( a ) CCR9 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( b ) CCR9 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( c ) CCR9 expression in the small intestine ( SI ) and colon of Rana catesbiana Shaw and Rana nigromaculata . ( d ) GPR15 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( e ) GPR15 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( f ) C10ORF99 expression in the small intestine ( SI ) and colon of Gallus gallus and Lonchura striata . ( g ) C10ORF99 expression in the small intestine ( SI ) and colon of Chrysemys picta bellii and Pelodiscus sinensis . ( h ) GPR15 and C10ORF99 expression in the small intestine ( SI ) and colon of Rana catesbeiana Shaw and Rana nigromaculata . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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

(a) CCR9 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (b) CCR9 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (c) CCR9 expression in the small intestine (SI) and colon of Rana catesbiana Shaw and Rana nigromaculata. (d) GPR15 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (e) GPR15 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (f) C10ORF99 expression in the small intestine (SI) and colon of Gallus gallus and Lonchura striata. (g) C10ORF99 expression in the small intestine (SI) and colon of Chrysemys picta bellii and Pelodiscus sinensis. (h) GPR15 and C10ORF99 expression in the small intestine (SI) and colon of Rana catesbeiana Shaw and Rana nigromaculata. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( i ) GPR15 expression in CD4 + T cell from chicken small intestine ( SI ) and colon . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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

(i) GPR15 expression in CD4+ T cell from chicken small intestine (SI) and colon. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


( j ) The migration of CD4 + cells from chicken small intestine ( SI ) and colon induced by ligand C10ORF99 . Data information : All data shown are representative of at least three independent technical replicates . Error bars indicate SD ( n = 3 ) . UD , undetermined , * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; NS : no significance ( t - test ) .

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

(j) The migration of CD4+ cells from chicken small intestine (SI) and colon induced by ligand C10ORF99. Data information: All data shown are representative of at least three independent technical replicates. Error bars indicate SD (n=3). UD, undetermined, ***P-value < 0.001; **P-value < 0.01; NS: no significance (t-test).


The expression levels of GPR15 , C10ORF99 , FOXP3 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

The expression levels of GPR15, C10ORF99, FOXP3 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


The expression levels of IL - 10 in mice fed with corn , chicken , and mixture of corn plus chicken , commercial food chow and a positive control ( propionate added to commercial food chow ) at different times ( weanling mice were defined as w - 1 ) in colonic tissue were determined by q - RT - PCR . Each symbol or bar represents pooled RNA from colon tissues from three mice . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

The expression levels of IL-10 in mice fed with corn, chicken, and mixture of corn plus chicken, commercial food chow and a positive control (propionate added to commercial food chow) at different times (weanling mice were defined as w-1) in colonic tissue were determined by q-RT-PCR. Each symbol or bar represents pooled RNA from colon tissues from three mice. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( f , g ) SCFAs and acetic acid content in dietary experiments . Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

(f, g) SCFAs and acetic acid content in dietary experiments. Faecal samples from mice on the indicated diets were analysed for SCFAs by gas chromatography. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( h ) TNF - α and IL - 6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups . IL - 6 and TNF - α protein production levels , as determined by performing enzyme - linked immunosorbent assays . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

(h) TNF-α and IL-6 levels in faecal samples from mice on the indicated diet demonstrated no colitis in all mouse groups. IL-6 and TNF-α protein production levels, as determined by performing enzyme-linked immunosorbent assays. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( i ) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

(i) Luciferase activities of constructs with the mouse C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells . Data information : All data shown are representative of at least three independent biological replicates . Error bars indicate SD ( n = 3 ) . * * * * P - value < 0 . 0001 * * * P - value < 0 . 001 ; * * P - value < 0 . 01 ; * P - value < 0 . 05 ; NS : no significance ( One - way ANOVA ) .

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

Luciferase activities of constructs with the Japanese quail C10ORF99 promoter in HEK293 cells. Data information: All data shown are representative of at least three independent biological replicates.Error bars indicate SD (n=3). ****P-value < 0.0001 ***P-value < 0.001; **P-value < 0.01; *P-value < 0.05; NS: no significance (One-way ANOVA).


( C ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 . C - terminally 6xHA tagged Rx and C - terminally 4xMyc - tagged NRC2EEE were co - expressed with either free GFP or C - terminally GFP - tagged CP . Free mCherry - 4xMyc and mCherry - 6xHA fusions were used as controls in the treatments without NRC2 and Rx , respectively . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisks indicate bands corresponding to the activated NRC2 complex . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 using different heavy and light sensor - helper combinations . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisk indicates bands corresponding to the activated NRC2 complex . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE and SDS - PAGE assays with inactive and activated Rx - NRC2 using different " heavy " and " light " helper combinations . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . A non - specific band was observed at ~ 110 kDa for the anti - FLAG antibody . Red asterisks indicate bands corresponding to the activated " heavy " NRC2 complexes . Blue asterisks indicate bands corresponding to " light " NRC2 complexes . Purple bands indicate intermediate molecular weight complexes combining " heavy " and " light " NRC2 . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different "heavy" and "light" helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated "heavy" NRC2 complexes. Blue asterisks indicate bands corresponding to "light" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining "heavy" and "light" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE and SDS - PAGE assays with inactive and activated NRC - dependent sensors and NRC2 . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisk indicates bands corresponding to the activated NRC2 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


BN - PAGE and SDS - PAGE assays with inactive and activated NRC - dependent sensors and NRC4AAA . Total protein was extracted with a HEPES - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisk indicates bands corresponding to the activated NRC4 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


C - terminally GFP - tagged NRC2EEE and C - terminally RFP - tagged Rx were co - expressed either with an EV - 4xMyc construct or a CP - 4xMyc construct in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines . ( A - B ) Single - plane confocal micrographs show the localization of both components of the inactive and active Rx - NRC2 system , together with PM marker RPW8 . 2 - BFP . Scale bars represent 10 µm . ( A ) NRC2EEE - GFP and Rx - RFP co - localize in the cytoplasm prior to activation . ( B ) Upon co - expression of CP and activation of the system , NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm .

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

C-terminally GFP-tagged NRC2EEE and C-terminally RFP-tagged Rx were co-expressed either with an EV-4xMyc construct or a CP-4xMyc construct in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines. (A-B) Single-plane confocal micrographs show the localization of both components of the inactive and active Rx-NRC2 system, together with PM marker RPW8.2-BFP. Scale bars represent 10 µm. (A) NRC2EEE-GFP and Rx-RFP co-localize in the cytoplasm prior to activation. (B) Upon co-expression of CP and activation of the system, NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm.


( C ) Membrane enrichment assays are consistent with microscopy , showing that inactive NRC2EEE - GFP is mostly present in the soluble ( cytoplasmic ) fraction , whereas activated NRC2EEE - GFP exhibits equal distribution across soluble and membrane fractions . Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP . T = total , S = soluble , M = membrane . ATPase was used as a membrane marker . Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining ( PS ) . Red asterisks indicate bands matching the expected MW for each protein . The experiment was repeated two times .

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

(C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.


( B ) BN - PAGE and SDS - PAGE assays with infected and uninfected leaves expressing Rx and NRC2 . Total protein was extracted with a Tris - HCl - based buffer , as described in materials and methods . Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left . Approximate molecular weights ( kDa ) of the proteins are shown on the right . Red asterisks indicate bands corresponding to the activated NRC2 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . The experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( A - B ) Single - plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection . C - terminally GFP - tagged NRC2EEE was co - infiltrated with either EV - RFP or Rx - RFP in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines and infected with PVX by agroinfection . Scale bars represent 10 µm . ( A ) In the absence of Rx , NRC2EEE - GFP is localized to the cytoplasm during PVX infection . ( B ) When Rx is present , NRC2EEE forms puncta associated with the PM during PVX infection .

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

(A-B) Single-plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection. C-terminally GFP-tagged NRC2EEE was co-infiltrated with either EV-RFP or Rx-RFP in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines and infected with PVX by agroinfection. Scale bars represent 10 µm. (A) In the absence of Rx, NRC2EEE-GFP is localized to the cytoplasm during PVX infection. (B) When Rx is present, NRC2EEE forms puncta associated with the PM during PVX infection.


( A ) MEFs were transfected with GFP - tagged wild - type ( WT ) or mutant Parkin expression plasmid followed by an 18 - h treatment of CCCP . Cells are immunostained with a Tom20 antibody to visualize mitochondria ( red ) . GFP - parkin - transfected cells are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial aggregates . Bar , 10 µm . ( B ) The average percentages of mitochondria - free cells from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 .

O O O B-CELL_LINE O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O

(A) MEFs were transfected with GFP-tagged wild-type (WT) or mutant Parkin expression plasmid followed by an 18-h treatment of CCCP. Cells are immunostained with a Tom20 antibody to visualize mitochondria (red). GFP-parkin-transfected cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of mitochondria-free cells from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01


( C ) MEFs were transfected and treated with CCCP as described in A , followed by an immunoblotting analysis with antibodies for cytochrome c , actin , and parkin . Note that levels of parkin R275W and R42P mutant were lower , as previously reported ( Wang et al . , 2005 ) .

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

(C) MEFs were transfected and treated with CCCP as described in A, followed by an immunoblotting analysis with antibodies for cytochrome c, actin, and parkin. Note that levels of parkin R275W and R42P mutant were lower, as previously reported (Wang et al., 2005).


CCCP - induced parkin - mitochondrial aggregate formation is an intermediate step for mitophagy . ( A ) MEFs expressing WT GFP - parkin were treated with CCCP or CCCP and nocodazole ( NOC , 10 µM ) for 8 , 16 , and 24 h as indicated . Cells were immunostained with anti - Tom20 to visualize mitochondria . GFP - parkin - positive cells are marked by dotted lines . Arrows indicate parkin - positive mitochondria or mitochondrial aggregates . Bar , 25 µm . ( B ) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar . * * , P < 0 . 01 .

B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O

CCCP-induced parkin-mitochondrial aggregate formation is an intermediate step for mitophagy. (A) MEFs expressing WT GFP-parkin were treated with CCCP or CCCP and nocodazole (NOC, 10 µM) for 8, 16, and 24 h as indicated. Cells were immunostained with anti-Tom20 to visualize mitochondria. GFP-parkin-positive cells are marked by dotted lines. Arrows indicate parkin-positive mitochondria or mitochondrial aggregates. Bar, 25 µm. (B) The average percentages of cells with mitochondrial aggregates or without mitochondria from three independent experiments from A are presented with standard deviation as error bar. **, P < 0.01.


( A ) MEFs were transfected with GFP wild - type ( WT ) , A240R , and T415N mutant parkin followed by CCCP treatment for 8 h . Cells were immunostained with antibodies for cytochrome c ( red ) and ubiquitin ( blue ) . Arrows indicate mitochondrial aggregates . Bar , 10 µm . ( B ) The average percentages of ubiquitin - positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar .

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

(A) MEFs were transfected with GFP wild-type (WT), A240R, and T415N mutant parkin followed by CCCP treatment for 8 h. Cells were immunostained with antibodies for cytochrome c (red) and ubiquitin (blue). Arrows indicate mitochondrial aggregates. Bar, 10 µm. (B) The average percentages of ubiquitin-positive mitochondrial aggregates from three independent experiments from A are presented with standard deviation as error bar.


( C and D ) MEFs were transfected and treated with CCCP as described in A . Cells were subjected to the fractionation to isolate crude mitochondria . Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin , parkin , cytochrome c , and cytosolic Hsp90 .

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

(C and D) MEFs were transfected and treated with CCCP as described in A. Cells were subjected to the fractionation to isolate crude mitochondria. Cytoplasmic and mitochondrial fractions were subjected to immunoblotting analysis using antibodies for ubiquitin, parkin, cytochrome c, and cytosolic Hsp90.


Parkin - mediated ubiquitination recruits p62 and HDAC6 . MEFs were transfected with WT , A240R , and T415N mutant GFP - parkin followed by CCCP treatment for 8 h . Cells were double immunostained with cytochrome c ( red ) and p62 antibody ( blue ) in A , and cytochrome c ( red ) and HDAC6 antibody in C . Arrows indicate mitochondrial aggregates . ( B and D ) The average percentages of p62 - or HDAC6 - positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar .

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

Parkin-mediated ubiquitination recruits p62 and HDAC6. MEFs were transfected with WT, A240R, and T415N mutant GFP-parkin followed by CCCP treatment for 8 h. Cells were double immunostained with cytochrome c (red) and p62 antibody (blue) in A, and cytochrome c (red) and HDAC6 antibody in C. Arrows indicate mitochondrial aggregates. (B and D) The average percentages of p62- or HDAC6-positive mitochondrial aggregates from three independent experiments are presented with standard deviation as error bar.


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


( E ) Wild - type and HDAC6 knockout ( KO ) MEFs were transfected with parkin - GFP or cotransfected with a Flag - tagged HDAC6 followed by CCCP treatment for 16 h as indicated . Cells are immunostained with Tom20 ( red ) and Flag ( blue ) antibodies . Bar , 25 µm .

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

(E) Wild-type and HDAC6 knockout (KO) MEFs were transfected with parkin-GFP or cotransfected with a Flag-tagged HDAC6 followed by CCCP treatment for 16 h as indicated. Cells are immunostained with Tom20 (red) and Flag (blue) antibodies. Bar, 25 µm.


( F ) Wild - type MEFs were transfected with control or HDAC6 siRNA and parkin - GFP , and treated with or without CCCP for 16 h . Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20 , parkin , HDAC6 , and actin .

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

(F) Wild-type MEFs were transfected with control or HDAC6 siRNA and parkin-GFP, and treated with or without CCCP for 16 h. Cell lysates were subjected to immunoblotting analysis using antibodies for Tom20, parkin, HDAC6, and actin.


MEFs were transfected with control or cortactin RNAi and parkin - GFP , and incubated with or without CCCP for 16 h as indicated . ( A ) Cells were subjected to immunostaining with antibodies for Tom20 and parkin . Bar , 10 µm .

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

MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated. (A) Cells were subjected to immunostaining with antibodies for Tom20 and parkin. Bar, 10 µm.


MEFs were transfected with control or cortactin RNAi and parkin - GFP , and incubated with or without CCCP for 16 h as indicated . ( B ) Cell lysates were subjected to immunoblotting with antibodies for Tom20 , parkin , cortactin , and actin .

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

MEFs were transfected with control or cortactin RNAi and parkin-GFP, and incubated with or without CCCP for 16 h as indicated.(B) Cell lysates were subjected to immunoblotting with antibodies for Tom20, parkin, cortactin, and actin.


( b ) A53T α - synuclein ( α - syn ) expression was induced in stable PC12 cells for 48 h , then switched off for 24 h . Cells were treated with DMSO ( control ) , or with 0 . 94 μM , 4 . 7 μM or 47 μM of SMER10 ; 0 . 86 μM , 4 . 3 μM or 43 μM of SMER18 ; or 0 . 9 μM , 4 . 7 μM or 47 μM of SMER28 added in the switch - off period . A53T α - syn levels were analyzed by immunoblotting with antibody against HA ( left ) and densitometry analysis relative to actin ( right ) . Error bars denote s . e . m . P = 0 . 0917 , P = 0 . 009 , P = 0 . 0001 ( for increasing concentrations of SMER10 ) ; P = 0 . 0068 , P = 0 . 0023 , P = 0 . 0002 ( for increasing concentrations of SMER18 ) ; P = 0 . 0016 , P 0 . 0001 , P 0 . 0001 ( for increasing concentrations of SMER28 ) .

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

(b) A53T α-synuclein (α-syn) expression was induced in stable PC12 cells for 48 h, then switched off for 24 h. Cells were treated with DMSO (control), or with 0.94 μM, 4.7 μM or 47 μM of SMER10; 0.86 μM, 4.3 μM or 43 μM of SMER18; or 0.9 μM, 4.7 μM or 47 μM of SMER28 added in the switch-off period. A53T α-syn levels were analyzed by immunoblotting with antibody against HA (left) and densitometry analysis relative to actin (right). Error bars denote s.e.m. P = 0.0917, P = 0.009, P = 0.0001 (for increasing concentrations of SMER10); P = 0.0068, P = 0.0023, P = 0.0002 (for increasing concentrations of SMER18); P = 0.0016, P 0.0001, P 0.0001 (for increasing concentrations of SMER28).


( c ) COS - 7 cells transfected with EGFP - HDQ74 construct were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( Rap ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , and we assessed the proportions of apoptotic or aggregate - containing transfected cells . Error bars : 95 % confidence interval . P 0 . 0001 ( Rap and SMER28 ) , P = 0 . 013 ( SMER10 ) , P = 0 . 019 ( SMER18 ) ( aggregation ) ; P 0 . 0001 ( Rap , SMER18 and SMER28 ) , P = 0 . 004 ( SMER10 ) ( cell death ) .

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

(c) COS-7 cells transfected with EGFP-HDQ74 construct were treated with DMSO (control), 0.2 μM rapamycin (Rap), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, and we assessed the proportions of apoptotic or aggregate-containing transfected cells. Error bars: 95% confidence interval. P 0.0001 (Rap and SMER28), P = 0.013 (SMER10), P = 0.019 (SMER18) (aggregation); P 0.0001 (Rap, SMER18 and SMER28), P = 0.004 (SMER10) (cell death).


( d ) Wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs were transfected with EGFP - HDQ74 and treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h , and we assessed the percentages of aggregate - containing transfected cells . The control ( DMSO - treated ) values were fixed at 1 for both cell lines . Error bars : 95 % confidence interval . P = 0 . 039 ( SMER10 ) , P 0 . 0001 ( SMER18 and SMER28 ) ( in Atg5 + / + cells ) ; P = 0 . 092 ( SMER10 ) , P = 0 . 271 ( SMER18 ) , P = 0 . 358 ( SMER28 ) ( in Atg5 − / − cells ) . Note that EGFP - HDQ74 aggregation was higher in Atg5 − / − than in Atg5 + / + cells ( Supplementary Fig . 3a ) .

O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-GENEPROD B-CELL_LINE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O O O O

(d) Wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs were transfected with EGFP-HDQ74 and treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h, and we assessed the percentages of aggregate-containing transfected cells. The control (DMSO-treated) values were fixed at 1 for both cell lines. Error bars: 95% confidence interval. P = 0.039 (SMER10), P 0.0001 (SMER18 and SMER28) (in Atg5+/+ cells); P = 0.092 (SMER10), P = 0.271 (SMER18), P = 0.358 (SMER28) (in Atg5−/− cells). Note that EGFP-HDQ74 aggregation was higher in Atg5−/− than in Atg5+/+ cells (Supplementary Fig. 3a).


e ) The percentages of EGFP - positive COS - 7 cells with EGFP - HDQ74 aggregates as in c , treated with DMSO ( control ) , 10 μM lactacystin ( proteasome inhibitor ) or both 10 μM lactacystin along with either 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 48 h , expressed as odds ratios . Error bars : 95 % confidence interval . P 0 . 0001 ( control versus Lact ) , P = 0 . 014 ( SMER10 versus Lact ) , P 0 . 0001 ( SMER18 versus Lact ) , P = 0 . 001 ( SMER28 versus Lact ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

e) The percentages of EGFP-positive COS-7 cells with EGFP-HDQ74 aggregates as in c, treated with DMSO (control), 10 μM lactacystin (proteasome inhibitor) or both 10 μM lactacystin along with either 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 48 h, expressed as odds ratios. Error bars: 95% confidence interval. P 0.0001 (control versus Lact), P = 0.014 (SMER10 versus Lact), P 0.0001 (SMER18 versus Lact), P = 0.001 (SMER28 versus Lact). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a ) COS - 7 cells transfected with EGFP - LC3 construct for 4 h were treated with DMSO ( control ) , 0 . 2 μM rapamycin ( positive control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 16 h , and analyzed by fluorescence microscopy . The effects of treatment on the percentage of EGFP - positive cells with > 5 EGFP - LC3 vesicles are shown . Error bars denote s . e . m . P 0 . 0001 ( all SMERs ) .

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

(a) COS-7 cells transfected with EGFP-LC3 construct for 4 h were treated with DMSO (control), 0.2 μM rapamycin (positive control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 16 h, and analyzed by fluorescence microscopy. The effects of treatment on the percentage of EGFP-positive cells with >5 EGFP-LC3 vesicles are shown. Error bars denote s.e.m. P 0.0001 (all SMERs).


( b ) HeLa cells stably expressing EGFP - LC3 were treated with DMSO ( control ) , 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 for 24 h . Confocal sections show cells containing EGFP - positive autophagic vesicles . Nuclei are stained with DAPI . Bar , 20 μM .

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

(b) HeLa cells stably expressing EGFP-LC3 were treated with DMSO (control), 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28 for 24 h. Confocal sections show cells containing EGFP-positive autophagic vesicles. Nuclei are stained with DAPI. Bar, 20 μM.


( c ) HeLa cells stably expressing EGFP - LC3 were treated for 4 h with DMSO ( control ) or 200 nM bafilomycin A1 ( Baf ) , or with 200 nM bafilomycin A1 and 47 μM SMER10 , 43 μM SMER18 or 47 μM SMER28 . Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1 . Levels of EGFP - LC3 - II were determined by immunoblotting with antibody against EGFP ( above ) and densitometry analysis relative to actin ( below ) . Error bars denote s . e . m . P = 0 . 0259 ( baf ) , P 0 . 0001 ( SMER10 ) , P = 0 . 0003 ( SMER18 and SMER28 ) versus control ; P = 0 . 0025 ( SMER10 ) , P = 0 . 0218 ( SMER18 ) , P = 0 . 0195 ( SMER28 ) versus bafilomycin A1 . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 .

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

(c) HeLa cells stably expressing EGFP-LC3 were treated for 4 h with DMSO (control) or 200 nM bafilomycin A1 (Baf), or with 200 nM bafilomycin A1 and 47 μM SMER10, 43 μM SMER18 or 47 μM SMER28. Cells were left untreated or pretreated with SMERs for 24 h before adding bafilomycin A1. Levels of EGFP-LC3-II were determined by immunoblotting with antibody against EGFP (above) and densitometry analysis relative to actin (below). Error bars denote s.e.m. P = 0.0259 (baf), P 0.0001 (SMER10), P = 0.0003 (SMER18 and SMER28) versus control; P = 0.0025 (SMER10), P = 0.0218 (SMER18), P = 0.0195 (SMER28) versus bafilomycin A1. ***P 0.001; **P 0.01; *P 0.05.


( a - c ) Flies treated with 100 μM SMER10 ( a ) , 200 μM SMER18 ( b ) or 100 μM SMER28 ( c ) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO ( control ) alone ( 2 d after eclosion ) . Rhabdomere counts from all three independent experiments are included . n = 600 ommatidia ( SMER10 ) , n = 1 , 500 ommatidia ( SMER18 ) and n = 600 ommatidia ( SMER28 ) . Mann - Whitney test values P 0 . 0001 ( all SMERs ) . Student ' s t - test ( 1 - tailed ) P = 0 . 005 ( SMER10 ) , P = 0 . 004 ( SMER18 ) , P = 0 . 03 ( SMER28 ) comparing means of distributions from independent experiments . These SMER concentrations cause no overt toxicity to flies ( see Supplementary Methods ) . Distributions of DMSO - treated flies may vary when SMERs are treated in different experiments at different times . For instance , an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time - dependent .

O O O O O B-ORGANISM O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O B-TISSUE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O

(a-c) Flies treated with 100 μM SMER10 (a), 200 μM SMER18 (b) or 100 μM SMER28 (c) show a shift in the distribution of the number of rhabdomeres compared with flies treated with DMSO (control) alone (2 d after eclosion). Rhabdomere counts from all three independent experiments are included. n = 600 ommatidia (SMER10), n = 1,500 ommatidia (SMER18) and n = 600 ommatidia (SMER28). Mann-Whitney test values P 0.0001 (all SMERs). Student's t-test (1-tailed) P = 0.005 (SMER10), P = 0.004 (SMER18), P = 0.03 (SMER28) comparing means of distributions from independent experiments. These SMER concentrations cause no overt toxicity to flies (see Supplementary Methods). Distributions of DMSO-treated flies may vary when SMERs are treated in different experiments at different times. For instance, an individual experiment may have lasted slightly longer or shorter and photoreceptor degeneration is progressive and time-dependent.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( a ) , 43 μM SMER18 ( b ) or 47 μM SMER28 ( c ) ) or both for the 8 h switch - off period - was analyzed by immunoblotting with antibody against HA ( above ) and densitometry analysis relative to actin ( below ) . The concentration of rapamycin is saturating for its effect on the clearance of A53T α - synuclein . Error bars denote s . e . m . P = 0 . 0025 ( Rap ) , P = 0 . 0018 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 0001 ( Rap or SMER10 versus SMER10 + Rap ) ( a ) ; P = 0 . 0069 ( Rap ) , P = 0 . 0498 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 0038 ( Rap versus SMER18 + Rap ) , P = 0 . 0007 ( SMER18 versus SMER18 + Rap ) ( b ) ; P 0 . 0001 ( Rap , SMER28 , Rap versus SMER28 + Rap , SMER28 versus SMER28 + Rap ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (a), 43 μM SMER18 (b) or 47 μM SMER28 (c)) or both for the 8 h switch-off period-was analyzed by immunoblotting with antibody against HA (above) and densitometry analysis relative to actin (below). The concentration of rapamycin is saturating for its effect on the clearance of A53T α-synuclein. Error bars denote s.e.m. P = 0.0025 (Rap), P = 0.0018 (SMER10), P 0.0001 (SMER10 + Rap), P 0.0001 (Rap or SMER10 versus SMER10 + Rap) (a); P = 0.0069 (Rap), P = 0.0498 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.0038 (Rap versus SMER18 + Rap), P = 0.0007 (SMER18 versus SMER18 + Rap) (b); P 0.0001 (Rap, SMER28, Rap versus SMER28 + Rap, SMER28 versus SMER28 + Rap) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates ( above ) and cell death ( below ) in COS - 7 cells , as in Figure 1c - treated with DMSO ( control ) , or with 0 . 2 μM rapamycin alone , SMER alone ( 140 μM SMER10 ( d ) , 43 μM SMER18 ( e ) or 47 μM SMER28 ( f ) ) or both for 24 h - were expressed as odds ratios . Error bars : 95 % confidence interval . Aggregation ( top panels ) : P = 0 . 248 ( Rap ) , P = 0 . 217 ( SMER10 ) , P 0 . 0001 ( SMER10 + Rap ) , P 0 . 001 ( Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 248 ( Rap ) , P = 0 . 543 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 008 ( Rap versus SMER18 + Rap ) , P = 0 . 002 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 248 ( Rap ) , P = 0 . 002 ( SMER28 ) , P 0 . 0001 ( SMER28 + Rap ) , P 0 . 0001 ( Rap versus SMER28 + Rap ) , P = 0 . 012 ( SMER28 versus SMER28 + Rap ) ( f ) . Cell death ( bottom panels ) : P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER10 , SMER10 + Rap , Rap or SMER10 versus SMER10 + Rap ) ( d ) ; P = 0 . 002 ( Rap ) , P = 0 . 948 ( SMER18 ) , P 0 . 0001 ( SMER18 + Rap ) , P = 0 . 015 ( Rap versus SMER18 + Rap ) , P 0 . 0001 ( SMER18 versus SMER18 + Rap ) ( e ) ; P = 0 . 002 ( Rap ) , P 0 . 0001 ( SMER28 , SMER28 + Rap , Rap or SMER28 versus SMER28 + Rap ) ( f ) . Note that we have treated cells for a shorter time in this experiment ( 24 h ) , compared with Figure 1c ( 48 h ) . This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates (above) and cell death (below) in COS-7 cells, as in Figure 1c-treated with DMSO (control), or with 0.2 μM rapamycin alone, SMER alone (140 μM SMER10 (d), 43 μM SMER18 (e) or 47 μM SMER28 (f)) or both for 24 h-were expressed as odds ratios. Error bars: 95% confidence interval. Aggregation (top panels): P = 0.248 (Rap), P = 0.217 (SMER10), P 0.0001 (SMER10 + Rap), P 0.001 (Rap or SMER10 versus SMER10 + Rap) (d); P = 0.248 (Rap), P = 0.543 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.008 (Rap versus SMER18 + Rap), P = 0.002 (SMER18 versus SMER18 + Rap) (e); P = 0.248 (Rap), P = 0.002 (SMER28), P 0.0001 (SMER28 + Rap), P 0.0001 (Rap versus SMER28 + Rap), P = 0.012 (SMER28 versus SMER28 + Rap) (f). Cell death (bottom panels): P = 0.002 (Rap), P 0.0001 (SMER10, SMER10 + Rap, Rap or SMER10 versus SMER10 + Rap) (d); P = 0.002 (Rap), P = 0.948 (SMER18), P 0.0001 (SMER18 + Rap), P = 0.015 (Rap versus SMER18 + Rap), P 0.0001 (SMER18 versus SMER18 + Rap) (e); P = 0.002 (Rap), P 0.0001 (SMER28, SMER28 + Rap, Rap or SMER28 versus SMER28 + Rap) (f). Note that we have treated cells for a shorter time in this experiment (24 h), compared with Figure 1c (48 h). This probably accounts for the failure of the protective trends of rapamycin and some of the SMERs to reach significance for aggregation on their own in some of the experiments. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


( a - c ) Clearance of A53T α - synuclein in stable PC12 cells as in Figure 1b - treated for 24 h with either DMSO ( control ) , or with 47 μM SMER10 and its analogs ( SMER10a - c ) ( a ) , 43 μM SMER18 and its analogs ( SMER18a - l ) ( b ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( c ) - was analyzed by immunoblotting with anti - HA antibody ( above ) and densitometry analysis relative to actin ( below ) . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars denote s . e . m . P = 0 . 0058 ( SMER10a ) , P = 0 . 6736 ( SMER10b ) , P = 0 . 9507 ( SMER10c ) , P = 0 . 0481 ( SMER10 ) ( a ) ; P = 0 . 0006 ( SMER18a ) , P = 0 . 0249 ( SMER18b ) , P = 0 . 0167 ( SMER18c ) , P = 0 . 0117 ( SMER18d ) , P = 0 . 0269 ( SMER18e ) , P = 0 . 0165 ( SMER18f ) , P = 0 . 0148 ( SMER18g ) , P = 0 . 0011 ( SMER18h ) , P = 0 . 7369 ( SMER18i ) , P = 0 . 0012 ( SMER18j ) , P = 0 . 1531 ( SMER18k ) , P = 0 . 0006 ( SMER18l ) , P = 0 . 0001 ( SMER18 ) ( b ) ; P = 0 . 0014 ( SMER28a ) , P = 0 . 0002 ( SMER28b ) , P = 0 . 0001 ( SMER28d ) , P = 0 . 0048 ( SMER28h ) , P = 0 . 0002 ( SMER28i ) , P = 0 . 0162 ( SMER28j ) , P 0 . 0001 ( SMER28c , SMER28e - g , SMER28k , SMER28 ) ( c ) .

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

(a-c) Clearance of A53T α-synuclein in stable PC12 cells as in Figure 1b-treated for 24 h with either DMSO (control), or with 47 μM SMER10 and its analogs (SMER10a-c) (a), 43 μM SMER18 and its analogs (SMER18a-l) (b), or 47 μM SMER28 and its analogs (SMER28a-k) (c)-was analyzed by immunoblotting with anti-HA antibody (above) and densitometry analysis relative to actin (below). All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars denote s.e.m. P = 0.0058 (SMER10a), P = 0.6736 (SMER10b), P = 0.9507 (SMER10c), P = 0.0481 (SMER10) (a); P = 0.0006 (SMER18a), P = 0.0249 (SMER18b), P = 0.0167 (SMER18c), P = 0.0117 (SMER18d), P = 0.0269 (SMER18e), P = 0.0165 (SMER18f), P = 0.0148 (SMER18g), P = 0.0011 (SMER18h), P = 0.7369 (SMER18i), P = 0.0012 (SMER18j), P = 0.1531 (SMER18k), P = 0.0006 (SMER18l), P = 0.0001 (SMER18) (b); P = 0.0014 (SMER28a), P = 0.0002 (SMER28b), P = 0.0001 (SMER28d), P = 0.0048 (SMER28h), P = 0.0002 (SMER28i), P = 0.0162 (SMER28j), P 0.0001 (SMER28c, SMER28e-g, SMER28k, SMER28) (c).


( d - f ) The percentage of EGFP - positive cells with EGFP - HDQ74 aggregates in COS - 7 cells as in Figure 1c - treated for 48 h with either DMSO ( control ) , or with 47 μM SMER10 and its analog ( SMER10a ) ( d ) , 43 μM SMER18 and its analogs ( SMER18a , SMER18c - h , SMER18j , SMER18l ) ( e ) , or 47 μM SMER28 and its analogs ( SMER28a - k ) ( f ) - were expressed as odds ratios . All the analogs were used in the cell culture medium at 1 : 400 dilution of 5 mg ml − 1 stock solution ( in DMSO ) . Error bars : 95 % confidence interval . P = 0 . 003 ( SMER10a ) , P = 0 . 004 ( SMER10 ) ( d ) ; P 0 . 0001 ( SMER18a , SMER18c , SMER18d , SMER18f , SMER18h ) , P = 0 . 009 ( SMER18e ) , P = 0 . 001 ( SMER18g ) , P = 0 . 382 ( SMER18j ) , P = 0 . 067 ( SMER18l ) , P = 0 . 031 ( SMER18 ) ( e ) ; P 0 . 0001 ( SMER28a , SMER28c , SMER28e - g , SMER28i - k , SMER28 ) , P = 0 . 574 ( SMER28b ) , P = 0 . 041 ( SMER28d ) , P = 0 . 002 ( SMER28h ) ( f ) . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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

(d-f) The percentage of EGFP-positive cells with EGFP-HDQ74 aggregates in COS-7 cells as in Figure 1c-treated for 48 h with either DMSO (control), or with 47 μM SMER10 and its analog (SMER10a) (d), 43 μM SMER18 and its analogs (SMER18a, SMER18c-h, SMER18j, SMER18l) (e), or 47 μM SMER28 and its analogs (SMER28a-k) (f)-were expressed as odds ratios. All the analogs were used in the cell culture medium at 1:400 dilution of 5 mg ml−1 stock solution (in DMSO). Error bars: 95% confidence interval. P = 0.003 (SMER10a), P = 0.004 (SMER10) (d); P 0.0001 (SMER18a, SMER18c, SMER18d, SMER18f, SMER18h), P = 0.009 (SMER18e), P = 0.001 (SMER18g), P = 0.382 (SMER18j), P = 0.067 (SMER18l), P = 0.031 (SMER18) (e); P 0.0001 (SMER28a, SMER28c, SMER28e-g, SMER28i-k, SMER28), P = 0.574 (SMER28b), P = 0.041 (SMER28d), P = 0.002 (SMER28h) (f). ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


B . RIBEYE mutations do not alter mouse survival . Data show surviving post - weaning mice ( at > P21 ) derived from matings between mice heterozygous for the RBEKI allele ( dark red ) or the RBEKO allele ( bright red ) normalized for the total number of mice ( absolute numbers per genotype / absolute number of all mice per respective breeding strategy ) The expected Mendelian ratio is indicated by the dashed line . Statistical analysis using the 2 - test revealed no statistical difference between wild - type and mutant mice ( RBEKI allele p = 0 . 33 ; RBEKO allele p = 0 . 11 ) .

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

B. RIBEYE mutations do not alter mouse survival. Data show surviving post-weaning mice (at >P21) derived from matings between mice heterozygous for the RBEKI allele (dark red) or the RBEKO allele (bright red) normalized for the total number of mice (absolute numbers per genotype/absolute number of all mice per respective breeding strategy) The expected Mendelian ratio is indicated by the dashed line. Statistical analysis using the 2-test revealed no statistical difference between wild-type and mutant mice (RBEKI allele p=0.33; RBEKO allele p=0.11).


C . Representative immunoblot of retina homogenates from homozygous RBEWT / WT , RBEKI / KI and RBEKO / KO mice probed with antibodies to CtBP2 ( to detect the B - domain of RIBEYE and CtBP2 ) and to EGFP ( to detect GCamP3 in the RIBEYE A - domain GCamP3 fusion protein referred to as RIB - G3 ) .

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

C. Representative immunoblot of retina homogenates from homozygous RBEWT/WT, RBEKI/KI and RBEKO/KO mice probed with antibodies to CtBP2 (to detect the B-domain of RIBEYE and CtBP2) and to EGFP (to detect GCamP3 in the RIBEYE A-domain GCamP3 fusion protein referred to as RIB-G3).


D . Measurements by RT - PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild - type mice ( RBEWT / WT ) , KI - mice expressing the RIBEYE - A - domain fusion protein with GCamP3 ( RBEKI / KI ) , and KO mice ( RBEKO / KO ) . These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina , and to examine whether the RIBEYE A - domain KO impairs CtBP2 expression in brain , which expresses little RIBEYE . Assays are based on a qRT - PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms ( RIBEYE and CtBP2 ) . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * * * , p < 0 . 001 ; n = 4 mice per genotype ) .

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

D. Measurements by RT-PCR of the relative abundance of CtBP2 and RIBEYE mRNAs using an assay that simultaneously measures both mRNAs in retina and in brain from control wild-type mice (RBEWT/WT), KI-mice expressing the RIBEYE-A-domain fusion protein with GCamP3 (RBEKI/KI), and KO mice (RBEKO/KO). These measurements were performed to test the relative expression of RIBEYE and CtBP2 in retina, and to examine whether the RIBEYE A-domain KO impairs CtBP2 expression in brain, which expresses little RIBEYE. Assays are based on a qRT-PCR strategy detecting mRNAs containing the spliced connection between exon 2 and 3 which is common to both transcript isoforms (RIBEYE and CtBP2). Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (***, p<0.001; n=4 mice per genotype).


E . Representative immunofluorescence labeling of cryostat sections from littermate wild - type and RBEKI / KI mice for EGFP ( green , to detect GCamP3 in RIB - G3 ) and SV2 ( red , to label synapses ) demonstrates expression of the A - domain / GCamP3 fusion protein in knockin mice ( OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , granule cell layer ) . Scale bar = 5 µm .

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

E. Representative immunofluorescence labeling of cryostat sections from littermate wild-type and RBEKI/KI mice for EGFP (green, to detect GCamP3 in RIB-G3) and SV2 (red, to label synapses) demonstrates expression of the A-domain/GCamP3 fusion protein in knockin mice (OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, granule cell layer). Scale bar = 5 µm.


F . Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT / WT and RBEKO / KO mice . Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies ; results are normalized to loading controls depending on molecular weights ( actin , fodrin , VCP , and GDI ) and to wild - type controls . Statistical analyses were performed using Student ' s t - test ; data are presented as mean ± SEM ( * , p < 0 . 05 ; n = 4 mice per genotype ) .

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

F. Levels of CtBP2 and of synaptic proteins in retina homogenates from littermate RBEWT/WT and RBEKO/KO mice. Levels were determined by quantitative immunoblotting using fluorescently labeled secondary antibodies; results are normalized to loading controls depending on molecular weights (actin, fodrin, VCP, and GDI) and to wild-type controls. Statistical analyses were performed using Student's t-test; data are presented as mean ± SEM (*, p<0.05; n=4 mice per genotype).


G . Immunofluoresence labeling for CtBP2 ( green , to label RIBEYE ) and mGluR6 ( red , to label photoreceptorsynapses ) in cryostat sections from littermate RBEWT / WT and RBEKO / KOmouseretinas demonstrates that the KO abolishes RIBEYE expression ( OPL , outer plexiform layer ) . Scale bars : 5 µm .

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

G. Immunofluoresence labeling for CtBP2 (green, to label RIBEYE) and mGluR6 (red, to label photoreceptorsynapses) in cryostat sections from littermate RBEWT/WT and RBEKO/KOmouseretinas demonstrates that the KO abolishes RIBEYE expression (OPL, outer plexiform layer). Scale bars: 5 µm.


A . Overview of the synaptic organization of the retina in RBEWT / WT and RBEKO / KOmice . Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B - domain ( CtBP2 ; green ) and SV2 . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer ; IPL , inner plexiform layer ; GCL , ganglion cell layer .

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

A. Overview of the synaptic organization of the retina in RBEWT/WT and RBEKO/KOmice. Cryostat sections from littermate mice were labeled by double immunofluorescence for the RIBEYE B-domain (CtBP2; green) and SV2. Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.


B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice for PSD95 ( green , to label postsynaptic specializations ) and mGluR6 ( red , to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses ) . Abbreviations are as in A . Scale bars : 20 µm ( left and right overview ) , 5 µm ( left panel magnification ) , 2 µm ( right panel magnification ) . C . Same as B , except that retinas were stained for PKCα ( green , to label bipolar neurons ) and PSD95 ( red , to label amacrine cells ) .

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

B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice for PSD95 (green, to label postsynaptic specializations) and mGluR6 (red, to label specifically postsynaptic sites formed by bipolar neurons in photoreceptorsynapses). Abbreviations are as in A. Scale bars: 20 µm (left and right overview), 5 µm (left panel magnification), 2 µm (right panel magnification).C. Same as B, except that retinas were stained for PKCα (green, to label bipolar neurons) and PSD95 (red, to label amacrine cells).


A and B . Double immunofluorescence staining of retinas from RBEWT / WT and RBEKO / KO mice , respectively , for PSD95 ( green , to label presynaptic photoreceptor terminals ) and Cav1 . 4 ( red , to label presynaptic Ca2 + - channel ) . Scale bar : 20 µm ; abbreviations : ONL , outer nuclear layer ; OPL , outer plexiform layer ; INL , inner nuclear layer . ( A1 to A4 ) Magnification of Cav1 . 4 staining in the outer plexiform layer ( OPL ) in the retina of RBEWT / WT and ( B1 to B4 ) RBEKO / KO mice . White arrow heads indicate Ca2 + - channel distribution at selected synapses . Images represent 2 µm z - stack projections . Scale bar : 2 µm .

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

A and B. Double immunofluorescence staining of retinas from RBEWT/WT and RBEKO/KO mice, respectively, for PSD95 (green, to label presynaptic photoreceptor terminals) and Cav1.4 (red, to label presynaptic Ca2+-channel). Scale bar: 20 µm; abbreviations: ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer. (A1 to A4) Magnification of Cav1.4 staining in the outer plexiform layer (OPL) in the retina of RBEWT/WT and (B1 to B4) RBEKO/KO mice. White arrow heads indicate Ca2+-channel distribution at selected synapses. Images represent 2 µm z-stack projections. Scale bar: 2 µm.


A . Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO / KOmice in which the synaptic ribbon is absent . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT / WTmice . Abbreviations : sr , synaptic ribbon ; pre , presynaptic ; post , postsynaptic . Scale bars : 500 nm .

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

A. Transmission EM demonstrates the absence of synaptic ribbons in presynaptic terminals of photoreceptorribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Asterisks in A2 and A3 denote photoreceptor active zones of RBEKO/KOmice in which the synaptic ribbon is absent. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to that of RBEWT/WTmice. Abbreviations: sr, synaptic ribbon; pre, presynaptic; post, postsynaptic. Scale bars: 500 nm.


B . Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO / KOmice , while synaptic ribbons are readily visible in the terminals of RBEWT / WTmice . Except for the absence of synaptic ribbons , the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT / WTmice . Abbreviations are the same as in A . Scale bars : 1 µm ( B1 , B2 , B4 , B5 ) ; 300 nm ( B3 , B6 ) .

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

B. Transmission EM demonstrates absence of synaptic ribbons in presynaptic terminals of bipolar cellribbon synapses in RBEKO/KOmice, while synaptic ribbons are readily visible in the terminals of RBEWT/WTmice. Except for the absence of synaptic ribbons, the ultrastructural appearance of the presynaptic terminals is comparable to RBEWT/WTmice. Abbreviations are the same as in A. Scale bars: 1 µm (B1, B2, B4, B5); 300 nm (B3, B6).


A and B . Representative transmission EM pictures of RBEWT / WT ( A ) and RBEKO / KO ( B ) photoreceptor ribbon synapses ( blue rectangles , area adjacent to synaptic junction used for measuring junction - associated vesicle density [ size = 200 nm x 300 nm ] ; black squares , are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [ size = 200 nm x 200 nm ] ) . C . Average vesicle ratio ( expressed as vesicle numbers in the area adjacent to synaptic junctions ( blue rectangle ) or the cytosol ( black square ) normalized to RBEWT / WT . D . Average number of docked vesicles within a 150 nm range in the vicinity of the active zone .

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

A and B. Representative transmission EM pictures of RBEWT/WT (A) and RBEKO/KO (B) photoreceptor ribbon synapses (blue rectangles, area adjacent to synaptic junction used for measuring junction-associated vesicle density [size = 200 nm x 300 nm]; black squares, are in the presynaptic cytosol used to measure cytosolic synaptic vesicles [size = 200 nm x 200 nm]).C. Average vesicle ratio (expressed as vesicle numbers in the area adjacent to synaptic junctions (blue rectangle) or the cytosol (black square) normalized to RBEWT/WT.D. Average number of docked vesicles within a 150 nm range in the vicinity of the active zone.


A . Experimental design and representative traces for analysis of synaptic transmission in retinas from wild - type ( black ) and RIBEYE KO mice ( red ) . Presynaptic bipolar neurons were depolarized in voltage - clamp mode from - 70 mV to - 10 mV for 0 . 5 s as indicated on top to open Ca2 + - channels and trigger release . Presynaptic Ca2 + - currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces ; dotted box displays an expansion of the initial Ca2 + - currents and EPSCs . B . RIBEYE KO does not alter presynaptic Ca2 + - currents . Summary graphs show average peak Ca2 + - currents and Ca2 + - current . C . RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization . Plot shows integrated EPSC charge as a function of time ; both initial and sustained neurotransmitter release are impaired . D . RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude . E . RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization . Summary graphs display integrated EPSC charges for the initial phase ( 0 - 50 ms ) and the sustained phase ( 50 - 500 ms ) . F . RIBEYE KO does not alter the kinetics of EPSCs . Summary graphs depict the mean synaptic delays ( left ) , rise times ( center ) and decay time constants ( right ) of EPSCs .

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

A. Experimental design and representative traces for analysis of synaptic transmission in retinas from wild-type (black) and RIBEYE KO mice (red). Presynaptic bipolar neurons were depolarized in voltage-clamp mode from -70 mV to -10 mV for 0.5 s as indicated on top to open Ca2+-channels and trigger release. Presynaptic Ca2+-currents and postsynaptic EPSCs were measured simultaneously as indicated in the traces; dotted box displays an expansion of the initial Ca2+-currents and EPSCs.B. RIBEYE KO does not alter presynaptic Ca2+-currents. Summary graphs show average peak Ca2+-currents and Ca2+-current.C. RIBEYE KO severely impairs EPSCs triggered by presynaptic depolarization. Plot shows integrated EPSC charge as a function of time; both initial and sustained neurotransmitter release are impaired.D. RIBEYE KO strongly reduces initial synchronous release induced by presynaptic depolarization as indicated by the summary graph of the peak EPSC amplitude.E. RIBEYE KO suppresses both the initial synchronous and the sustained phase of release induced by presynaptic depolarization. Summary graphs display integrated EPSC charges for the initial phase (0-50 ms) and the sustained phase (50-500 ms).F. RIBEYE KO does not alter the kinetics of EPSCs. Summary graphs depict the mean synaptic delays (left), rise times (center) and decay time constants (right) of EPSCs.


A . Representative mEPSCs recorded from AII cells in wild - type ( black ) and RIBEYE KO mice ( red ) . The insets in dashed boxes show mEPSCs on expanded scales , while the inset in a continuous box on the right shows the averaged mEPSC waveforms ( average of 100 - 200 isolated mini events ) of the example cells . Calibration bars apply to wild - type and mutant EPSCs . B . RIBEYE KO does not alter fundamental parameters of mEPSCs . Summary graphs show mean mEPSC frequency , amplitude , integrated charge , rise time , and decay time constant . Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses .

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

A. Representative mEPSCs recorded from AII cells in wild-type (black) and RIBEYE KO mice (red). The insets in dashed boxes show mEPSCs on expanded scales, while the inset in a continuous box on the right shows the averaged mEPSC waveforms (average of 100-200 isolated mini events) of the example cells. Calibration bars apply to wild-type and mutant EPSCs.B. RIBEYE KO does not alter fundamental parameters of mEPSCs. Summary graphs show mean mEPSC frequency, amplitude, integrated charge, rise time, and decay time constant. Note that the only excitatory inputs into AII amacrine cells is provided by bipolar cell synapses.


C and D . RIBEYE KO renders mEPSCs sensitive to the slow Ca2 + - buffer EGTA ( C , representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA - AM [ 0 . 2 mM ] ; D , summary graphs of the mean mEPSC frequency ( left ) and amplitude ( right ) after incubation in DMSO or EGTA - AM ) .

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

C and D. RIBEYE KO renders mEPSCs sensitive to the slow Ca2+-buffer EGTA (C, representative mEPSCs traces recorded from AII cells after 30 minutes incubation in DMSO or EGTA-AM [0.2 mM]; D, summary graphs of the mean mEPSC frequency (left) and amplitude (right) after incubation in DMSO or EGTA-AM).


E and F . mEPSCs in AII amacrine cells are largely triggered by Ca2 + - influx via presynaptic L - type Ca2 + - channels ( E , representative mEPSCs traces recorded in regular [ 2 mM ] or reduced extracellular Ca2 + - concentration [ 0 . 3 mM ] , or in the presence of nimodipine [ 50 µM ] in regular Ca2 + ; F , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E ) .

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

E and F. mEPSCs in AII amacrine cells are largely triggered by Ca2+-influx via presynaptic L-type Ca2+-channels (E, representative mEPSCs traces recorded in regular [2 mM] or reduced extracellular Ca2+-concentration [0.3 mM], or in the presence of nimodipine [50 µM] in regular Ca2+; F, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for E).


G and H . mEPSCs in AII amacrine cells are suppressed by the fast Ca2 + - buffer BAPTA ( G , representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA - AM [ 30 µM ] ; H , summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G ) .

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

G and H. mEPSCs in AII amacrine cells are suppressed by the fast Ca2+-buffer BAPTA (G, representative mEPSCs traces recorded after 30 min incubations with DMSO or BAPTA-AM [30 µM]; H, summary graphs of the mean mEPSC frequency and amplitude under the conditions described for G).


Autophagy components promote CHIKV infection and control virus ‐ induced cell death in HeLa cells . Cells were mock infected or infected and immunoblotted for actin , p62 ( A ) or LC3 ( B ) .

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Autophagy components promote CHIKV infection and control virus‐induced cell death in HeLa cells. Cells were mock infected or infected and immunoblotted for actin, p62 (A) or LC3 (B).


Cells were infected for 15 h and labeled using antibodies to p62 and capsid ( C ) .

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Cells were infected for 15 h and labeled using antibodies to p62 and capsid (C).


Cells were transfected with GFP ‐ LC3 ‐ B , infected for 15 h and labeled with anti ‐ capsid antibody ( D ) .

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Cells were transfected with GFP‐LC3‐B, infected for 15 h and labeled with anti‐capsid antibody (D).


Mice were infected for 3 days , then whole ‐ cell lysates of muscle were immunoblotted for LC3 or actin ( E ) and muscle sections were labeled with p62 and capsid antibodies ( F ) .

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Mice were infected for 3 days, then whole‐cell lysates of muscle were immunoblotted for LC3 or actin (E) and muscle sections were labeled with p62 and capsid antibodies (F).


Cells were treated with DMSO ( CTRL ) or rapamycin and then infected . Viral replication ( G ) and production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) .

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Cells were treated with DMSO (CTRL) or rapamycin and then infected. Viral replication (G) and production (H) were assessed and whole‐cell lysates were immunoblotted for capsid or actin (I).


Cells were treated with CTRL , Beclin1 ( left ) or Atg7 ( right ) siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( K ) and production ( L ) were assessed and whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for Beclin1 , Atg7 , capsid and actin ( M ) . Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean + s . d . ( n = 3 ) . Scale bar , 10 μm . CHIKV , Chikungunya virus ; CTRL , control ; GFP , green fluorescent protein ; DMSO , dimethylsulphoxide ; siRNA , short interfering RNA .

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Cells were treated with CTRL, Beclin1 (left) or Atg7 (right) siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (K) and production (L) were assessed and whole‐cell lysates of siRNA‐treated cells were immunoblotted for Beclin1, Atg7, capsid and actin (M). Images and graphs shown are representative of at least three independent experiments and data presented in graphs correspond to mean+s.d. (n=3). Scale bar, 10 μm. CHIKV, Chikungunya virus; CTRL, control; GFP, green fluorescent protein; DMSO, dimethylsulphoxide; siRNA, short interfering RNA.


Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells . Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid ( A ) , to p62 , NDP52 and capsid ( B ) , to NDP52 , LC3 ‐ C or capsid ( C ) , to p62 , ubiquitin ( FK2 ) and capsid ( D ) , or to NDP52 , ubiquitin ( FK2 ) , and capsid ( E ) .

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Autophagy receptors p62 and NDP52 localize to distinct pools of capsid and have distinct effects on CHIKV infection in Hela cells. Cells were infected for 15 h and labeled using antibodies to NDP52 and capsid (A), to p62, NDP52 and capsid (B), to NDP52, LC3‐C or capsid (C), to p62, ubiquitin (FK2) and capsid (D), or to NDP52, ubiquitin (FK2), and capsid (E).


Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( F ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( G ) , and viral production ( H ) were assessed and whole ‐ cell lysates were immunoblotted for p62 , capsid or actin ( I ) .

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Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (F), viral replication (left) and qRT‐PCR (right) (G), and viral production (H) were assessed and whole‐cell lysates were immunoblotted for p62, capsid or actin (I).


Cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) , viral replication ( left ) and qRT ‐ PCR ( right ) ( K ) and production were assessed ( L ) .

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Cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J), viral replication (left) and qRT‐PCR (right) (K) and production were assessed (L).