ( green ) . C Quantification of mCherry and c - Fos double positive cells out of total mCherry - positive cells in the vHIP CA1 regions . Scale bar , 50 μm . Mock ( n = 5 mice ) vs . ZIKV ( n = 5 mice ) , p = 0 . 0013 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE B-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O B-ORGANISM O O O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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 confocal images of the vHIP stained with mCherry (red) and c-Fos (green). C Quantification of mCherry and c-Fos double positive cells out of total mCherry-positive cells in the vHIP CA1 regions. Scale bar, 50 μm. Mock (n= 5 mice) vs. ZIKV (n= 5 mice), p=0.0013. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


F Representative EPSC traces of the pyramidal neurons in the mPFC . G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC ( PL and IL ) . Mock ( n = 17 neurons from 5 mice ) vs . ZIKV ( n = 19 neurons from 5 mice ) , p = 0 . 0122 . Data information : Data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

O O B-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O B-CELL_TYPE O O B-ORGANISM O O O B-ORGANISM O O O O B-CELL_TYPE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O 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 Representative EPSC traces of the pyramidal neurons in the mPFC. G Quantification of EPSC amplitude of the pyramidal neurons in the mPFC (PL and IL). Mock (n=17 neurons from 5 mice) vs. ZIKV (n=19 neurons from 5 mice), p=0.0122. Data information: Data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


B Representative examples of GFP - labeled presynaptic boutons . Scale bars : 2 . 5 μm . C Quantification of presynaptic bouton density and weighted size in the mPFC ( PL and IL ) . Mock : n = 57 axons from 6 mice ; ZIKV : n = 63 axons from 7 mice . D Quantification of presynaptic bouton weighted size in the mPFC ( PL and IL ) . Mock : n = 2974 boutons from 6 mice ; ZIKV : n = 3486 boutons from 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O B-SUBCELLULAR O O B-ORGANISM O B-ORGANISM O O O O B-SUBCELLULAR O O B-ORGANISM O O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY O O B-TISSUE O B-TISSUE O B-TISSUE O O O O O O O B-SUBCELLULAR O O B-ORGANISM O B-ORGANISM O O O O B-SUBCELLULAR O O B-ORGANISM O O O O O O O O O O O O O O O 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 examples of GFP-labeled presynaptic boutons. Scale bars: 2.5 μm. C Quantification of presynaptic bouton density and weighted size in the mPFC (PL and IL). Mock: n= 57 axons from 6 mice; ZIKV: n= 63 axons from 7 mice. D Quantification of presynaptic bouton weighted size in the mPFC (PL and IL). Mock: n= 2974 boutons from 6 mice; ZIKV: n= 3486 boutons from 7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


H Comparisons of percentages of vHIP - innervated mPFC ( PL and IL ) neuron subtypes . Mock , n = 4 mice ; ZIKV , n = 7 mice . P = 0 . 5343 in CaMKIIa , 0 . 0117 in PV , 0 . 7491 in CAL , 0 . 4484 in SST , 0 . 1519 in other uncharacterized neuron subtypes . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

O O O O O B-TISSUE O O B-TISSUE O B-TISSUE O B-TISSUE O B-CELL_TYPE O O O O O O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O 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 Comparisons of percentages of vHIP-innervated mPFC (PL and IL) neuron subtypes. Mock, n=4 mice; ZIKV, n=7 mice. P= 0.5343 in CaMKIIa, 0.0117 in PV, 0.7491 in CAL, 0.4484 in SST, 0.1519 in other uncharacterized neuron subtypes. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


J Representative confocal images of CTB488 - labeled tdTomato - positive neurons in the vHIP . White arrowheads indicate CTB488 single positive neurons . Scale bars : 200 μm . K Quantification of the percentage of tdTomato ; CTB488 double - positive cells out of total CTB488 - positive cells . Mock , n = 7 mice ; ZIKV , n = 7 mice . Data information : All data were presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , Two - tailed unpaired t - test unless described otherwise .

O O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O O B-GENEPROD O O B-CELL_TYPE O O B-TISSUE O O O O B-SMALL_MOLECULE O O B-CELL_TYPE O O O O O O O O O O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O B-ORGANISM O O O O O O O O O O O O O O O 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 Representative confocal images of CTB488-labeled tdTomato-positive neurons in the vHIP. White arrowheads indicate CTB488 single positive neurons. Scale bars: 200 μm. K Quantification of the percentage of tdTomato;CTB488 double-positive cells out of total CTB488-positive cells. Mock, n= 7 mice; ZIKV, n=7 mice. Data information: All data were presented as mean ± SEM. *P<0.05, **P<0.01, Two-tailed unpaired t-test unless described otherwise.


C Representative animal tracks in three - chamber test . D Discrimination indices of the social memory session of three - chamber test . mCherry mock , n = 8 mice ; mCherry ZIKV , n = 10 mice ; hM4Di ZIKV n = 10 mice , mCherry mock vs . mCherry ZIKV p = 0 . 0041 ; mCherry ZIKV vs . hM4Di ZIKV p = 0 . 0115 ; and mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O B-ORGANISM O O O B-ORGANISM O B-GENEPROD O O O B-GENEPROD B-ORGANISM O O O O O O B-GENEPROD B-ORGANISM O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


E Representative confocal images of mCherry ( red ) and c - Fos ( green ) double - labeled cells in the vHIP CA1 regions . Scale bars , 50 μm . F . Quantification of the percentage of c - fos ; mCherry double positive cells out of total mCherry cells . mCherry mock , n = 6 mice ; mCherry ZIKV , n = 4 mice ; hM4Di ZIKV n = 6 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-ORGANISM O B-GENEPROD B-ORGANISM O O O O B-ORGANISM O O B-ORGANISM O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O 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 Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia . Select compounds were injected 24 hours before and on the day of LPS administration , using routes and doses specified in the methods . C57bl / 6 female mice were injected with a high - lethality dose of E . coli LPS ( 38 - 40 μg / g ) followed by a subcutaneous injection of sterile saline . Significant differences in concentration between drug and vehicle - treated pre - and post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . Percent survival was compared using a log rank Mantel Cox test

O O O O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O B-DISEASE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O O O O O 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-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O

Therapeutic leads generated using PDN were directly tested for survival benefit using a murine model of endotoxemia. Select compounds were injected 24 hours before and on the day of LPS administration, using routes and doses specified in the methods. C57bl/6 female mice were injected with a high-lethality dose of E. coli LPS (38-40 μg/g) followed by a subcutaneous injection of sterile saline. Significant differences in concentration between drug and vehicle-treated pre- and post-pubertal mice are labeled with **** (p<0.0001), *** (p<0.001, ** (p<0.01), or * (p<0.05). Percent survival was compared using a log rank Mantel Cox test


D ) In the 6 months old p32cKO heart , p62 and multi - ubiquitin staining patterns were co - localized . Scale bar , 5 µm . E ) In the 6 months old p32cKO heart , the staining patterns of p62 and the mitochondrial marker protein , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm .

O O O O O O O B-GENEPROD B-TISSUE O B-GENEPROD O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-TISSUE O O O O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.


( F ) Western blot analysis of ubiquitinated proteins using an anti - multi - ubiquitin antibody in the 6 months old WT and p32cKO heart ( n = 4 mice per group ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-TISSUE O O O O B-ORGANISM 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-GENEPROD O O O O O O O O O O

(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( B ) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart . Tissues were excited at a wavelength of 540 ( upper panel ) or 470 ( lower panel ) and emission spectra were collected with a confocal microscope at wavelengths ( band path ) of 580 - 630 nm ( upper panel ) or 510 - 560 nm ( lower panel ) . Enlarged view of the white squares is shown right . Scale bars , 20 µm . The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM ( three months old mice , n = 11 ; 6 months old mice , n = 9 - 14 , 9 months old mice , n = 9 - 15 , 12 months old mice , n = 11 - 21 ) . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 005 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O 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) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.


( D ) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts . Quantification is shown in the lower panel ( n = 4 mice per group ) . GAPDH was used as an internal control . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( A ) LC - MS / MS metabolomic analysis of NAD + , NADH , NADP + , nicotinamide and NAAD in the 6 months old WT and p32cKO hearts . NAD + and NADP + showed significantly different levels between WT and p32cKO hearts ( n = 12 ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * p < 0 . 001 , * * * p < 0 . 0002 .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD B-TISSUE O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.


( B ) Real - time PCR analysis of RNA expression of NAD - synthesizing enzymes in the 6 months old WT and p32cKO hearts . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT mice vs p32cKO mice ( n = 6 ) , * * * p < 0 . 005 , * p < 0 . 05 . The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows . NAM : nicotinamide , NA : nicotinic acid , QA : quinolic acid , NMN : nicotinamide mononucleotide .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O

(B) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.


( G ) Nmnat3 mRNA expression in 3T3 - L1 cells after 1 mM CAP treatment for 72 h . The HIF1α inhibitor ( 20 or 50 µM ) was added 2 h before the CAP treatment . Error bars are presented as mean ± SD of three independent experiments . Student ' s t - test was performed on WT cells vs WT cells treated CAP and ( or ) HIF1α inhibitor , * * p < 0 . 01 , * p < 0 . 05 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.


( I ) Nmnat3 mRNA expression in 3T3 - L1 cells after 150 µM CoCl2 treatment for 72 h ( n = 3 ) . Error bars are presented as mean ± SD of three independent experiments . Statistical significance was assessed by Student ' s t - test , * * p < 0 . 01 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.


( A ) Lysosomal acidification is impaired in p32KO MEFs . Representative images of WT and p32KO MEFs , stained with dextran - Oregon Green ( 488 ) and dextran - TMRM . Scale bar , 5 µm . Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification , and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification . Dextran - Oregon Green is quenched under acidic pH , and thus an increased green : red ratio denotes impaired lysosomal acidification . The average ± SEM of the green : red ratio in at least 30 cells in two independent experiments is presented in the plot ( normalized to WT green / red = 1 ) . Statistical significance was assessed by Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD B-CELL_TYPE O O O O O O B-GENEPROD B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-CELL_TYPE O B-SUBCELLULAR B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.


( E ) Nmnat3 ( v1 ) overexpression in p32KO MEFs rescued lysosomal function . LysoTracker Red - stained p32KO MEFs were quantified by flow cytometry . Error bars are presented as mean ± SEM of 4 independent experiments . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 001 .

O O O B-GENEPROD O O O O O B-GENEPROD B-CELL_TYPE O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.


( A ) WT MEFs were stained with dextran - Oregon Green and dextran - TMRM to examine lysosomal acidification after 10 nM FK866 treatment . FK866 decreased lysosomal acidification , which was rescued by 1 mM NMN pretreatment . The stained MEFs were quantified using a microscope ( BZ - X800 , KEYENCE ) . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

O O O O B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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) WT MEFs were stained with dextran-Oregon Green and dextran-TMRM to examine lysosomal acidification after 10 nM FK866 treatment. FK866 decreased lysosomal acidification, which was rescued by 1 mM NMN pretreatment. The stained MEFs were quantified using a microscope (BZ-X800, KEYENCE). Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( F ) To examine autophagosome and autolysosome formation , we used DAPRed , which indicates autophagosomes , and DALGreen , which indicates autolysosomes . After starvation , co - localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells . Scale bars , 10 µm . Quantification is shown on the right . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_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 O O O O O O O O O

(F) To examine autophagosome and autolysosome formation, we used DAPRed, which indicates autophagosomes, and DALGreen, which indicates autolysosomes. After starvation, co-localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells. Scale bars, 10 µm. Quantification is shown on the right. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( D ) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs , the membrane pellet ( bound ) and supernatant ( free ) were subjected to immunoblotting with GAPDH and PGK1 antibodies .

O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O

(D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies.


( I ) After addition of GAP , NAD and Pi , ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin / luciferase kit . Iodoacetate ( IA ; 4 µM ) was used to inhibit GAPDH activity . Three independent experiments were performed . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * p < 0 . 002 .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002.


C Representative plot showing gating strategy for CD4 + T cell phenotypes . Effector Memory RA ( TEMRA ) : CD45RA + CD62L - ; Effector Memory ( EM ) : CD45RA - CD62L - ; Central Memory ( CM ) : CD45RA - CD62L + ; Stem Cell Memory ( TSCM ) : CD45RA + CD62L + .

O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O O

C Representative plot showing gating strategy for CD4+ T cell phenotypes. Effector Memory RA (TEMRA): CD45RA+ CD62L-; Effector Memory (EM): CD45RA-CD62L-; Central Memory (CM): CD45RA-CD62L+; Stem Cell Memory (TSCM): CD45RA+ CD62L+.


D Percentage of GFP + cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor ( HD ; n = 11 ) or patient ( Pt ; n = 1 ) derived CD4 + T cells , measured by FACS analysis . Median ± IQR .

O O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY O O O O O O

D Percentage of GFP+ cells within T cell subpopulations 17 days after CD40LG editing of healthy male donor (HD; n=11) or patient (Pt; n=1) derived CD4+ T cells, measured by FACS analysis. Median ± IQR.


E Percentage of GFP + cells or HDR in T cells from ( D ) . Median ± IQR .

O O O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O

E Percentage of GFP+ cells or HDR in T cells from (D). Median ± IQR.


F Population composition in male HD or patient derived untreated ( n = 9 HD UT , 1 Pt UT ) or bulk edited T cells ( Treat ) from ( D ) . For each subpopulation , the comparisons between HD Treated vs UT were performed by using LME models , with random effects defined to account for the same donor and for different number of replicates per donor within group . All p - values were adjusted with Bonferroni ' s correction to account for multiple testing . In all LME models , the percentages were used in square root scale to meet the assumption of normality of the residuals of the model . Mean ± SEM .

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-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Population composition in male HD or patient derived untreated (n=9 HD UT, 1 Pt UT) or bulk edited T cells (Treat) from (D). For each subpopulation, the comparisons between HD Treated vs UT were performed by using LME models, with random effects defined to account for the same donor and for different number of replicates per donor within group. All p-values were adjusted with Bonferroni's correction to account for multiple testing. In all LME models, the percentages were used in square root scale to meet the assumption of normality of the residuals of the model. Mean ± SEM.


G Percentage of TCRBV families detected by spectratyping . UT , sorted edited ( GFP + ) , sorted non - edited ( GFP - ) CD4 + T cells derived from male HD ( n = 1 ) or patient ( Pt ; n = 1 ) were analyzed at 17 days after CD40LG editing .

O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE 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-GENEPROD O O

G Percentage of TCRBV families detected by spectratyping. UT, sorted edited (GFP+), sorted non-edited (GFP-) CD4+ T cells derived from male HD (n=1) or patient (Pt; n=1) were analyzed at 17 days after CD40LG editing.


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by Relative Fluorescence Intensity ( RFI , normalized to T0 ; left ) and percentage ( right ) on UT ( n = 3 ) , edited ( GFP + ) or unedited ( GFP - ) HD or Pt CD4 + T cells from ( D ) ( n = 9 HD , 1 Pt ) . Longitudinal comparisons between HD GFP + vs GFP - were performed with an LME model , accounting for multiple donors and separately for RFI and % CD40L + cells ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to 8 hours time - point ( * * * * p < 0 . 0001 and * p = 0 . 0450 , respectively ) . # measured on the small fraction of CD40L + cells . Median ± IQR .

O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O 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-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by Relative Fluorescence Intensity (RFI, normalized to T0; left) and percentage (right) on UT (n=3), edited (GFP+) or unedited (GFP-) HD or Pt CD4+ T cells from (D) (n=9 HD, 1 Pt). Longitudinal comparisons between HD GFP+ vs GFP- were performed with an LME model, accounting for multiple donors and separately for RFI and %CD40L+ cells (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to 8 hours time-point (****p<0.0001 and *p=0.0450, respectively). #measured on the small fraction of CD40L+ cells. Median ± IQR.


I Representative plots showing CD40L and GFP expression in UT or bulk edited ( Tretated ) CD4 + T cells derived from male HD or Pt from ( D ) at 8 hr after PMA / Ionomycin stimulation .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

I Representative plots showing CD40L and GFP expression in UT or bulk edited (Tretated) CD4+ T cells derived from male HD or Pt from (D) at 8 hr after PMA/Ionomycin stimulation.


K Left : IgG positive spots resulting from B - T cell co - culture . Right : IgG + secreting B cells , evaluated by ELISPOT assay . B cells were isolated from PB of HD and co - cultured with male HD or Pt sorted GFP + , GFP - and UT T cells , resting ( R ) or stimulated with beads ( B ) or PMA / Ionomycin ( PI ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 1 for each group ) .

O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE I-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O B-TISSUE O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

K Left: IgG positive spots resulting from B-T cell co-culture. Right: IgG+ secreting B cells, evaluated by ELISPOT assay. B cells were isolated from PB of HD and co-cultured with male HD or Pt sorted GFP+, GFP- and UT T cells, resting (R) or stimulated with beads (B) or PMA/Ionomycin (PI). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=1 for each group).


L Left : Histograms representing proliferation results from B - T cell co - culture . Right : Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co - cultured with HD or Pt T cells from ( K ) . B cells cultured alone ( - ) or in presence of sCD40L ( + ) were used as negative and positive controls , respectively ( n = 2 for each group ) . Mean ± SEM .

O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O B-CELL_TYPE I-CELL_TYPE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L Left: Histograms representing proliferation results from B-T cell co-culture. Right: Analysis of B cell proliferative capacity by Cell Trace dilution assay in allogeneic sorted B cells isolated from PB of HD and co-cultured with HD or Pt T cells from (K). B cells cultured alone (-) or in presence of sCD40L (+) were used as negative and positive controls, respectively (n=2 for each group). Mean ± SEM.


B Percentage of NGFR + cells or HDR at 17 days after CD40LG editing of male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells ( n = 7 for each group ) . Median ± IQR .

O O O B-GENEPROD O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O

B Percentage of NGFR+ cells or HDR at 17 days after CD40LG editing of male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells (n=7 for each group). Median ± IQR.


C Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by RFI ( normalized to T0 ) on UT ( n = 2 ) , edited ( NGFR + ) or unedited ( NGFR - ) HD or Pt derived CD4 + T cells ( n = 4 HD , 1 Pt ) . Median ± IQR .

O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O

C Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by RFI (normalized to T0) on UT (n=2), edited (NGFR+) or unedited (NGFR-) HD or Pt derived CD4+ T cells (n=4 HD, 1 Pt). Median ± IQR.


D Representative plots showing CD40L and NGFR expression in UT or bulk edited ( Treated ) CD4 + T cells derived from male HD or Pt from ( C ) before and 8 hr after Pma / Ionomycin stimulation .

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O

D Representative plots showing CD40L and NGFR expression in UT or bulk edited (Treated) CD4+ T cells derived from male HD or Pt from (C) before and 8 hr after Pma/Ionomycin stimulation.


E Population composition in male HD bulk edited , sorted NGFR + or sorted NGFR - CD4 + T cells from ( B ) ( n = 7 for each group ) . Friedman test ( to account for the same donor ) with p - values adjusted with Bonferroni ' s correction to account for multiple testing . Mean ± SEM .

O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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 Population composition in male HD bulk edited, sorted NGFR+ or sorted NGFR- CD4+ T cells from (B) (n=7 for each group). Friedman test (to account for the same donor) with p-values adjusted with Bonferroni's correction to account for multiple testing. Mean ± SEM.


F Human CD45 + cell engraftment in PB after transplantation of male HD UT ( n = 5 ) , bulk edited ( NGFR bulk ; n = 5 ) or sorted NGFR + ( n = 5 ) CD4 + T cells . Longitudinal comparisons were performed with an LME model , followed by an appropriate post - hoc analysis ( see Appendix Supplementary Statistical Methods ) . The reported statistical comparisons refer only to the overall difference among groups . Median ± IQR .

O B-ORGANISM B-GENEPROD O O O O B-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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 Human CD45+ cell engraftment in PB after transplantation of male HD UT (n=5), bulk edited (NGFR bulk; n=5) or sorted NGFR+ (n=5) CD4+ T cells. Longitudinal comparisons were performed with an LME model, followed by an appropriate post-hoc analysis (see Appendix Supplementary Statistical Methods). The reported statistical comparisons refer only to the overall difference among groups. Median ± IQR.


G Percentage of HDR within human cells over time , measured by ddPCR in PB of mice from ( F ) ( n = 5 for each group , measured in engrafted mice with sufficient blood material ) . Longitudinal comparisons were performed with an LME model with time treated as a continuous variable ( see Appendix Supplementary Statistical Methods ) . UT group was not included in the analysis . Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection , highlighting a constant ( and significantly different , * * * * p < 0 . 0001 ) behavior of the groups over time . Median ± IQR .

O O O B-EXP_ASSAY O B-ORGANISM O O O O O O B-EXP_ASSAY O O O B-ORGANISM O O O O O O O O O O O O O O O B-ORGANISM O O B-TISSUE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Percentage of HDR within human cells over time, measured by ddPCR in PB of mice from (F) (n=5 for each group, measured in engrafted mice with sufficient blood material). Longitudinal comparisons were performed with an LME model with time treated as a continuous variable (see Appendix Supplementary Statistical Methods). UT group was not included in the analysis. Effect of the time and the eventual different effect of the time in the groups were not retained in the final model after backward variable selection, highlighting a constant (and significantly different, ****p<0.0001) behavior of the groups over time. Median ± IQR.


H Time course of CD40L surface expression after PMA / Ionomycin stimulation measured by MFI on pooled CD4 + T cells retrieved from spleens of mice from ( F ) ( n = 1 for each group , except for NGFR - unedited group , n = 3 ) . Median ± IQR .

O O O O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O

H Time course of CD40L surface expression after PMA/Ionomycin stimulation measured by MFI on pooled CD4+ T cells retrieved from spleens of mice from (F) (n=1 for each group, except for NGFR- unedited group, n=3). Median ± IQR.


I Dot plot depicting the ratio between expression of CD40LG edited mRNA ( codon - usage optimized , CO ) and CD40LG wild - type mRNA ( WT ) in cells edited with three different donor templates from Fig EV2H . Three independent experiments ( n = 3 EF1a . GFP , 1 IRES . NGFR , 3 IRES . GFP ) . Median ± IQR .

O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O

I Dot plot depicting the ratio between expression of CD40LG edited mRNA (codon-usage optimized, CO) and CD40LG wild-type mRNA (WT) in cells edited with three different donor templates from Fig EV2H. Three independent experiments (n=3 EF1a.GFP, 1 IRES.NGFR, 3 IRES.GFP). Median ± IQR.


J Time course of expression of WT - CD40LG mRNA or CO - CD40LG mRNA , measured as fold change ( FC ) on IPO8 housekeeping gene and normalized to T0 . Sorted edited ( + ) and sorted unedited ( - ) CD4 + T cells were analyzed before and 3 , 6 , 12 hr after Actinomycin D treatment ( n = 1 for each group ) .

O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O

J Time course of expression of WT-CD40LG mRNA or CO-CD40LG mRNA, measured as fold change (FC) on IPO8 housekeeping gene and normalized to T0. Sorted edited (+) and sorted unedited (-) CD4+ T cells were analyzed before and 3, 6, 12 hr after Actinomycin D treatment (n=1 for each group).


B Representative plots showing hEGFRt expression in bulk edited CD4 + T cells derived from male HD before and 8 hr after Pma / Ionomycin stimulation . Cells were edited with the three constructs depicted in ( A ) .

O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

B Representative plots showing hEGFRt expression in bulk edited CD4+ T cells derived from male HD before and 8 hr after Pma/Ionomycin stimulation. Cells were edited with the three constructs depicted in (A).


C Representative plots showing EGFR expression in bulk edited CD4 + T cells derived from male HD at 3 days after treatment with immunotoxin ( left ) , antibody ( middle ) or toxin ( right ) .

O O O O B-GENEPROD O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O 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 Representative plots showing EGFR expression in bulk edited CD4+ T cells derived from male HD at 3 days after treatment with immunotoxin (left), antibody (middle) or toxin (right).