D ChIP assays to detect ub - H2B binding at E - box , TSS gene of tim Pdp1ε in not RNAi ( timG4 / + ; Udcr2 / UnotRNAi ) control ( timG4 / + ; Udcr2 / + ) ( n = 3 ) . Student ' s t - , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Statistical difference is measured using two - way ANOVA , P < 0 . 001 , significant effect of genotypes were found for tim E - box ( P < 0 . 01 ) , Pdp1ε E - box ( P < 0 . 05 ) , tim TSS ( P < 0 . 05 ) Pdp1ε TSS ( P < 0 . 001 ) tim gene ( P < 0 . 01 ) . G4 , GAL4 ; U , UAS .

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

D ChIP assays to detect ub-H2B binding at E-box, TSS and gene body of tim and Pdp1ε in not RNAi (timG4/+;Udcr2/UnotRNAi) and control (timG4/+;Udcr2/+) (n=3). Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Statistical difference is measured using two-way ANOVA, P<0.001, significant effect of genotypes were found for tim E-box (P<0.01), Pdp1ε E-box (P<0.05), tim TSS (P<0.05) and Pdp1ε TSS (P<0.001) and tim gene body (P<0.01). G4, GAL4; U, UAS.


E , F period of DD locomotor rhythm of flies when knocking down Nipped - A over - expressing not . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

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

E, F The period of DD locomotor rhythm of flies when knocking down Nipped-A and over-expressing not. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


G period of DD locomotor rhythm of flies co - expressing Nipped - A RNAi not RNAi . Digits on bar are number of flies tested . of rhythmicity is indicated above bars . Statistical difference is measured using one - way ANOVA , P < 0 . 001 , Tukey ' s multiple comparison , * * P < 0 . 01 , * * * / # # # / + + + / $ $ $ P < 0 . 001 , * compared with G4 control , # compared with UAS control , + compared with Nipped - A RNAi flies , $ compared with not overexpression flies . bar indicates UAS or GAL4 controls . Dashed bar indicates flies with genetically manipulated not . Gray bar indicates flies with Nipped - A knocked down . Checked bar indicates flies with both genetically manipulated not Nipped - A knocked down .

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

G The period of DD locomotor rhythm of flies co-expressing Nipped-A RNAi and not RNAi. Digits on the bar are the number of flies tested. Percentage of rhythmicity is indicated above the bars. Statistical difference is measured using one-way ANOVA, P<0.001, Tukey's multiple comparison test, **P<0.01, ***/###/+++/$$$P<0.001, * compared with the G4 control, # compared with the UAS control, + compared with the Nipped-A RNAi flies, $ compared with the not overexpression flies. White bar indicates UAS or GAL4 controls. Dashed bar indicates flies with genetically manipulated not. Gray bar indicates flies with Nipped-A knocked down. Checked bar indicates flies with both genetically manipulated not and Nipped-A knocked down.


( A ) siRNA - treated BMDMs were infected for 16 hours with S . Typhimurium ( orgA - ) LDH IL - 1β release were measured .

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

(A) siRNA-treated BMDMs were infected for 16 hours with S. Typhimurium (orgA-) and LDH and IL-1β release were measured.


( B ) ( LDH ) IL - 1β release evaluation in WT , Irgm2 - / - , GBPChr3 - / - Casp11 - / - BMDMs infected for 16 hours with different Gram - negative ( MOI 25 ) .

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

(B) Cell death (LDH) and IL-1β release evaluation in WT, Irgm2-/-, GBPChr3-/- and Casp11-/- BMDMs infected for 16 hours with different Gram-negative bacteria (MOI 25).


( C ) Western blot examination of processed caspase - 1 ( p20 ) gasdermin - D ( p30 ) in supernatants pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) GAPDH in lysates of WT Irgm2 - / - BMDMs infected for 16 h with different Gram - negative strains .

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

(C) Western blot examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2-/- BMDMs infected for 16 h with different Gram-negative bacterial strains.


( D ) IL - 1β ( % LDH ) evaluation in immortalized WT , Irgm2 - / - , Casp11 - / - Casp11 - / - Irgm2 - / - ( referred as sgIrgm2 ) BMDMs after 16 hours of E . coli , S . Typhimurium orgA - OMV treatment .

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

(D) IL-1β and cell death (% LDH) evaluation in immortalized WT, Irgm2-/-, Casp11-/- and Casp11-/-Irgm2-/- (referred as sgIrgm2) BMDMs after 16 hours of E. coli, S. Typhimurium orgA- and OMV treatment.


( E ) ( % LDH ) evaluation in IFNγ - primed WT , Irgm2 - / - Casp11 - / - BMDMs 4 hours after electroporation or not with 1µg of E . coli LPS .

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

(E) Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2-/- and Casp11-/- BMDMs 4 hours after electroporation or not with 1µg of E. coli LPS.


( F - H ) Survival of WT , Casp11 - / - Irgm2 - / - mice primed with 100µg poly ( I : C ) for 6 hours injected ( i . p . ) with 5mg / k - 1 LPS or 5 25 µg of OMVs ( n = 6 per condition ) .

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

(F-H) Survival of WT, Casp11-/- and Irgm2-/- mice primed with 100µg poly(I:C) for 6 hours and injected (i.p.) with 5mg/k-1 LPS or 5 and 25 µg of OMVs (n=6 animals per condition).


( A ) Measure of LDH IL - 1β release in WT , GBPChr3 - / - Casp11 - / - BMDMs were Irgm2 was knocked down 16 hours after exposure to 2 , 5 µg / 2 . 105 of OMVs . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(A) Measure of LDH and IL-1β release in WT, GBPChr3-/- and Casp11-/- BMDMs were Irgm2 was knocked down 16 hours after exposure to 2,5 µg/2.105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( B ) ( LDH ) IL - 1β release evaluation in Irgm2 - silenced WT GBPChr3 - / - BMDMs infected for 16 hours with either S . Tm orgA - or F . novicida ( MOI 25 ) . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

(B) Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3-/- BMDMs infected for 16 hours with either S.Tm orgA- or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences.


( C ) Florescence microscopy associated quantifications of GBP - 2 ( green ) recruitments to intracellular S . Tm orgA - - mCherry ( MOI 10 , red ) in IFNγ - primed WT Irgm2 - / - BMDMs . Nucleus was stained with Hoechst ( blue ) . Confocal shown are from one experiment are representative of n = 3 independent experiments ; scale bars 5 µm . For quantifications , of GBP - associated was quantified ' n = ' refers to number of intracellular counted in each experiment ; quantifications from n = 3 independent experiments were then plotted expressed as mean ± .

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

(C) Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA--mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2-/-BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n=3 independent experiments; scale bars 5 µm. For quantifications, the percentage of GBP-associated bacteria was quantified and 'n=' refers to the number of intracellular bacteria counted in each experiment; quantifications from n=3 independent experiments were then plotted and expressed as mean ± SEM.


( D ) Confocal microscopy associated quantifications of caspase - 11 - C254G - GFP ( green ) recruitment to S . Tm - mCherry ( orgA - , red ) in IFNγ - primed iWT , iIrgm2 - / - , iGBPChr3 - / - iGBPChr3 - / - / sgIrgm2 BMDMs after 8 hours of infection . Nucleus ( blue ) was stained with Hoescht ; scale bar 5µm . For quantifications , of positive for caspase - 11 - C254G - GFP was determined by combining from n = 3 independent experiments expressed as mean ± . * * * p ≤ 0 . 001 for indicated comparisons using t - with bonferroni correction .

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

(D) Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S.Tm-mCherry (orgA-, red) in IFNγ-primed iWT, iIrgm2-/-, iGBPChr3-/- and iGBPChr3-/-/sgIrgm2 BMDMs after 8 hours of infection. Nucleus (blue) was stained with Hoescht; 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 ≤ 0.001 for the indicated comparisons using t-test with bonferroni correction.


( A ) GFP - Trap coupled to mass spectrometry strategy used . volcano plot represents 3 independent combined experiments . Threshold selection of enriched ( red dots ) in Irgm2 - GFP fraction was set at 2 fold enrichment ( x axis ) p - value < 0 . 05 ( y axis ) . Blacks Grey dots indicate that did not reach a p - value < 0 . 05 using t - with bonferroni correction . Labelled represent top 6 enriched in Irgm2 fraction .

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

(A) GFP-Trap coupled to mass spectrometry strategy used. The volcano plot represents 3 independent combined experiments. Threshold selection of enriched proteins (red dots) in Irgm2-GFP fraction was set at 2 fold enrichment (x axis) and p-value <0.05 (y axis). Blacks and Grey dots indicate proteins that did not reach a p-value <0.05 using t-test with bonferroni correction. Labelled proteins represent the top 6 enriched proteins in the Irgm2 fraction.


( B ) Green Florescent ( GFP ) - Trap of presence of Gate16 in Irgm2 - GFP - enriched fraction from lysates of IFNγ - primed iIrgm2 - / - BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP ( Irgm2 - GFP ) or GFP alone . Arrows show presence or not of Gate16 , Irgm2 - GFP GAPDH in Irgm2 - GFP - enriched fractions , flow - through total lysates . * means non - specific

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

(B) Green Florescent Protein (GFP)-Trap assay of the presence of Gate16 in Irgm2-GFP-enriched fraction from the lysates of IFNγ-primed iIrgm2-/- BMDMs complemented with lentiviral constructs coding for a fusion of Irgm2 withGFP (Irgm2-GFP) or GFP alone. Arrows show the presence or not of Gate16, Irgm2-GFP and GAPDH in the Irgm2-GFP-enriched fractions, flow-through and total cell lysates. * means non-specific band


( C ) LDH IL - 1β release from siRNA - treated WT BMDMs , then exposed to 2 . 5 µg / 2 . 105 of OMVs for 16 hours .

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

(C) LDH and IL-1β release from siRNA-treated WT BMDMs, and then exposed to 2.5 µg/2.105 cells of OMVs for 16 hours.


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

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD 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 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 processed caspase - 1 ( p20 ) gasdermin - D ( p30 ) in supernatants pro - caspase - 1 ( p45 ) , pro - gasdermin - D ( p55 ) , pro - caspase - 11 , Gate16 GAPDH in lysates of Gate16 - silenced WT , Irgm2 - / - GBPChr3 - / - BMDMs exposed to 2 , 5 . 105µg / 2 . 105 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 B-GENEPROD I-GENEPROD I-GENEPROD 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 B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-GENEPROD O O O B-GENEPROD O O O B-CELL_TYPE 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 microscopy 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 8 hours of infection . Nucleus ( blue ) was stained with Hoechst ; scale bar 5µm . For quantifications , of positive for caspase - 11 - C254G - GFP was determined by combining from n = 3 independent experiments expressed as mean ± . * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 for indicated comparisons using t - with bonferroni correction . Si Scramble ( siScr . ) refers to RNAi pools with non - targeting sequences .

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

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

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

O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE 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 B-GENEPROD O B-SMALL_MOLECULE 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 IL - 1B release from siRNA - treated WT or GBP1 / 2 / 5 - / - U937 Monocytic , primed with IFNγ ( 10UI / mL ) PAM3CSK4 ( 100ng / mL ) 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 I-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 B-GENEPROD O O O O O B-SMALL_MOLECULE 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 of differentiation ( left ) proliferation ( right ) in intestine after CDK8 deletion as in ( A ) . Olfm4 , Lysozyme , PAS Dclk1 was used to reveal , respectively , , Paneth , goblet tuft . β - Catenin allows detection of cancer ( vs localisation ) . proliferation was assessed by PCNA , Ki - 67 BrdU ( 2h ) . Scatter plots represent of stained by each antibody ( relative to occupied by hematoxylin ) . For Paneth , BrdU , PCNA Ki67 , only crypts were analysed . For goblet , crypts villi were analysed . For Tuft , Dclk1 - positive were counted in 50 villi . Colour code depicts small intestine ( green ) , proximal colon ( blue ) , distant colon ( red ) . Mean ± is shown . P - value of unpaired two - t - is indicated ( ns , not significant ; p > 0 . 05 ) . Scale bars , 25μm ( Olfm4 , Lysozyme , Dclk1 β - Catenin ) 50μm ( PAS , BrdU , Ki - 67 PCNA ) . ( n = 9 biological replicates )

O O B-EXP_ASSAY O O O B-EXP_ASSAY 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 B-GENEPROD O O O O O O O O B-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD B-SMALL_MOLECULE O O O O O O O O B-EXP_ASSAY O O O O O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD O O B-TISSUE O O O O B-CELL_TYPE O B-TISSUE I-TISSUE O O O O B-CELL_TYPE O B-GENEPROD 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 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 B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-GENEPROD I-GENEPROD I-GENEPROD I-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 of mouse colon after AOM / DSS treatment . of number of neoplastic lesions ( n = 10 for Cdk8 + / + , n = 7 for Cdk8 - / - mice ) . P - value of unpaired t - is indicated : ns , not significant ( p > 0 . 05 ) . Mean ± SD is shown .

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 B-GENEPROD 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

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

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 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

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 with β - Catenin of tumour - free regions with β - catenin localisation ( left ) tumour regions with β - catenin localisation ( right ) . Scale bars , 50μm .

O O O 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 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 loss of Cdk8 exon 2 in Cdk8 - / - Cdk8 - / - / Cdk19 - / - organoids after 7 days of OH - tamoxifen treatment .

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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 .

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


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

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

O 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 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 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

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

O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O O B-GENEPROD O B-CELL_TYPE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O 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 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 extracted from Wt Cdk8 - / - / Cdk19 - / - organoids . Included are CFTR positive negative control samples obtained from Cystic Fibrosis Foundation ( CFF ) . Amidoblack was used as loading control .

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

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

O O B-EXP_ASSAY I-EXP_ASSAY 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 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 B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD 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 confocal microscopy of Calcein green - labeled WT Cdk8 - / - / Cdk19 - / - organoids treated with forskolin . Scale bars , 100 μm . F 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 . surface of organoids at different time points relative to at t = 0 ( 100 % ) was measured ( mean ± SD , n = 8 biological replicates ) . Linear regression lines are shown .

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

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

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

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

O O O B-EXP_ASSAY B-SMALL_MOLECULE I-SMALL_MOLECULE O B-ORGANISM B-TISSUE I-TISSUE 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 B-SMALL_MOLECULE 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 B-ORGANISM O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE 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 , VmPE - 1 in cotyledons of V . mungo seedlings ( Attached cotyledons ) or axis - removed cotyledons of V . mungo seeds ( Detached cotyledons ) . Attached or detached cotyledons were prepared as described in Materials methods . 10 pairs of cotyledons were homogenized with 3 ml of 50 mM Tris - Cl ( pH 7 . 4 ) , 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 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 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 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 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 . Microsome PSV fractions were prepared from cotyledons of day 3 seedlings as described in Materials methods . Extracts from dry seeds day 3 cotyledons , microsomes , PSV fractions ( 20 μg each ) were separated by SDS - PAGE . in gel were analyzed by Coomassie Brilliant blue ( A ) , or by immunoblotting with anti - SH - EP antibody ( B ) or anti - α - amylase antibody ( C ) . Bracket indicates 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 B-SUBCELLULAR I-SUBCELLULAR O O O O B-TISSUE O O O O O O O O O O O O O O O O B-TISSUE O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O B-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 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 showing immunogold localization of α - amylase in attached cotyledons ( A - E ) , detached cotyledons ( F ) , or control for immunogold labeling ( G ) . ( A ) Anti - α - amylase antibody immunogold - stained PSV , but not SG . ( B C ) Golgi PSVs were both immunogold - labeled with anti - α - amylase antibody . ( D ) Immunogold localization of α - amylase ( 15 - nm particles ) SH - EP ( 10 - nm particles ) . α - Amylase SH - EP were localized in PSVs 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 of detached cotyledon without first antibody ( anti - α - amylase antibody ) . No gold particles were observed in . CW , wall ; G , Golgi ; KV , KDEL - cysteine proteinase - accumulating vesicle ; LV , lytic vacuole ; Mt , mitochondrion ; PSV , storage vacuole ; SG , starch granule . Bars , 200 nm .

B-EXP_ASSAY O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-TISSUE O O O O O O O B-TISSUE 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 B-SUBCELLULAR 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 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 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 B-TISSUE 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 B-SUBCELLULAR O O O B-SUBCELLULAR O B-SUBCELLULAR O B-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 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 showing ultrastructures of cotyledon of germinatedV . mungo seeds ( A - G ) , immunogold localization of α - amylase in cotyledon ( H ) , ultrastructure of of detached cotyledons ( I ) . Toluidine blue ( TB ) of sections from cotyledons of day 3 V . mungo seedlings . Conversion from TB - stained ( region I ) to TB - stainless ( region III ) was accompanied with that of PSV to LV . ( B ) SGs PSVs in TB - stained ( A , region I ) . PSV was filled with storage . Arrowheads indicate border between SGs cytoplasm . ( C ) SGs 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 cytoplasm . ( D ) SG LV in TB - stainless ( region III in A ) . PSV was converted to LV in . areas around SG with LED were enlarged . ( E ) Ultrastructure of TB - stainless . SGs wrapped with a LED contacted with LVs ( arrowheads ) . Vesicles with similar density to LVs were observed . ( F ) LED membranes around SGs fused with LV membranes ( arrow ) . ( G ) SGs were observed in LVs . 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 peripheral region of SGs , which is inserted into inside of LV . ( I ) PSVs were not converted to LVs , SGs were not taken up into PSVs in of detached cotyledons . Neither low density areas around SGs nor vesicles with similar density to LV was observed in . LV , lytic vacuole ; Mt , mitochondrion ; PSV , storage vacuole ; SG , starch granule . Bars : ( A ) 50 μm ; ( E G ) 2 μm ; ( D , F , I ) 1 μm ; ( B , C , H ) 200 nm .

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

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

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

B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O O O O O O B-TISSUE O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O B-TISSUE O O O B-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 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 representative of IF - of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) DAPI ( blue ) . Scale bar , 200 μm . F of numbers of Iba - 1 + on meningeal regions . Mock n = 5 ; ZIKV n = 7 . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - . data are presented as mean ± .

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

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

O O O B-EXP_ASSAY O 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 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 B-TISSUE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-TISSUE 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 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 representative of IF - of choroid plexus of E14 . 5 embryonic brains with antibodies against Iba - 1 ( green ) , CD68 ( red ) DAPI ( blue ) . Scale bar , 50 μm . L of CD68 + ; Iba - 1 + out of total Iba - 1 + . Data information : * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - . data are presented as mean ± .

O O O B-EXP_ASSAY O 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 B-SMALL_MOLECULE 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 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

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 tracks in sociability . C of time subject mice spent in each chamber in sociability session of three - chamber . 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 . D of time spent investigating stranger mouse 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 tracks in social memory . G of time subject mice spent in each in 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 . H of time spent investigating familiar mouse 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 social novelty session of three - chamber . Mock ( n = 11 ) vs . ZIKV ( n = 16 ) . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 , two - unpaired t - unless described otherwise ; n represent biological replicates .

O O O O O O O O O O B-ORGANISM 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 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 B-ORGANISM 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 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 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-ORGANISM 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 B-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

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 C . Data information : Data were collected from PL IL regions of mPFC , were presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 . Two - unpaired t - 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 B-TISSUE I-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

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 F . Data information : Data were collected from PL IL regions of mPFC , were presented as mean ± . * p < 0 . 05 , * * p < 0 . 01 . Two - unpaired t - 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 B-TISSUE I-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

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 of PV + , SST + CAL + in prelimbic ( PL , upper panel ) infralimbic ( IL , lower panel ) cortex . Scale bars : 200 μm .

O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O B-TISSUE O B-TISSUE 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 of mPFC stained with antibodies against vGLUT1 . Scale bars , 5 μm . I of vGLUT1 + numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 . Two - unpaired t - unless described otherwise .

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

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 of mPFC stained with antibodies against vGAT . Scale bars , 5 μm . K of vGAT + numbers out of 100 μm2 region . Mock , n = 4 mice ; ZIKV , n = 4 mice . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 . Two - unpaired t - 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 of vHIP stained with mCherry ( red ) c - Fos ( green ) . C of mCherry c - Fos double positive out of total mCherry - positive in 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 ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

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

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 pyramidal neurons in mPFC . G of EPSC amplitude of pyramidal neurons in mPFC ( PL 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 ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

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

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 of presynaptic bouton density weighted in mPFC ( PL IL ) . Mock : n = 57 axons from 6 mice ; ZIKV : n = 63 axons from 7 mice . D of presynaptic bouton weighted in mPFC ( PL IL ) . Mock : n = 2974 boutons from 6 mice ; ZIKV : n = 3486 boutons from 7 mice . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - 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 B-SUBCELLULAR I-SUBCELLULAR O B-EXP_ASSAY O B-TISSUE O B-TISSUE I-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 B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY O B-TISSUE O B-TISSUE I-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

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 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 : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

O O O O O B-TISSUE O O B-TISSUE O B-TISSUE I-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

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 of CTB488 - labeled tdTomato - positive neurons in vHIP . arrowheads indicate CTB488 single positive neurons . Scale bars : 200 μm . K of of tdTomato ; CTB488 double - positive out of total CTB488 - positive . Mock , n = 7 mice ; ZIKV , n = 7 mice . Data information : data were presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , Two - unpaired t - unless described otherwise .

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

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 tracks in three - chamber . D Discrimination indices of social memory session of three - chamber . 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 ; mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : data are presented as mean ± . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc .

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

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

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

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 on day of LPS administration , using routes doses specified in methods . C57bl / 6 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 between drug vehicle - treated pre - post - pubertal mice are labeled with * * * * ( p < 0 . 0001 ) , * * * ( p < 0 . 001 , * * ( p < 0 . 01 ) , or * ( p < 0 . 05 ) . survival was compared using a log rank Mantel Cox

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 B-SMALL_MOLECULE 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 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-EXP_ASSAY 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 6 months old p32cKO heart , p62 multi - ubiquitin patterns were co - localized . Scale bar , 5 µm . E ) In 6 months old p32cKO heart , patterns of p62 mitochondrial marker , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm .

O O O O O O B-GENEPROD B-TISSUE O B-GENEPROD O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-GENEPROD B-TISSUE O O O B-GENEPROD B-SUBCELLULAR 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 of ubiquitinated using an anti - multi - ubiquitin antibody in 6 months old WT p32cKO heart ( n = 4 mice per group ) . Error bars are presented as mean ± SD . Student ' s t - was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-SMALL_MOLECULE 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 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 nucleus in 6 months old p32cKO heart . Tissues were excited at a wavelength of 540 ( upper panel ) or 470 ( lower panel ) 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 squares is shown right . Scale bars , 20 µm . of of autofluorescence of DAPI is presented in plot in right panel as mean ± ( 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 - , * * * p < 0 . 005 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O B-SUBCELLULAR 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 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 B-EXP_ASSAY O B-SMALL_MOLECULE 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

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

O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD B-TISSUE 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 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 of NAD + , NADH , NADP + , nicotinamide NAAD in 6 months old WT p32cKO hearts . NAD + NADP + showed significantly different between WT p32cKO hearts ( n = 12 ) . Error bars are presented as mean ± SD . Student ' s t - 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 B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE 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 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 of expression of NAD - synthesizing enzymes in 6 months old WT p32cKO hearts . Error bars are presented as mean ± SD . Student ' s t - was performed on WT mice vs p32cKO mice ( n = 6 ) , * * * p < 0 . 005 , * p < 0 . 05 . right panel shows NAD synthesis pathway mRNA expression 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 B-EXP_ASSAY O B-SMALL_MOLECULE 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 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 B-SMALL_MOLECULE O O O B-EXP_ASSAY 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 after 1 mM CAP treatment for 72 h . HIF1α inhibitor ( 20 or 50 µM ) was added 2 h before CAP treatment . Error bars are presented as mean ± SD of three independent experiments . Student ' s t - was performed on WT vs WT treated CAP ( 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 B-SMALL_MOLECULE O O O O 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 O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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 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 - , * * 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 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

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

(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 ± of 4 independent experiments . Statistical significance was assessed by Student ' s t - , * * * 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

(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 dextran - TMRM to examine lysosomal acidification after 10 nM FK866 treatment . FK866 decreased lysosomal acidification , which was rescued by 1 mM NMN pretreatment . stained MEFs were quantified using a microscope ( BZ - X800 , KEYENCE ) . Error bars are presented as mean ± 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 I-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 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

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

O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR 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 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

(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 lysosomal fraction of WT MEFs , pellet ( bound ) supernatant ( free ) were subjected to immunoblotting with GAPDH PGK1 antibodies .

O O O O O O O O B-SMALL_MOLECULE O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD I-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 Pi , ATP production in 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 ± 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 I-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY 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

(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 . Effector Memory RA ( TEMRA ) : CD45RA + CD62L - ; Effector Memory ( EM ) : CD45RA - CD62L - ; Central Memory ( CM ) : CD45RA - CD62L + ; Memory ( TSCM ) : CD45RA + CD62L + .

O O O O O O O B-GENEPROD O B-CELL_TYPE 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 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 of GFP + within T subpopulations 17 days after CD40LG editing of healthy donor ( HD ; n = 11 ) or ( Pt ; n = 1 ) derived CD4 + T , measured by FACS . Median ± IQR .

O O B-GENEPROD O O B-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 B-GENEPROD O B-CELL_TYPE O O O B-EXP_ASSAY 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 of GFP + or HDR in T from ( D ) . Median ± IQR .

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

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

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 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 ( RFI , normalized to T0 ; left ) ( right ) on UT ( n = 3 ) , edited ( GFP + ) or unedited ( GFP - ) HD or Pt CD4 + T from ( D ) ( n = 9 HD , 1 Pt ) . Longitudinal comparisons between HD GFP + vs GFP - were performed with an LME model , accounting for multiple donors separately for RFI % CD40L + ( see Appendix Supplementary Statistical Methods ) . reported statistical comparisons refer only to 8 hours time - point ( * * * * p < 0 . 0001 * p = 0 . 0450 , respectively ) . # measured on small fraction of CD40L + . 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 B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O B-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 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 B-GENEPROD 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 GFP expression in UT or bulk edited ( Tretated ) CD4 + T derived from HD or Pt from ( D ) at 8 hr after PMA / Ionomycin stimulation .

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

O O O B-SUBCELLULAR O O O O O O B-CELL_TYPE O O O O O O B-SUBCELLULAR O O B-CELL_TYPE O O O B-EXP_ASSAY O B-CELL_TYPE O O O B-TISSUE O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O B-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 O O O O O O 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).