Figure 7C Representative animal tracks in three - chamber test . D Discrimination indices of the social memory session of three - chamber test . mCherry mock , n = 8 mice ; mCherry ZIKV , n = 10 mice ; hM4Di ZIKV n = 10 mice , mCherry mock vs . mCherry ZIKV p = 0 . 0041 ; mCherry ZIKV vs . hM4Di ZIKV p = 0 . 0115 ; and mCherry mock vs . hM4Di ZIKV p = 0 . 7076 . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test . E Representative confocal images of mCherry ( red ) and c - Fos ( green ) double - labeled cells in the vHIP CA1 regions . Scale bars , 50 μm . F . Quantification of the percentage of c - fos ; mCherry double positive cells out of total mCherry cells . mCherry mock , n = 6 mice ; mCherry ZIKV , n = 4 mice ; hM4Di ZIKV n = 6 mice . Data information : All data are presented as mean ± SEM . * P < 0 . 05 , * * P < 0 . 01 , * * * * P < 0 . 0001 , one - way ANOVA with Tukey post hoc test .

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Figure 7C Representative animal tracks in three-chamber test. D Discrimination indices of the social memory session of three-chamber test. mCherry mock, n=8 mice; mCherry ZIKV, n=10 mice; hM4Di ZIKV n=10 mice, mCherry mock vs. mCherry ZIKV p=0.0041; mCherry ZIKV vs. hM4Di ZIKV p=0.0115; and mCherry mock vs. hM4Di ZIKV p=0.7076. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.E Representative confocal images of mCherry (red) and c-Fos (green) double-labeled cells in the vHIP CA1 regions. Scale bars, 50 μm. F. Quantification of the percentage of c-fos;mCherry double positive cells out of total mCherry cells. mCherry mock, n=6 mice; mCherry ZIKV, n=4 mice; hM4Di ZIKV n=6 mice. Data information: All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey post hoc test.


Figure 1 ( C ) Co - immunoprecipitation of Cdc4 using either unphosphorylated or Clb5 - Cdk1 - phosphorylated Far1 ( 85 - 150 ) as bait . The experiment was repeated twice , a representative example is shown . ( D ) Time - lapse fluorescence images showing the expression and degradation of wild - type and S87A S91A mutant Far1 ( 1 - 150 ) - GFP and nuclear - cytoplasmic shuttling of Whi5 - mCherry . Nuclear export of Whi5 - mCherry takes place at 0 min ( Start point ) and was used to synchronize the cells for quantitative analysis . ( E , F ) Mean ± SEM fluorescence intensities of the indicated Far1 ( 1 - 150 ) - GFP mutants over the cell cycle . ( G ) Time from Start to degradation of 50 % of the indicated Far1 - GFP sensor in single cells . The bars show median ± 95 % confidence intervals . The numbers above the plot show median values . * * * * indicates p - value < 0 . 0001 , * * * < 0 . 001 and ns > 0 . 05 for pair - wise comparisons with wild - type using Mann Whitney U test . ( H ) In vitro kinase assay showing the effect of N130A , L131A , L135A and L136A mutations on Far1 ( 1 - 150 ) - GFP phosphorylation by Clb5 - Cdk1 . WT / mut shows the decrease in phosphorylation rate of the mutant compared to wild - type . The experiment was repeated twice , a representative example is shown .

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Figure 1(C) Co-immunoprecipitation of Cdc4 using either unphosphorylated or Clb5-Cdk1-phosphorylated Far1(85-150) as bait. The experiment was repeated twice, a representative example is shown.(D) Time-lapse fluorescence images showing the expression and degradation of wild-type and S87A S91A mutant Far1(1-150)-GFP and nuclear-cytoplasmic shuttling of Whi5-mCherry. Nuclear export of Whi5-mCherry takes place at 0 min (Start point) and was used to synchronize the cells for quantitative analysis.(E, F) Mean±SEM fluorescence intensities of the indicated Far1(1-150)-GFP mutants over the cell cycle.(G) Time from Start to degradation of 50% of the indicated Far1-GFP sensor in single cells. The bars show median ±95% confidence intervals. The numbers above the plot show median values. **** indicates p-value <0.0001, *** <0.001 and ns >0.05 for pair-wise comparisons with wild-type using Mann Whitney U test.(H) In vitro kinase assay showing the effect of N130A, L131A, L135A and L136A mutations on Far1(1-150)-GFP phosphorylation by Clb5-Cdk1. WT/mut shows the decrease in phosphorylation rate of the mutant compared to wild-type. The experiment was repeated twice, a representative example is shown.


Figure 2 ( A ) Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild - type or docking site mutant cyclin - Cdk1 complexes . Far1 ( 1 - 150 mut ) is L131A L135A L136A mutant , Sic1ΔC is the non - inhibitory mutant of Sic1 ( deletion of 216 - 284 ) . The experiment was repeated twice , a representative example is shown . ( B ) Mean ± SEM fluorescence intensities of wild - type and NLxxxL motif mutant Far1 ( 1 - 150 ) - GFP in either wild - type or clb5 ( hpm ) clb6 ( hpm ) strain over the cell cycle .

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Figure 2(A) Autoradiograph of a cyclin specificity analysis of the indicated substrates with wild-type or docking site mutant cyclin-Cdk1 complexes. Far1(1-150 mut) is L131A L135A L136A mutant, Sic1ΔC is the non-inhibitory mutant of Sic1 (deletion of 216-284). The experiment was repeated twice, a representative example is shown.(B) Mean±SEM fluorescence intensities of wild-type and NLxxxL motif mutant Far1(1-150)-GFP in either wild-type or clb5(hpm) clb6(hpm) strain over the cell cycle.


Figure 3 ( C ) Plot showing the mean ± SEM fluorescence intensities of Far1 ( 1 - 150 ) - GFP constructs with the indicated docking site . In rxl mutant , the NLxxxL motif is replaced with a mutated non - functional RxL motif ( VKATAF ) . ( D ) Kinetic characterization of Far1 ( 1 - 150 WT ) - GFP and Far1 ( 1 - 150 RxL ) - GFP phosphorylation by Clb5 - Cdk1 . The data is from two experimental replicates . ( E ) Mean ± SEM fluorescence intensities of wild - type or RxL - dependent Far1 ( 1 - 150 ) - GFP in wild - type or the indicated cyclin hpm mutant strains . ( F ) Pheromone sensitivity halo assay with PGAL1 - 3HA - CLB5 sic1Δ strains carrying either wild - type FAR1 , far1Δ , far1 ( L135A ) or far1 ( RxL ) . Different concentrations of α - factor were pipetted on the paper discs . On glucose plates , α - factor triggers cell cycle arrest . CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing , which is rescued by the presence of α - factor , which leads to Far1 - depedent inhibition of excess Clb5 - Cdk1 activity . The experiment was repeated twice , a representative example is shown .

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Figure 3(C) Plot showing the mean±SEM fluorescence intensities of Far1(1-150)-GFP constructs with the indicated docking site. In rxl mutant, the NLxxxL motif is replaced with a mutated non-functional RxL motif (VKATAF).(D) Kinetic characterization of Far1(1-150 WT)-GFP and Far1(1-150 RxL)-GFP phosphorylation by Clb5-Cdk1. The data is from two experimental replicates.(E) Mean±SEM fluorescence intensities of wild-type or RxL-dependent Far1(1-150)-GFP in wild-type or the indicated cyclin hpm mutant strains.(F) Pheromone sensitivity halo assay with PGAL1-3HA-CLB5 sic1Δ strains carrying either wild-type FAR1, far1Δ, far1(L135A) or far1(RxL). Different concentrations of α-factor were pipetted on the paper discs. On glucose plates, α-factor triggers cell cycle arrest. CLB5 overexpression from PGAL1 in sic1Δ strain causes lethality presumably by inhibition of replication licensing, which is rescued by the presence of α-factor, which leads to Far1-depedent inhibition of excess Clb5-Cdk1 activity. The experiment was repeated twice, a representative example is shown.


Figure 4 ( B ) The degradation of Far1 ( 1 - 150 ) - GFP with either wild - type or all serine - based phosphorylation sites and the indicated docking motifs was measured in time - lapse microscopy . Plot shows mean ± SEM of GFP fluorescence intensities from Start . ( C ) Phos - tag SDS - PAGE autoradiographs showing the Cks1 - dependency of Clb5 - Cdk1 - mediated multisite phosphorylation of Far1 ( 1 - 150 ) with either NLxxxL ( WT ) motif or RxL motif . The experiment was performed twice , a representative example is shown . ( D ) Plot showing the kinetics of Clb5 - Cdk1 - mediated phosphorylation of Far1 ( 85 - 150 ) with either wild - type ( NLxxxL ) or RxL docking motif . The data is from two replicates . ( F ) Mean ± SEM GFP fluorescence intensities of the indicated Far1 ( 1 - 85 ) constructs , synchronized at the time of Start . ( G ) Plot showing the timing of 50 % degradation of the indicated Far1 ( 85 - 150 ) constructs in single cells . The numbers above the plot show median values . The bars show median ± 95 % CI . * * * * and ns indicate p - values < 0 . 0001 and > 0 . 05 by Mann - Whitney test for pair - wise comparisons with WT .

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Figure 4(B) The degradation of Far1(1-150)-GFP with either wild-type or all serine-based phosphorylation sites and the indicated docking motifs was measured in time-lapse microscopy. Plot shows mean±SEM of GFP fluorescence intensities from Start.(C) Phos-tag SDS-PAGE autoradiographs showing the Cks1-dependency of Clb5-Cdk1-mediated multisite phosphorylation of Far1(1-150) with either NLxxxL (WT) motif or RxL motif. The experiment was performed twice, a representative example is shown.(D) Plot showing the kinetics of Clb5-Cdk1-mediated phosphorylation of Far1(85-150) with either wild-type (NLxxxL) or RxL docking motif. The data is from two replicates.(F) Mean±SEM GFP fluorescence intensities of the indicated Far1(1-85) constructs, synchronized at the time of Start.(G) Plot showing the timing of 50% degradation of the indicated Far1(85-150) constructs in single cells. The numbers above the plot show median values. The bars show median ±95% CI. **** and ns indicate p-values <0.0001 and >0.05 by Mann-Whitney test for pair-wise comparisons with WT.


Figure 5 ( C ) Plot showing the mean ± SEM fluorescence intensities of the Sic1 - based threshold sensors shown in ' A ' containing the indicated cyclin docking motif . ( D ) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50 % of peak level was measured in single cells . The black lines show median ± 95 % CI . The numbers above the plot show median degradation timings of the indicated sensors . ( E ) In vitro phosphorylation kinetics of Sic1 ( T33 + 16T45 rxl llpp ΔC ) with the indicated cyclin docking motif by Clb5 - Cdk1 . The data is from two replicate experiments . ( F ) Analysis of multisite phosphorylation of Sic1 ( T33 + 16T45 rxl llpp ΔC ) with different docking motifs using Phos - tag SDS - PAGE autoradiography . The experiment was performed twice , a representative example is shown . ( G ) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos - tag gels as shown in panel ' F ' .

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Figure 5(C) Plot showing the mean±SEM fluorescence intensities of the Sic1-based threshold sensors shown in 'A' containing the indicated cyclin docking motif.(D) The time from Start to the fluorescence levels of the threshold sensors with indicated docking motifs decreasing to 50% of peak level was measured in single cells. The black lines show median±95% CI. The numbers above the plot show median degradation timings of the indicated sensors.(E) In vitro phosphorylation kinetics of Sic1(T33+16T45 rxl llpp ΔC) with the indicated cyclin docking motif by Clb5-Cdk1. The data is from two replicate experiments.(F) Analysis of multisite phosphorylation of Sic1(T33+16T45 rxl llpp ΔC) with different docking motifs using Phos-tag SDS-PAGE autoradiography. The experiment was performed twice, a representative example is shown.(G) The fraction of maximally phosphorylated form from total phosphorylation signal was quantified from Phos-tag gels as shown in panel 'F'.


Figure 6 ( B ) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation . The experiments were repeated twice , representative examples are shown . ( C ) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5 - Cdk1 , as shown by the in vitro kinase assay . The experiments were repeated twice , representative examples are shown . ( D ) Autoradiograph of Clb5 - Cdk1 - mediated phosphorylation of indicated Fin1 mutants . The experiments were repeated twice , representative examples are shown . ( G ) The effect of different docking motifs on the degradation of Far1 ( 1 - 150 SP ) - GFP , where all TP sites are mutated to SP . Plot shows mean ± SEM of fluorescence intensities in the cell cycle . ( H ) The time from Start to the degradation of 50 % of Far1 ( 1 - 150 SP ) - GFP with the indicated docking motif was measured in single cells . The numbers above the plot show median values for the sensors , the black lines show median ± 95 % CI . * * * * and ns denote p - value < 0 . 0001 and > 0 . 05 , respectively , for pair - wise comparisons with SP WT using Mann - Whitney U test .

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Figure 6(B) Autoradiograph showing the cyclin specificity and docking dependency of Lif1 and Slx4 phosphorylation. The experiments were repeated twice, representative examples are shown.(C) NLxxxL is necessary for efficient phosphorylation of Lif1 and Slx4 by Clb5-Cdk1, as shown by the in vitro kinase assay. The experiments were repeated twice, representative examples are shown.(D) Autoradiograph of Clb5-Cdk1-mediated phosphorylation of indicated Fin1 mutants. The experiments were repeated twice, representative examples are shown.(G) The effect of different docking motifs on the degradation of Far1(1-150 SP)-GFP, where all TP sites are mutated to SP. Plot shows mean±SEM of fluorescence intensities in the cell cycle.(H) The time from Start to the degradation of 50% of Far1(1-150 SP)-GFP with the indicated docking motif was measured in single cells. The numbers above the plot show median values for the sensors, the black lines show median±95% CI. **** and ns denote p-value <0.0001 and >0.05, respectively, for pair-wise comparisons with SP WT using Mann-Whitney U test.


Figure 7 ( A ) Time - lapse microscopy was used to measure cyclin - Citrine levels during mitotic exit in unperturbed cell cycles . The cells were synchronized by the nuclear import of 50 % of Whi5 - mCherry at mitotic exit . The plot shows mean ± SEM fluorescence intensities averaged over a population of cells . The vertical line at - 14 minutes marks the estimated time of anaphase entry . ( B ) Cyclin - Citrine degradation relative to the nuclear import of 50 % Whi5 - mCherry was followed in single cells . Plot shows the time from nuclear import of 50 % Whi5 - mCherry to the degradation of 50 % of the indicated cyclin in individual cells . The error bars show 95 % CI of the mean . ( E ) The effect of cyclin specificity on the phosphorylation of Fin1 , Cnn1 and Spc110 was studied in a kinase assay . The plots show the phosphorylation by Clb5 - , Clb3 - and Clb2 - Cdk1 , the error bars show standard deviation ( n = 2 ) . ( F ) Images from a time - lapse microscopy experiment of an exemplar cell expressing Spc42 - mCherry for detection of SPBs and EGFP - Fin1 . Cells were imaged every 2 minutes , the white arrow indicates onset of anaphase . ( G ) The accumulation of EGFP - Fin1 to SPBs in anaphase was measured in experiments described in ' F ' . The plot shows mean EGFP fluorescence intensities at SPBs around the time of anaphase onset . The error bars are ± SEM .

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Figure 7(A) Time-lapse microscopy was used to measure cyclin-Citrine levels during mitotic exit in unperturbed cell cycles. The cells were synchronized by the nuclear import of 50% of Whi5-mCherry at mitotic exit. The plot shows mean ±SEM fluorescence intensities averaged over a population of cells. The vertical line at -14 minutes marks the estimated time of anaphase entry.(B) Cyclin-Citrine degradation relative to the nuclear import of 50% Whi5-mCherry was followed in single cells. Plot shows the time from nuclear import of 50% Whi5-mCherry to the degradation of 50% of the indicated cyclin in individual cells. The error bars show 95% CI of the mean.(E) The effect of cyclin specificity on the phosphorylation of Fin1, Cnn1 and Spc110 was studied in a kinase assay. The plots show the phosphorylation by Clb5-, Clb3- and Clb2-Cdk1, the error bars show standard deviation (n=2).(F) Images from a time-lapse microscopy experiment of an exemplar cell expressing Spc42-mCherry for detection of SPBs and EGFP-Fin1. Cells were imaged every 2 minutes, the white arrow indicates onset of anaphase.(G) The accumulation of EGFP-Fin1 to SPBs in anaphase was measured in experiments described in 'F'. The plot shows mean EGFP fluorescence intensities at SPBs around the time of anaphase onset. The error bars are ±SEM.


Figure 1Jacob expression in human AD . The ratio of normalized levels of pJacob / Jacob is significantly reduced in the temporal cortex of AD patients as compared to the age - matched control group . All samples are normalized to Histone3 ( H3 ) . N = 11 - 12 different subjects . ( G , H ) Jacob shRNA knockdown prevents Aβ - induced CREB shutoff . ( G ) Representative confocal images of hippocampal neurons transfected with Jacob - shRNA or scrambled ( scr ) shRNA control ( both expressing GFP ) and treated with oligomeric preparations of Aβ1 - 42 or Aβ3 ( pE ) - 42 . ( H ) Neurons transfected with a Jacob knockdown construct did not display reduction of pCREB immunofluorescence intensity after treatment with Aβ1 - 42 or Aβ3 ( pE ) - 42 . Bar plot of mean nuclear pCREB intensity normalized to untreated control . Scale bar : 10 µm . N = 29 - 39 nuclei from 2 independent experiments . ( J ) Representative confocal images of slices immunolabeled against pCREB , co - labeled with NeuN and DAPI . Scale bar : 100 µm . ( K ) Bar plot of pCREB , N = 17 - 21 slices . ( L , M ) The quantification of pCREB intensity in NeuN - positive cells revealed a statistically significant decrease in pCREB immunoreactivity in TBA2 . 1 but not in double transgenic animals ( TBA2 . 1 , - / - ) . ( L ) Representative confocal images of CA1 cryosections from 13 weeks old mice stained for NeuN , DAPI and pCREB . Scale bar : 100 µm . Data represented as cumulative frequency distribution . ( M ) Bar plot of average hippocampal pCREB nuclear immunoreactivity normalized to WT . N = 34 - 38 hippocampal sections from 10 - 11 animals . ( P ) Double transgenic TBA2 . 1 Jacob / Nsmf knockout ( TBA2 . 1 , - / - ) mice display significantly lower neuronal loss compared to TBA2 . 1 mice . The number of NeuN positive cells was normalized to WT group . N = 36 - 47 CA1 images analyzed from 10 - 12 animals per genotype . Dotted line marks value of the normalized control group - 100 % . ( Q ) Jacob knockout rescues decrease in the BdnfIV gene transcription . Bar plot of mean BdnfIV transcript levels in hippocampal homogenates normalized to β - actin as reference transcript . N = 5 - 10 hippocampi .

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Figure 1Jacob expression in human AD. The ratio of normalized levels of pJacob/Jacob is significantly reduced in the temporal cortex of AD patients as compared to the age-matched control group. All samples are normalized to Histone3 (H3). N=11-12 different subjects.(G, H) Jacob shRNA knockdown prevents Aβ-induced CREB shutoff. (G) Representative confocal images of hippocampal neurons transfected with Jacob-shRNA or scrambled (scr) shRNA control (both expressing GFP) and treated with oligomeric preparations of Aβ1-42 or Aβ3(pE)-42. (H) Neurons transfected with a Jacob knockdown construct did not display reduction of pCREB immunofluorescence intensity after treatment with Aβ1-42 or Aβ3(pE)-42. Bar plot of mean nuclear pCREB intensity normalized to untreated control. Scale bar: 10 µm. N=29-39 nuclei from 2 independent experiments.(J) Representative confocal images of slices immunolabeled against pCREB, co-labeled with NeuN and DAPI. Scale bar: 100 µm. (K) Bar plot of pCREB, N=17-21 slices.(L, M) The quantification of pCREB intensity in NeuN-positive cells revealed a statistically significant decrease in pCREB immunoreactivity in TBA2.1 but not in double transgenic animals (TBA2.1, -/-). (L) Representative confocal images of CA1 cryosections from 13 weeks old mice stained for NeuN, DAPI and pCREB. Scale bar: 100 µm. Data represented as cumulative frequency distribution. (M) Bar plot of average hippocampal pCREB nuclear immunoreactivity normalized to WT. N=34-38 hippocampal sections from 10-11 animals.(P) Double transgenic TBA2.1 Jacob/Nsmf knockout (TBA2.1, -/-) mice display significantly lower neuronal loss compared to TBA2.1 mice. The number of NeuN positive cells was normalized to WT group. N=36-47 CA1 images analyzed from 10-12 animals per genotype. Dotted line marks value of the normalized control group -100%. (Q) Jacob knockout rescues decrease in the BdnfIV gene transcription. Bar plot of mean BdnfIV transcript levels in hippocampal homogenates normalized to β-actin as reference transcript. N=5-10 hippocampi.


Figure 2 ( A ) Pull - down assays confirm a direct interaction of MBP - Jacob - 45 - 532 and His - SUMO - CREB . ( C ) The C - terminus ( CREB - 166 - 341 - tagRFP ) but not the N - terminus ( CREB - 1 - 165 - tagRFP ) of CREB closely associates with Jacob - GFP in FRET saturation experiments . ( D ) Both the N - ( Jacob - 1 - 228 - GFP ) and the C - terminus ( Jacob - 262 - 532 - GFP ) of Jacob are in close proximity to CREB - tagRFP , however , the Jacob - 1 - 228 - GFP association with CREB is significantly stronger . FRET efficiency is presented in arbitrary units from 5 - 6 independent experiments . ( G ) A proximity ligation assay revealed an interaction between Jacob and CREB in primary neurons . Dashed ROIs indicate neuronal nuclei . ( M ) Co - immunoprecipitation experiments to map the binding region of Jacob to the LIM1 domain of LMO4 revealed the association with Jacob - 179 - 246 - GFP , but not with Jacob - 45 - 172 - GFP ( CREB - binding region ) . A Jacob - LMO4 - binding mutant expressed in the nucleus does not induce CREB shutoff . Representative confocal images of hippocampal neurons transfected with ΔMyr - Jacob - GFP ( Jacob targeted to the nucleus ) or ΔMyr - Jacob - L175A - V176A - GFP . Scale bar : 10 µm . Lookup table indicates the pixel intensities from 0 to 255 . ( R , T ) The mean of nuclear ( T ) CREB immunoreactivity in Jacob - expressing neurons was normalized to non - transfected controls . N = 23 - 33 neuronal nuclei from two independent cell cultures .

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

Figure 2(A) Pull-down assays confirm a direct interaction of MBP-Jacob-45-532 and His-SUMO-CREB.(C) The C-terminus (CREB-166-341-tagRFP) but not the N-terminus (CREB-1-165-tagRFP) of CREB closely associates with Jacob-GFP in FRET saturation experiments. (D) Both the N- (Jacob-1-228-GFP) and the C-terminus (Jacob-262-532-GFP) of Jacob are in close proximity to CREB-tagRFP, however, the Jacob-1-228-GFP association with CREB is significantly stronger. FRET efficiency is presented in arbitrary units from 5-6 independent experiments.(G) A proximity ligation assay revealed an interaction between Jacob and CREB in primary neurons. Dashed ROIs indicate neuronal nuclei.(M) Co-immunoprecipitation experiments to map the binding region of Jacob to the LIM1 domain of LMO4 revealed the association with Jacob-179-246-GFP, but not with Jacob-45-172-GFP (CREB-binding region).A Jacob-LMO4-binding mutant expressed in the nucleus does not induce CREB shutoff. Representative confocal images of hippocampal neurons transfected with ΔMyr-Jacob-GFP (Jacob targeted to the nucleus) or ΔMyr-Jacob-L175A-V176A-GFP. Scale bar: 10 µm. Lookup table indicates the pixel intensities from 0 to 255. (R, T) The mean of nuclear (T) CREB immunoreactivity in Jacob-expressing neurons was normalized to non-transfected controls. N =23-33 neuronal nuclei from two independent cell cultures.


Figure 3 ( B , C ) FRET saturation experiments with ( B ) LMO4 - GFP and CREB - 1 - 165 - tagRFP or ( C ) CREB - 166 - 341 - tagRFP and CREB - GFP and LIM1 - 1 - 80 - tagRFP or LIM2 - 81 - 165 - tagRFP revealed an association between the LIM1 domain of LMO4 and the C - terminus of CREB . N = 8 independent experiments . ( D ) Recombinant His - SUMO - CREB directly binds to GST - LMO4 in pull - down experiments . ( H - K ) Knockdown of LMO4 reduces nuclear pCREB immunoreactivity . ( H , J ) Representative confocal images of hippocampal neurons transfected with LMO4 shRNA construct or scrambled control ( both expressing GFP under CMV promoter as a transfection control ) . Scale bar : 10µm . Dot plots representing the mean of nuclear ( I ) pCREB or ( K ) CREB staining intensity normalized to scrambled control . N = 30 - 37 nuclei analyzed from at least 3 independent cell cultures . The N - terminus of Jacob displaces LMO4 from CREB . ( N ) GST - LMO4 coupled to beads was preincubated with His - SUMO - CREB and subsequently incubated with an increasing amount of MBP - Jacob - 45 - 228 .

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

Figure 3(B, C) FRET saturation experiments with (B) LMO4-GFP and CREB-1-165-tagRFP or (C) CREB-166-341-tagRFP and CREB-GFP and LIM1-1-80-tagRFP or LIM2-81-165-tagRFP revealed an association between the LIM1 domain of LMO4 and the C-terminus of CREB. N=8 independent experiments.(D) Recombinant His-SUMO-CREB directly binds to GST-LMO4 in pull-down experiments.(H-K) Knockdown of LMO4 reduces nuclear pCREB immunoreactivity. (H, J) Representative confocal images of hippocampal neurons transfected with LMO4 shRNA construct or scrambled control (both expressing GFP under CMV promoter as a transfection control). Scale bar: 10µm. Dot plots representing the mean of nuclear (I) pCREB or (K) CREB staining intensity normalized to scrambled control. N=30-37 nuclei analyzed from at least 3 independent cell cultures.The N-terminus of Jacob displaces LMO4 from CREB. (N) GST-LMO4 coupled to beads was preincubated with His-SUMO-CREB and subsequently incubated with an increasing amount of MBP-Jacob-45-228.


Figure 4 ( A ) Pull - down experiments confirmed a direct interaction between recombinant MBP - Jacob - 45 - 532 and His - PP1γ . ( B ) Mapping of the mCherry - PP1γ interaction region within the Jacob sequence revealed binding of a C - terminal fragment ( Jacob - 310 - 250 - GFP ) as well as the N - terminal part ( Jacob - 173 - 246 - GFP ) where the region between 213 - 246 aa is sufficient for immunoprecipitation . The pink boxes in the schematic indicate binding regions . Treatment of hippocampal primary neurons expressing phosphodeficient Jacob in the nucleus with OA rescues Jacob - induced CREB shutoff . Confocal images of pCREB immunostaining in DIV15 neurons overexpressing ΔMyr - Jacob - S180A - GFP with and without OA treatment . Scale bar : 20 μm . Lookup table indicates the pixel intensities from 0 to 255 . N = 14 - 17 nuclei analyzed from two independent cell cultures . ( K ) Treatment with staurosporine decreases Jacob phosphorylation level ( S180 ) , but increases its association with LMO4 - tagRFP . Immunoblot of HEK293T cells extracts transfected with LMO4 - tagRFP and ΔMyr - Jacob - GFP or GFP alone . ( L ) Treatment with staurosporine decreases the association of Jacob with CREB . Immunoblot of HEK293T cells extract transfected with CREB - tagRFP and ΔMyr - Jacob - GFP or GFP as a control . (

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

Figure 4(A) Pull-down experiments confirmed a direct interaction between recombinant MBP-Jacob-45-532 and His-PP1γ.(B) Mapping of the mCherry-PP1γ interaction region within the Jacob sequence revealed binding of a C-terminal fragment (Jacob-310-250-GFP) as well as the N-terminal part (Jacob-173-246-GFP) where the region between 213-246 aa is sufficient for immunoprecipitation. The pink boxes in the schematic indicate binding regions.Treatment of hippocampal primary neurons expressing phosphodeficient Jacob in the nucleus with OA rescues Jacob-induced CREB shutoff. Confocal images of pCREB immunostaining in DIV15 neurons overexpressing ΔMyr-Jacob-S180A-GFP with and without OA treatment. Scale bar: 20 μm. Lookup table indicates the pixel intensities from 0 to 255. N=14-17 nuclei analyzed from two independent cell cultures.(K) Treatment with staurosporine decreases Jacob phosphorylation level (S180), but increases its association with LMO4-tagRFP. Immunoblot of HEK293T cells extracts transfected with LMO4-tagRFP and ΔMyr-Jacob-GFP or GFP alone. (L) Treatment with staurosporine decreases the association of Jacob with CREB. Immunoblot of HEK293T cells extract transfected with CREB-tagRFP and ΔMyr-Jacob-GFP or GFP as a control. (


Figure 5Nitarsone disrupts binding of Jacob to LMO4 in a concentration - dependent manner . ( H ) MBP immunoreactivity normalized to input . N = 6 - 12 independent experiments . ( N , O ) ( N ) 5 µM Nitarsone reduced Jacob - LMO4 complex formation in Aβ - treated ( 500 nM ) primary neurons as revealed by proximity ligation assay . Scale bar : 5 µm . ( O ) Bar plot of normalized PLA signal intensity . N = 20 - 24 neuronal nuclei from 3 cultures .

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

Figure 5Nitarsone disrupts binding of Jacob to LMO4 in a concentration-dependent manner. (H) MBP immunoreactivity normalized to input. N=6-12 independent experiments.(N, O) (N) 5 µM Nitarsone reduced Jacob-LMO4 complex formation in Aβ-treated (500 nM) primary neurons as revealed by proximity ligation assay. Scale bar: 5 µm. (O) Bar plot of normalized PLA signal intensity. N=20-24 neuronal nuclei from 3 cultures.


Figure 6 ( A , B ) Co - application of Nitarsone prevents Aβ - induced CREB shutoff . DIV16 hippocampal cultures were treated with 5 μM Nitarsone , 500 nM Aβ1 - 42 , 5 μM Nitarsone with 500 nM Aβ1 - 42 or vehicle control for 48h and stained for pCREB , MAP2 , and DAPI . ( A ) Representative confocal images . Scale bar : 10 µm . ( B ) Nuclear pCREB immunoreactivity normalized to control . N = 63 - 67 nuclei from three independent cultures . ( E , F ) Treatment with Nitarsone rescues Aβ1 - 42 - induced decrease of synaptic GluR1 - immunoreactivity within Shank3 . DIV16 dissociated , hippocampal cultures were treated with 5 μM Nitarsone , 500 nM Aβ1 - 42 , 5 μM Nitarsone with 500 nM Aβ1 - 42 or vehicle control for 48h and stained for Shank3 , surface GluR1 , and MAP2 . ( E ) Representative confocal images of dendritic segments . Scale bar : 5 µm . ( F ) GluR1 - immunoreactivity within Shank3 signal . N = 39 - 61 of dendritic segments from four independent cell cultures . Nitarsone administration rescues mEPSC amplitude . ( G ) Analog traces of mEPSCs recorded in DIV16 hippocampal neurons treatd with 500 nM Aβ1 - 42 , 5 μM Nitarsone , 5 μM Nitarsone with 500 nM Aβ1 - 42 or vehicle control for 48h .

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

Figure 6(A, B) Co-application of Nitarsone prevents Aβ-induced CREB shutoff. DIV16 hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for pCREB, MAP2, and DAPI. (A) Representative confocal images. Scale bar: 10 µm. (B) Nuclear pCREB immunoreactivity normalized to control. N=63-67 nuclei from three independent cultures.(E, F) Treatment with Nitarsone rescues Aβ1-42-induced decrease of synaptic GluR1-immunoreactivity within Shank3. DIV16 dissociated, hippocampal cultures were treated with 5 μM Nitarsone, 500 nM Aβ1-42, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h and stained for Shank3, surface GluR1, and MAP2. (E) Representative confocal images of dendritic segments. Scale bar: 5 µm. (F) GluR1-immunoreactivity within Shank3 signal. N=39-61 of dendritic segments from four independent cell cultures.Nitarsone administration rescues mEPSC amplitude. (G) Analog traces of mEPSCs recorded in DIV16 hippocampal neurons treatd with 500 nM Aβ1-42, 5 μM Nitarsone, 5 μM Nitarsone with 500 nM Aβ1-42 or vehicle control for 48h.


Figure 7 ( B , C ) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of TBA2 . 1 mice . ( B ) Representative confocal images of cryosections from 11 weeks old mice stained for NeuN , DAPI , and pCREB . Scale bar : 10 µm ( C ) Bar plot of pCREB nuclear staining intensity . N = 21 - 34 hippocampal sections from 6 - 9 animals . ( D , E ) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of 5xFAD mice . ( D ) Representative confocal images of CA1 cryosections from 18 weeks old mice stained for NeuN , DAPI , and pCREB . Scale bar : 10 µm . ( E ) Cumulative frequency distribution of pCREB nuclear staining intensity . N = 22 - 27 hippocampal sections from 6 - 7 animals . ( K , L ) Nitarsone reduces number of varicosities in SLM of CA1 of TBA2 . 1 mice . ( K ) Representative , confocal images of dendrites filled with biocytin stained from 13 weeks old mice . Arrows indicate varicosities . Scale bar : 1 μm . ( L ) Number of dendritic swellings per 10 μm . N = 19 - 22 dendrites from 2 animals per genotype .

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

Figure 7(B, C) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of TBA2.1 mice. (B) Representative confocal images of cryosections from 11 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm (C) Bar plot of pCREB nuclear staining intensity. N=21-34 hippocampal sections from 6-9 animals.(D, E) Nitarsone rescues the reduction of pCREB immunoreactivity in NeuN positive cells in the CA1 region of 5xFAD mice. (D) Representative confocal images of CA1 cryosections from 18 weeks old mice stained for NeuN, DAPI, and pCREB. Scale bar: 10 µm. (E) Cumulative frequency distribution of pCREB nuclear staining intensity. N=22-27 hippocampal sections from 6-7 animals.(K, L) Nitarsone reduces number of varicosities in SLM of CA1 of TBA2.1 mice. (K) Representative, confocal images of dendrites filled with biocytin stained from 13 weeks old mice. Arrows indicate varicosities. Scale bar: 1 μm. (L) Number of dendritic swellings per 10 μm. N=19-22 dendrites from 2 animals per genotype.


Figure 8 ( A ) Nitarsone rescues late CA1 - LTP impairment in TBA2 . 1 mice . Insets show representative fEPSPs analog traces at indicated time points : 1 = baseline , 2 = late LTP . ( B ) Averaged fEPSP slopes recorded during the last 30 min . N = 14 - 18 slices from 5 - 6 mice . ( C ) Nitarsone rescues late CA1 - LTP impairment in 5xFAD mice . Insets show representative fEPSPs analog traces at indicated time points : 1 = baseline , 2 = late LTP . ( D ) Averaged fEPSP slopes recorded during the last 30 min . N = 17 - 18 slices from 6 mice . ( H - J ) Nitarsone rescues short - term memory impairment in Y - maze object recognition task in ( I ) TBA2 . 1 N = 9 - 14 and ( J ) 5xFAD mice . N = 9 -

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

Figure 8(A) Nitarsone rescues late CA1-LTP impairment in TBA2.1 mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (B) Averaged fEPSP slopes recorded during the last 30 min. N=14-18 slices from 5-6 mice. (C) Nitarsone rescues late CA1-LTP impairment in 5xFAD mice. Insets show representative fEPSPs analog traces at indicated time points: 1 = baseline, 2 = late LTP. (D) Averaged fEPSP slopes recorded during the last 30 min. N=17-18 slices from 6 mice.(H-J) Nitarsone rescues short-term memory impairment in Y-maze object recognition task in (I) TBA2.1 N=9-14 and (J) 5xFAD mice. N=9-11


Figure 1B - D . Mice were analyzed by Kaplan - Meier survival plot ( B ) , the percentage of body weight ( C ) , and the clinical scores ( D ) of WT and GPx8 - / - mice with colitis induced by 4 % DSS for 6 days ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( E ) Images and statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . , 5 - 6 mice were used for each experiment . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections . The infiltrated immune cells at the transmural extensions were indicated by arrowhead . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . 5 - 6 mice were used for each experiment . ( G ) Colon sections were stained with markers for macrophages ( F4 / 80 ) and anti - GPx8 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Colons treated with 4 % DSS for 5 days were collected on day 14 . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates ( n = 10 per group ) . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing ( n = 5 per group ) and are presented by a heat map , ( I ) , and principal coordinates analysis , ( J ) . Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions . ( K ) α - diversity indicated bacteria species in each mouse depicted by richness ( total operational taxonomic units , OTUs ) , the Chao1 richness estimator ( Chao1 ) , and the abundance - based coverage estimator ( ACE ) metrics . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 1B-D. Mice were analyzed by Kaplan-Meier survival plot (B), the percentage of body weight (C), and the clinical scores (D) of WT and GPx8-/- mice with colitis induced by 4% DSS for 6 days (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (E) Images and statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. , 5-6 mice were used for each experiment. (F) Images and semiquantitative scoring of hematoxylin and eosin staining of colon sections. The infiltrated immune cells at the transmural extensions were indicated by arrowhead. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. 5-6 mice were used for each experiment. (G) Colon sections were stained with markers for macrophages (F4/80) and anti-GPx8. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Colons treated with 4% DSS for 5 days were collected on day 14. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates (n = 10 per group). Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. Bray Curtis dissimilarity of fecal microbiota were measured by bacterial 16S RNA sequencing (n = 5 per group) and are presented by a heat map, (I), and principal coordinates analysis, (J).Gut microbiome was shaped by Gpx8 deficiency under standard housing conditions. (K) α-diversity indicated bacteria species in each mouse depicted by richness (total operational taxonomic units, OTUs), the Chao1 richness estimator (Chao1), and the abundance-based coverage estimator (ACE) metrics. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 2B . Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages ( F4 / 80 ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . ( C ) The percentage of body weight . ( D ) The clinical scores of mice . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( E ) Statistical analysis of colon length . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( F ) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( G ) Colon sections stained with anti - F4 / 80 . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) . After undergoing macrophage depletion , C57BL / 6 mice received macrophages derived from WT ( WT BMDMs ) or GPx8 - / - mice ( GPx8 - / - BMDMs ) were under DSS - induced colitis ( n = 6 - 9 per group ) . D ) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model . ( H ) Production of IL - 1β , IL - 6 and IL - 18 in colon tissue lysates . Data information : Data are presented as the mean ± SEM . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s one ‐ tailed t ‐ test ) .

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

Figure 2B. Colons from mice treated with clodronate or control liposomes and subjected to adoptive cell transfer after macrophage depletion were stained with markers for macrophages (F4/80).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). (C) The percentage of body weight. (D) The clinical scores of mice. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (E) Statistical analysis of colon length. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (F) Images and semiquantitative scoring of hematoxylin and eosin staining colon sections. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (G) Colon sections stained with anti-F4/80. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).After undergoing macrophage depletion, C57BL/6 mice received macrophages derived from WT (WT BMDMs) or GPx8-/- mice (GPx8-/- BMDMs) were under DSS-induced colitis (n = 6-9 per group). D) Colon samples from BMDMs transplanted mice were collected on day 7 under colitis model. (H) Production of IL-1β, IL-6 and IL-18 in colon tissue lysates. Data information: Data are presented as the mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's one‐tailed t‐test).


Figure 3A . Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency . BMDMs were primed for 6 h with 0 . 5 μg / ml LPS and then transfected with LPS ( TF LPS ) for 8 h , stimulated with ATP ( 5 mM ) for 3 h , MSU ( 150 μg / mL ) for 8 h , or nigericin ( 5 μM ) for 3 h . Activation of inflammasome was demonstrated by secreted IL - 1β . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . B . GPx8 deficiency enhanced noncanonical inflammasome activation . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . C . Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs . BMDMs were primed for 6 h with 1 μg / ml poly ( I : C ) and then transfected with LPS for 8 h . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( D ) Activation of noncanonical inflammasome in BMDMs with empty vector ( EV ) or hGPx8 was verified by IL - 1β ( left panel ) and LDH release ( right panel ) . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Over - activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically - expressing human GPx8 ( hGPx8 ) . ( E ) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR . Data information : data are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . F . GPx8 deficiency enhanced caspase - 11 cleavage and activation . Levels of processed caspase - 1 and - 11 in the supernatants ( Sup ) and full - length caspase - 1 , - 11 and GAPDH in the cell lysates ( Cell ext ) were determined by immunoblotting . Fold changes of protein expression were normalized with internal controls and are indicated below the blots . G . RNA expression of caspase - 11 was not altered by GPx8 deficiency . Caspase - 11 RNA expression of BMDMs isolated from GPx8 - / - mice was measured after LPS priming . Data information : ata are representative of at least 3 independent experiments and presented as the mean ± SD , n = 4 - 5 , technical repeats . H . GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo . Colon samples from 5 WT or GPx8 - / - mice treated with DSS for 5 days were collected on day 7 . Levels of full - length and processed caspase - 1 , - 11 and gasdermin D ( GSDMD ) in tissue lysates were individually analyzed by immunoblots . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( I ) Survival of mice primed with ultra pure LPS ( E . coli O55 : B5 , 400 μg / kg ) and re - challenged 6 h later with LPS ( 40 mg / kg ) ( n = 8 per group ) . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( J ) Survival of mice primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 10 mg / kg ) ( n = 9 per group ) . GPx8 - / - mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome . ( K ) IL - 1β production levels in mice sera were measured 2 h after being primed with poly ( I : C ) ( LMW 1 mg / kg ) and re - challenged 6 h later with LPS ( 1 mg / kg ) ( n = 8 - 9 per group ) . In ( K ) , data are presented as the mean ± SEM . NS , no significance . P > 0 . 05 ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 3A. Canonical NLRP3 inflammasome activation was not altered by GPx8 deficiency. BMDMs were primed for 6 h with 0.5 μg/ml LPS and then transfected with LPS (TF LPS) for 8 h, stimulated with ATP (5 mM) for 3 h, MSU (150 μg/mL) for 8 h, or nigericin (5 μM) for 3 h. Activation of inflammasome was demonstrated by secreted IL-1β. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).B. GPx8 deficiency enhanced noncanonical inflammasome activation. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).C. Upregulation of noncanonical inflammasome activation by GPx8 deficiency occurred independently of TLRs. BMDMs were primed for 6 h with 1 μg/ml poly(I:C) and then transfected with LPS for 8 h. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (D) Activation of noncanonical inflammasome in BMDMs with empty vector (EV) or hGPx8 was verified by IL-1β (left panel) and LDH release (right panel). Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Over-activation of noncanonical inflammasome induced by GPx8 deficiency in BMDMs can be rescued by ectopically-expressing human GPx8 (hGPx8). (E) Expression of hGPx8 mRNA in BMDMs was confirmed by quantitative reverse transcriptase PCR. Data information: data are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.F. GPx8 deficiency enhanced caspase-11 cleavage and activation. Levels of processed caspase-1 and -11 in the supernatants (Sup) and full-length caspase-1, -11 and GAPDH in the cell lysates (Cell ext) were determined by immunoblotting. Fold changes of protein expression were normalized with internal controls and are indicated below the blots.G. RNA expression of caspase-11 was not altered by GPx8 deficiency. Caspase-11 RNA expression of BMDMs isolated from GPx8-/- mice was measured after LPS priming. Data information: ata are representative of at least 3 independent experiments and presented as the mean ± SD, n=4-5, technical repeats.H. GPx8 deficiency enhanced noncanonical inflammasome activation and pyroptosis in vivo. Colon samples from 5 WT or GPx8-/- mice treated with DSS for 5 days were collected on day 7. Levels of full-length and processed caspase-1, -11 and gasdermin D (GSDMD) in tissue lysates were individually analyzed by immunoblots.GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (I) Survival of mice primed with ultra pure LPS (E. coli O55:B5, 400 μg/kg) and re-challenged 6 h later with LPS (40 mg/kg) (n = 8 per group).GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (J) Survival of mice primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (10 mg/kg) (n = 9 per group).GPx8-/- mice were more susceptible than WT mice to endotoxic shock by activation of the noncanonical inflammasome. (K) IL-1β production levels in mice sera were measured 2 h after being primed with poly(I:C) (LMW 1 mg/kg) and re-challenged 6 h later with LPS (1 mg/kg) (n = 8-9 per group). In (K), data are presented as the mean ± SEM. NS, no significance. P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 4A . Interaction of GPx8 with human caspase - 4 ( Casp4 ) or mouse caspase - 11 ( Casp11 ) was confirmed by co - IP assays . 293T cells were co - transfected with vectors expressing FLAG tagged Casp4 or Casp11 , in addition to GPx8 expressing vectors . Cell lysate containing Casp4 - or Casp11 - FLAG proteins was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 and anti - FLAG Ab ( left panel ) . GPx8 and its interacting proteins were precipitated by anti - GPx8 Ab and analyzed by the indicated Abs ( right panel ) . B . Co - localization of Myc - tagged GPx8 ( red ) and endogenous caspase - 4 ( green ) in THP - 1 cells . C . Interaction between GPx8 and caspase - 4 in THP - 1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay ( PLA ) ( red dots , labeled by arrowheads ) . GPx8 - Myc stably expressing THP - 1 were used to detect the interaction between GPx8 - Myc and endogenous caspase - 4 . Untreated cells ( Ctrl ) or cells primed with LPS ( primed LPS ) were incubated with anti - Myc and anti - caspase - 4 Abs , according to the manufacturer ' s instructions , and the nucleus was stained with DAPI ( blue ) . Results were quantified by counting at least 5 different fields with an average of 300 cells . Reactions without primary Abs were used as the control for the PLA assay ( PLA Ctrl ) . Data information : All data are representative of at least 3 independent experiments . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( D ) Complexes of GPx8 and caspase - 4 were demonstrated by co - IP assays . Cysteine - dependent covalent interactions of GPx8 and caspase - 4 ( C258S ) were analyzed under non - reducing and reducing conditions . Disulfide - linked complexes of Gpx8 and caspase - 4 are indicated . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( E ) Interactions of GPx8 and caspase - 4 in response to H2O2 treatment were demonstrated by co - IP assays of lysates prepared from 293T cells treated with H2O2 ( 200 μM ) . 293T cells were co - transfected with vectors expressing FLAG tagged caspase - 4 ( WT or C258S ) in addition to GPx8 expressing vectors . Cell lysate was immunoprecipitated by anti - FLAG Ab and subsequently analyzed by Western blots using anti - GPx8 or anti - FLAG Ab . ( F ) C79S , C108S , and C2S2 mutants of GPx8 abolished most of the binding to caspase - 4 by co - IP assays . C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . THP - 1 cells stably expressing empty vector ( EV ) , WT , or mutant GPx8 proteins were confirmed by immunoblotting ( G ) . C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation . ( H and I ) Cells were treated with 12 - O - tetradecanoylphorbol 13 - actate ( PMA ) ( 0 . 1 μM ) and differentiated for 3 days then primed and transfected with LPS . Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL - 1β ( H ) and LDH release ( I ) . Data information : All data are representative of at least 3 independent experiments . In H and I , data are presented as the mean ± SD , n = 4 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . J . E . coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase - 4 ( C258S ) to form a disulfide - linked complex ( oxidized GPx8 : oGPx8 ; reduced GPx8 : rGPx8 ) . Lanes 2 and 3 , GPx8 pretreated with 1 and 5 mM H2O2 , respectively . Lanes 4 and 5 , Gpx8 pretreated with 50 and 200 mM DTT , respectively . Disulfide - linked complexes of recombinant Gpx8 and caspase - 4 ( C258S ) are indicated . K . GPx8 inhibited the activation of caspase - 4 by decreasing the oligomerization of caspase - 4 . Oxidized GPx8 and caspase - 4 ( C258S ) were co - incubated for 30 min . Different amounts of LPS were added to the complex and incubated for another 30 min . Oligomerized caspase - 4 was analyzed by native blue PAGE according to the manufacturer ' s instructions . Mock : without GPx8 ; oxidized GPx8 : oGPx8 ; oxidized GPx8C79S : oC79S . Data information : All data are representative of at least 3 independent experiments . In K , quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers / total proteins . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 4A. Interaction of GPx8 with human caspase-4 (Casp4) or mouse caspase-11 (Casp11) was confirmed by co-IP assays. 293T cells were co-transfected with vectors expressing FLAG tagged Casp4 or Casp11, in addition to GPx8 expressing vectors. Cell lysate containing Casp4- or Casp11-FLAG proteins was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 and anti-FLAG Ab (left panel). GPx8 and its interacting proteins were precipitated by anti-GPx8 Ab and analyzed by the indicated Abs (right panel).B. Co-localization of Myc-tagged GPx8 (red) and endogenous caspase-4 (green) in THP-1 cells.C. Interaction between GPx8 and caspase-4 in THP-1 cells in the presence or absence of priming LPS was demonstrated by the in situ proximity ligation assay (PLA) (red dots, labeled by arrowheads). GPx8-Myc stably expressing THP-1 were used to detect the interaction between GPx8-Myc and endogenous caspase-4. Untreated cells (Ctrl) or cells primed with LPS (primed LPS) were incubated with anti-Myc and anti-caspase-4 Abs, according to the manufacturer's instructions, and the nucleus was stained with DAPI (blue). Results were quantified by counting at least 5 different fields with an average of 300 cells. Reactions without primary Abs were used as the control for the PLA assay (PLA Ctrl). Data information: All data are representative of at least 3 independent experiments. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (D) Complexes of GPx8 and caspase-4 were demonstrated by co-IP assays. Cysteine-dependent covalent interactions of GPx8 and caspase-4 (C258S) were analyzed under non-reducing and reducing conditions. Disulfide-linked complexes of Gpx8 and caspase-4 are indicated.293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (E) Interactions of GPx8 and caspase-4 in response to H2O2 treatment were demonstrated by co-IP assays of lysates prepared from 293T cells treated with H2O2 (200 μM).293T cells were co-transfected with vectors expressing FLAG tagged caspase-4 (WT or C258S) in addition to GPx8 expressing vectors. Cell lysate was immunoprecipitated by anti-FLAG Ab and subsequently analyzed by Western blots using anti-GPx8 or anti-FLAG Ab. (F) C79S, C108S, and C2S2 mutants of GPx8 abolished most of the binding to caspase-4 by co-IP assays.C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. THP-1 cells stably expressing empty vector (EV), WT, or mutant GPx8 proteins were confirmed by immunoblotting (G).C79 of GPx8 was essential for its inhibitory effect on noncanonical inflammasome activation. (H and I) Cells were treated with 12-O-tetradecanoylphorbol 13-actate (PMA) (0.1 μM) and differentiated for 3 days then primed and transfected with LPS. Activation of the noncanonical inflammasome by different amounts of cytoplasmic LPS was verified by IL-1β (H) and LDH release (I). Data information: All data are representative of at least 3 independent experiments. In H and I, data are presented as the mean ± SD, n=4, technical repeats. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).J. E. coli purified oxidized GPx8 interacts with mammalian cell purified FLAG tagged caspase-4 (C258S) to form a disulfide-linked complex (oxidized GPx8: oGPx8; reduced GPx8: rGPx8). Lanes 2 and 3, GPx8 pretreated with 1 and 5 mM H2O2, respectively. Lanes 4 and 5, Gpx8 pretreated with 50 and 200 mM DTT, respectively. Disulfide-linked complexes of recombinant Gpx8 and caspase-4 (C258S) are indicated.K. GPx8 inhibited the activation of caspase-4 by decreasing the oligomerization of caspase-4. Oxidized GPx8 and caspase-4 (C258S) were co-incubated for 30 min. Different amounts of LPS were added to the complex and incubated for another 30 min. Oligomerized caspase-4 was analyzed by native blue PAGE according to the manufacturer's instructions. Mock: without GPx8; oxidized GPx8: oGPx8; oxidized GPx8C79S: oC79S. Data information: All data are representative of at least 3 independent experiments. In K, quantification of oligomers was calculated from 3 independent experiments and is presented as the mean ± SD of percent of oligomers/total proteins. *P < 0.05; **P < 0.01; *** P < 0.001 (Student's two‐tailed t‐test).


Figure 5A . Casp4C118S mutant partially abolished the covalent disulfide - link with GPx8 . Interacting GPx8 were co - immunoprecipitated with FLAG - tagged caspase - 4 ( WT , C118S , C329S mutants ) and analyzed under reducing ( + DTT ) or non - reducing ( - DTT ) conditions . Disulfide - linked complexes of GPx8 and caspase - 4 are indicated . Fold change of complexes was normalized with internal controls and indicated below blots . C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . THP - 1 cells stably expressing empty vector ( EV ) , FLAG - tagged caspase - 4 ( WT , C258S , C118S mutants ) , or GPx8 ( WT and C79S mutants ) were confirmed by immunoblotting ( B ) . Data information : All data shown are representative of at least 3 independent experiments . C118 of caspase - 4 was required for the GPx8 - mediated inhibitory effect . ( C - D ) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS . THP - 1 cells were differentiated , primed , and transfected with LPS . Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL - 1β ( C ) and LDH ( D ) . In C and D , data are presented as the mean ± SD , n = 4 , technical repeats . * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 5A. Casp4C118S mutant partially abolished the covalent disulfide-link with GPx8. Interacting GPx8 were co-immunoprecipitated with FLAG-tagged caspase-4 (WT, C118S, C329S mutants) and analyzed under reducing (+DTT) or non-reducing (-DTT) conditions. Disulfide-linked complexes of GPx8 and caspase-4 are indicated. Fold change of complexes was normalized with internal controls and indicated below blots.C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. THP-1 cells stably expressing empty vector (EV), FLAG-tagged caspase-4 (WT, C258S, C118S mutants), or GPx8 (WT and C79S mutants) were confirmed by immunoblotting (B). Data information: All data shown are representative of at least 3 independent experiments.C118 of caspase-4 was required for the GPx8-mediated inhibitory effect. (C-D) Casp4C118S completely abolished the inhibition of noncanonical inflammasome signaling in response to intracellular LPS. THP-1 cells were differentiated, primed, and transfected with LPS. Activation of the noncanonical inflammasome by different quantities of cytoplasmic LPS is confirmed by the production of IL-1β (C) and LDH (D). In C and D, data are presented as the mean ± SD, n=4, technical repeats. ***P < 0.001 (Student's two‐tailed t‐test).


Figure 6A . The caspase - 4 / 11 and - 1 inhibitor , VX - 765 , inhibited noncanonical inflammasome activation . BMDMs isolated from mice were primed with LPS for 5 - 6 h , treated with 20 μM VX - 765 , and transfected with LPS for 12 h . Data information data are presented as the mean ± SD , n = 5 , technical repeats . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and analyzed by a Kaplan - Meier survival plot ( B ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . Data information : , data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model and the percentage of body weight ( C ) and clinical scores ( D ) of mice with colitis induced by 4 % DSS for 6 days ( n = 10 - 11 per group ) . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Full - length and cleavage forms of caspase - 11 , caspase - 1 , and GSDMD ( E ) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) . Mice were injected intraperitoneally with VX - 765 ( 20 mg / kg ) under colitis model Colon samples from mice treated with 4 % DSS for 5 days ( n = 6 - 10 per group ) . Production of IL - 1β and IL - 6 ( F ) in colon tissue lysates was assessed on day 14 after treatment . data are presented as the mean ± SEM . NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ( Student ' s two ‐ tailed t ‐ test ) .

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

Figure 6A. The caspase-4/11 and -1 inhibitor, VX-765, inhibited noncanonical inflammasome activation. BMDMs isolated from mice were primed with LPS for 5-6 h, treated with 20 μM VX-765, and transfected with LPS for 12 h. Data information data are presented as the mean ± SD, n=5, technical repeats. *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and analyzed by a Kaplan-Meier survival plot (B) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). Data information: , data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model and the percentage of body weight (C) and clinical scores (D) of mice with colitis induced by 4% DSS for 6 days (n = 10-11 per group). data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Full-length and cleavage forms of caspase-11, caspase-1, and GSDMD (E) in colon tissue lysates were analyzed by immunoblots on day 7 after DSS treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).Mice were injected intraperitoneally with VX-765 (20 mg/kg) under colitis model Colon samples from mice treated with 4% DSS for 5 days (n = 6-10 per group). Production of IL-1β and IL-6 (F) in colon tissue lysates was assessed on day 14 after treatment. data are presented as the mean ± SEM. NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001 (Student's two‐tailed t‐test).


Figure 7A . GPx8 was predominantly expressed in macrophages of UC patients . Colon biopsies of non - inflamed regions from UC patients were stained with the pan - macrophage marker , CD68 , or the monocyte / macrophage activating marker , CD163 , combined with anti - GPx8 . Quantification of double - positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double - positive cells . The total cell numbers are the sum of double - and single - positive cells . B . Relative expression levels of GPx8 , GPx7 and caspase - 4 in non - inflamed biopsy specimens from healthy controls ( Ctrl ) ( n = 14 ) and UC patients ( n = 25 ) are demonstrated by immunoblots . C . Data are normalized to the mean of healthy controls and presented as the mean ± SEM . Mann Whitney test for GPx8 and Student ' s two ‐ tailed t ‐ test for GPx7 and caspase - 4 expression analysis . Data information : NS : not significant ( P > 0 . 05 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 . D . Paired GPx8 and caspase - 4 expression from each individual are linked by lines and presented as the mean ± SEM . The p - value was analyzed by the Fisher ' s exact test . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

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

Figure 7A. GPx8 was predominantly expressed in macrophages of UC patients. Colon biopsies of non-inflamed regions from UC patients were stained with the pan-macrophage marker, CD68, or the monocyte/macrophage activating marker, CD163, combined with anti-GPx8. Quantification of double-positive cells was calculated from 3 individual patients and is presented as the mean ± SEM of the percentage of double-positive cells. The total cell numbers are the sum of double- and single-positive cells.B. Relative expression levels of GPx8, GPx7 and caspase-4 in non-inflamed biopsy specimens from healthy controls (Ctrl) (n = 14) and UC patients (n = 25) are demonstrated by immunoblots. C. Data are normalized to the mean of healthy controls and presented as the mean ± SEM. Mann Whitney test for GPx8 and Student's two‐tailed t‐test for GPx7 and caspase-4 expression analysis. Data information: NS: not significant (P > 0.05). *P < 0.05; **P < 0.01; ***P < 0.001.D. Paired GPx8 and caspase-4 expression from each individual are linked by lines and presented as the mean ± SEM. The p-value was analyzed by the Fisher's exact test. *P < 0.05; **P < 0.01; ***P < 0.001.


Figure 1A A bright field image of a wild - type adult C . elegans . The position of the AIY interneurons in the head is indicated with green , and the gut is outlined with dashed lines . A ' A cartoon diagram of the C . elegans head modified from wormatlas with permission . The AIY processes and soma are indicated in grey , and the presynaptic sites are marked with green ( Altun et al . , 2002 - 2019 ) . B - G ' Confocal images of the AIY labeled with synaptic vesicle marker GFP : : RAB - 3 ( B - G , green ) and active zone marker GFP : : SYD - 1 ( B ' - G ' , pseudo - red ) in wild - type ( B , B ' ) , cwn - 2 ( ok895 ) ( C , C ' ) , cfz - 2 ( ok1201 ) ( D , D ' ) , pop - 1 ( hu9 ) ( E , E ' ) , and sys - 1 RNAi ( G , G ' ) animals . H - H ' Quantification of the fluorescence intensity of AIY RAB - 3 ( H ) and SYD - 1 ( H ' ) for the indicated genotypes . I - M ' Confocal images of AIY labeled with mCherry : : RAB - 3 ( pseudo - GFP ) ( I - M ) , or GFP : : RAB - 3 ( I ' - M ' ) of tissue - specific RNAi treatment for control ( I - I ' ) , cwn - 2 ( J - J ' ) , cfz - 2 ( K - K ' ) , sys - 1 ( L - L ' ) and pop - 1 ( M - M ' ) . N - N ' Quantification of the fluorescence intensity of AIY mCherry : : RAB - 3 or GFP : : RAB - 3 for the indicated RNAi treatment .

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

Figure 1A A bright field image of a wild-type adult C. elegans. The position of the AIY interneurons in the head is indicated with green, and the gut is outlined with dashed lines. A' A cartoon diagram of the C. elegans head modified from wormatlas with permission. The AIY processes and soma are indicated in grey, and the presynaptic sites are marked with green (Altun et al., 2002-2019).B-G' Confocal images of the AIY labeled with synaptic vesicle marker GFP::RAB-3 (B-G, green) and active zone marker GFP::SYD-1 (B'-G', pseudo-red) in wild-type (B, B'), cwn-2(ok895) (C, C'), cfz-2(ok1201) (D, D'), pop-1(hu9) (E, E'), and sys-1 RNAi (G, G') animals. H-H' Quantification of the fluorescence intensity of AIY RAB-3 (H) and SYD-1 (H') for the indicated genotypes.I-M' Confocal images of AIY labeled with mCherry::RAB-3 (pseudo-GFP) (I-M), or GFP::RAB-3 (I'-M') of tissue-specific RNAi treatment for control (I-I'), cwn-2(J-J'), cfz-2(K-K'), sys-1(L-L') and pop-1(M-M'). N-N' Quantification of the fluorescence intensity of AIY mCherry::RAB-3 or GFP::RAB-3 for the indicated RNAi treatment.


Figure 2A Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 in wild - type , egl - 3 ( n589 ) and aex - 5 ( sa23 ) mutants . C Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated RNAi treatment . Candidates are intestinal expressed nlp or flp . Confocal images of AIY labeled with GFP : : RAB - 3 in wild - type ( E ) with a wild type nlp - 40 transgene . Confocal images of AIY labeled with nlp - 40 ( tm4085 ) ( F ) , nlp - 40 ( vj3 ) ( G ) , nlp - 40 ( tm4085 ) ( H ) or nlp - 40 ( vj3 ) ( I ) with a wild type nlp - 40 transgene . J Quantification of the fluorescence intensity of AIY GFP : : RAB - 3 for the indicated genotypes . Transgenic data are averaged from at least two independent lines . K - L . Confocal images of AIY synaptic marker GFP : : RAB - 3 for the indicated intestinal - specific RNAi treatment . M Quantification for ( K ) and ( L ) .

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

Figure 2A Quantification of the fluorescence intensity of AIY GFP::RAB-3 in wild-type , egl-3(n589) and aex-5(sa23) mutants.C Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated RNAi treatment. Candidates are intestinal expressed nlp or flp.Confocal images of AIY labeled with GFP::RAB-3 in wild-type (E) with a wild type nlp-40 transgene.Confocal images of AIY labeled with nlp-40(tm4085) (F), nlp-40(vj3) (G), nlp-40(tm4085) (H) or nlp-40(vj3) (I) with a wild type nlp-40 transgene. J Quantification of the fluorescence intensity of AIY GFP::RAB-3 for the indicated genotypes. Transgenic data are averaged from at least two independent lines.K-L. Confocal images of AIY synaptic marker GFP::RAB-3 for the indicated intestinal-specific RNAi treatment. M Quantification for (K) and (L).


Figure 3A - C Quantification of the fluorescence intensity of AIY presynaptic marker GFP : : RAB - 3 ( A , B ) and nlp - 40 mRNA level ( C ) for the indicated genotypes . The AIY synaptic region was quantified for the synaptic intensity analysis . D - I Confocal images of the transcriptional nlp - 40 reporter ( Pnlp - 40 : : GFP ) in wild - type ( D ) , cwn - 2 ( ok895 ) ( E ) , cfz - 2 ( ok1201 ) ( F ) , pop - 1 ( hu9 ) ( G ) , and control or sys - 1 RNAi ( H and I ) animals at adult Day 1 stage . J Quantification of the Pnlp - 40 : : GFP fluorescence intensity for the indicated genotypes . K - P Confocal images of an intestinal - specific reporter ERM - 1 : : GFP in wild type ( K ) , cwn - 2 ( ok895 ) ( L ) , cfz - 2 ( ok1201 ) ( M ) , pop - 1 ( hu9 ) ( N ) , and control or sys - 1 RNAi ( O and P ) animals at the adult Day1 stage . Q Quantification of the ERM - 1 : : GFP fluorescence intensity for the indicated genotype .

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

Figure 3A-C Quantification of the fluorescence intensity of AIY presynaptic marker GFP::RAB-3 (A, B) and nlp-40 mRNA level (C) for the indicated genotypes. The AIY synaptic region was quantified for the synaptic intensity analysis.D-I Confocal images of the transcriptional nlp-40 reporter (Pnlp-40::GFP) in wild-type (D), cwn-2(ok895) (E), cfz-2(ok1201) (F), pop-1(hu9) (G), and control or sys-1 RNAi (H and I) animals at adult Day 1 stage. J Quantification of the Pnlp-40::GFP fluorescence intensity for the indicated genotypes.K-P Confocal images of an intestinal-specific reporter ERM-1::GFP in wild type (K), cwn-2(ok895) (L), cfz-2(ok1201) (M), pop-1(hu9) (N), and control or sys-1 RNAi (O and P) animals at the adult Day1 stage. Q Quantification of the ERM-1::GFP fluorescence intensity for the indicated genotype.