Figure 3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(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.


Figure 4 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(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.


Figure 5BN - 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 .

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

Figure 5BN-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.


Figure 6C - 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6C-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.


Figure 7 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(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.


Figure 8 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8(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.


Figure 1 . ( 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1.(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).


Figure 2 . 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 .

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

Figure 2.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.


Figure 3 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(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.


Figure 4 . 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 . 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.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.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.(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.


Figure 5 . 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 .

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

Figure 5.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.


figf1 ( 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 ) . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(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).(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.


figf2 ( 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 . ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf2(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.(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.


figf3 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(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.


figf4 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(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.


figf5 ( 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(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.


Figure 1B . 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 ) . 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 . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1B. 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).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.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.


Figure 2A . 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 ) .

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Figure 2A. 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).


Figure 3A 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 .

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Figure 3A 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.


Figure 4A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. 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).


Figure 5A 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A 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.


Figure 6A . 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A. 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.


Figure 7A . 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 ) . 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 ) .

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Figure 7A. 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).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).


Figure 1Autophagy 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 ) . 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 ) . 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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Figure 1Autophagy 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).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).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.


Figure 2Autophagy 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 ) . 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 ) . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or 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 . C , capsid ; CHIKV , Chikungunya virus ; CTRL , control ; NDP52 , nuclear dot protein 52 kDa ; qRT ‐ PCR , quantitative reverse transcription polymerase chain reaction ; siRNA , short interfering RNA .

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Figure 2Autophagy 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).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).Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or 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. C, capsid; CHIKV, Chikungunya virus; CTRL, control; NDP52, nuclear dot protein 52 kDa; qRT‐PCR, quantitative reverse transcription polymerase chain reaction; siRNA, short interfering RNA.


Figure 3p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells . Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2 , capsid or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid ( A , B ) . Cells were transfected with Ub ‐ HA and capsid ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ HA antibody ( C ) . Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using antibodies to p62 , FK2 or capsid ( D , E ) . Cells were infected for 3 h and then transfected with empty plasmid ( CTRL ) or p62WT ‐ 3XFLAG or p62deltaUBA ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . with anti ‐ FLAG antibody . Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid ( F ) . Cells were treated with CTRL or p62 siRNA , then infected for 24 h . Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls . Immunoprecipitated proteins were revealed using anti ‐ capsid antibody ( G ) . Cells were infected for 15 h and labeled with antibodies to p62 , LAMP ‐ 1 or capsid ( H ) . Cells were infected for 9 , 15 or 24 h and treated with DMSO ( CTRL ) or bafilomycin 3 h before the end of experiment . Whole ‐ cell lysates were immunoblotted for capsid or actin ( I ) . HeLa cells were transfected with empty plasmid ( CTRL ) , control protein ( cherry ‐ 3XFLAG ) or capsid ‐ 3XFLAG at fixed ( 750 ng ) or at the indicated concentrations . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( J , K ) . Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( L ) . Cells were infected with CHIKV for 3 h and transfected with empty plasmid ( CTRL ) , p62WT ‐ 3XFLAG , p62deltaUBA ‐ 3XFLAG or p62deltaLIR ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 24 h post transfection ( M ) .

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Figure 3p62 associates with ubiquitinated capsid and controls cell death induced by cytotoxic capsid in HeLa cells. Cells were infected for 24 h and immunoprecipitation experiments were performed using antibodies to FK2, capsid or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to FK2 or capsid (A,B).Cells were transfected with Ub‐HA and capsid‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐HA antibody (C).Immunoprecipitation experiments were performed using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using antibodies to p62, FK2 or capsid (D,E).Cells were infected for 3 h and then transfected with empty plasmid (CTRL) or p62WT‐3XFLAG or p62deltaUBA‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. with anti‐FLAG antibody. Immunoprecipitated proteins were revealed using antibodies to FLAG or capsid (F).Cells were treated with CTRL or p62 siRNA, then infected for 24 h. Immunoprecipitation experiments were performed using antibodies to FK2 or nonspecific IgG controls. Immunoprecipitated proteins were revealed using anti‐capsid antibody (G).Cells were infected for 15 h and labeled with antibodies to p62, LAMP‐1 or capsid (H).Cells were infected for 9, 15 or 24 h and treated with DMSO (CTRL) or bafilomycin 3 h before the end of experiment. Whole‐cell lysates were immunoblotted for capsid or actin (I).HeLa cells were transfected with empty plasmid (CTRL), control protein (cherry‐3XFLAG) or capsid‐3XFLAG at fixed (750 ng) or at the indicated concentrations. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (J,K).Cells were treated with CTRL or p62 siRNA and transfected with NT or capsid‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (L).Cells were infected with CHIKV for 3 h and transfected with empty plasmid (CTRL), p62WT‐3XFLAG, p62deltaUBA‐3XFLAG or p62deltaLIR‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 24 h post transfection (M).


Figure 4Human NDP52 binds nsP2 , localizes near CHIKV RCs , and restricts cell shutoff promoting cell survival in HeLa cells . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants ( top ) . Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone ( empty vector ) or to NDP52 ( bottom ) ( A ) . Cells were infected for 15 h , then labeled using antibodies to NDP52 and nsP2 ( B ) , or to TGN46 , nsP2 and nsP3 ( C ) . Cells were treated with control ( CTRL ) ( left ) or NDP52 ( right ) siRNA , infected for 15 h , then labeled using antibodies to NDP52 , nsP2 and puromycin . Quantitative analysis was performed by counting the percentage of infected cells with TGN ‐ associated RCs ( n = 30 cells per experiment ) ( D ) . Cells were transfected with empty plasmid ( CTRL ) or nsP2wt ‐ 3XFLAG or nsP2R606A ‐ 3XFLAG . Cell mortality ( that is , % of dead cells ) was measured 24 h post transfection ( E ) . Cells were treated with control ( CTRL ) or NDP52 siRNA , then mock infected ( M ) or infected ( C ) for 24 h . Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52 . The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 ( F ) . Cells were treated with control ( CTRL ) or NDP52 siRNA , infected for 15 and 24 h and treated with puromycin . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for NDP52 , capsid or actin . Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin ( G ) . Cells were treated with CTRL or NDP52 siRNA , then transfected with NT or nsP2 ‐ 3XFLAG . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) was measured 48 h post transfection ( H ) .

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Figure 4Human NDP52 binds nsP2, localizes near CHIKV RCs, and restricts cell shutoff promoting cell survival in HeLa cells. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to NDP52 or the indicated NDP52 deletion mutants (top). Yeast cells expressing BD fused to nsP2 or the indicated nsP2 deletion mutants were cotransformed with a plasmid encoding the Gal4 AD fused alone (empty vector) or to NDP52 (bottom) (A).Cells were infected for 15 h, then labeled using antibodies to NDP52 and nsP2 (B), or to TGN46, nsP2 and nsP3 (C).Cells were treated with control (CTRL) (left) or NDP52 (right) siRNA, infected for 15 h, then labeled using antibodies to NDP52, nsP2 and puromycin. Quantitative analysis was performed by counting the percentage of infected cells with TGN‐associated RCs (n=30 cells per experiment) (D).Cells were transfected with empty plasmid (CTRL) or nsP2wt‐3XFLAG or nsP2R606A‐3XFLAG. Cell mortality (that is, % of dead cells) was measured 24 h post transfection (E).Cells were treated with control (CTRL) or NDP52 siRNA, then mock infected (M) or infected (C) for 24 h. Cytoplasmic and nuclear fractions were isolated and immunoblotted for nsP2 and NDP52. The amounts of the cytoplasmic and nuclear fractions of nsP2 were quantified by densitometry and expressed as the ratio of nuclear to cytoplasmic fraction of nsP2 (F).Cells were treated with control (CTRL) or NDP52 siRNA, infected for 15 and 24 h and treated with puromycin. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for NDP52, capsid or actin. Puromycin incorporation into newly synthesized protein was revealed using antibodies to puromycin (G).Cells were treated with CTRL or NDP52 siRNA, then transfected with NT or nsP2‐3XFLAG. Cell mortality (that is, fold change relative to mock‐infected cells) was measured 48 h post transfection (H).


Figure 5NDP52 promotes CHIKV infection in a species ‐ specific manner . MEF cells were treated with CTRL or NDP52 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( A ) and viral production ( B ) were measured . Yeast cells expressing Gal4 DNA BD fused alone ( empty vector ) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 ( C ) . MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) or hNDP52 ‐ 3XFLAG . Immunoprecipitation experiments were performed 24 h p . i . using antibodies to FLAG or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ nsP2 antibody ( D ) . MEF cells were infected for 3 h and transfected with empty plasmid ( CTRL ) , hNDP52 , hLC3 ‐ C or hNDP52 and hLC3 ‐ C . Viral production ( E ) and cell mortality ( F ) were assessed 24 h post transfection . Isogenic wt or Atg5 − / − MEFs were infected for 24 h and cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( G ) and viral production ( H ) were assessed . MEF cells were infected and immunoprecipitation experiments were performed 24 h p . i . using antibodies to p62 or nonspecific IgG controls . Immunoprecipitated proteins were revealed with anti ‐ capsid antibody ( I ) . MEF cells were treated with CTRL or p62 siRNA , then infected for 24 h . Cell mortality ( that is , fold change relative to mock ‐ infected cells ) ( J ) and viral production were assessed . Whole ‐ cell lysates of siRNA ‐ treated cells were immunoblotted for p62 or actin ( K ) .

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Figure 5NDP52 promotes CHIKV infection in a species‐specific manner. MEF cells were treated with CTRL or NDP52 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (A) and viral production (B) were measured. Yeast cells expressing Gal4 DNA BD fused alone (empty vector) or to nsP2 were cotransformed with a plasmid encoding the Gal4 AD fused to hNDP52 or mouse NDP52 (C).MEF cells were infected for 3 h and transfected with empty plasmid (CTRL) or hNDP52‐3XFLAG. Immunoprecipitation experiments were performed 24 h p.i. using antibodies to FLAG or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐nsP2 antibody (D).MEF cells were infected for 3 h and transfected with empty plasmid (CTRL), hNDP52, hLC3‐C or hNDP52 and hLC3‐C. Viral production (E) and cell mortality (F) were assessed 24 h post transfection. Isogenic wt or Atg5−/− MEFs were infected for 24 h and cell mortality (that is, fold change relative to mock‐infected cells) (G) and viral production (H) were assessed. MEF cells were infected and immunoprecipitation experiments were performed 24 h p.i. using antibodies to p62 or nonspecific IgG controls. Immunoprecipitated proteins were revealed with anti‐capsid antibody (I).MEF cells were treated with CTRL or p62 siRNA, then infected for 24 h. Cell mortality (that is, fold change relative to mock‐infected cells) (J) and viral production were assessed. Whole‐cell lysates of siRNA‐treated cells were immunoblotted for p62 or actin (K).


Figure 1A . Endogenous Cyclin B1 - YFP localisation during mitosis live in RPE1 cells ( still from Movie EV1 ) . Immunofluorescence images ( B ) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC . C . Immunofluorescence quantifications ( C ) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC . Each dot represents a kinetochore , data is from 40 kinetochore pairs ( 13 cells , max 5 kinetochore pairs / cell ) . D , E . Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole - arrested cells ( noco ) treated with the MPS1 inhibitors , AZ - 3146 ( 5μM ) or Reversine ( 500nM ) , either before ( D ) or after ( E ) mitotic entry . F . Immunofluorescence images of LacI - MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1G . Live imaging of Cyclin B1 - mCherry ( CycB1 - mCh ) in LacO - U2OS cells transfected with LacI - MAD1 - FL ( full length : aa1 - 718 ) or various LacI - MAD1 truncations ( amino acid numbers indicated ) . H , I . Immunofluorescence images ( H ) and quantifications ( I ) of Cyclin B1 and MAD1 kinetochore levels in control ( MAD1 - WT ) or MAD1β HeLa cells ( two independent clones : C13 or C24 ) treated with nocodazole . J , K . Immunofluorescence images ( J ) and quantification ( K ) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline - inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole . Cells were selected that had full MAD1 knockout in the doxycycline treatment ( this constituted approximately 30 % of cells ) . L . Relative kinetochore volumes occupied by Cyclin B1 and MAD1 ( relative to CenpC ) in nocodazole - arrested MAD1α and MAD1β cells ( calculated from experiments shown in ( H , I ) .

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Figure 1A. Endogenous Cyclin B1-YFP localisation during mitosis live in RPE1 cells (still from Movie EV1).Immunofluorescence images (B) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC.C. Immunofluorescence quantifications (C) of relative Cyclin B1 and MAD1 levels at unattached and attached kinetochores in cells arrested in STLC. Each dot represents a kinetochore, data is from 40 kinetochore pairs (13 cells, max 5 kinetochore pairs/cell).D,E. Quantification of relative kinetochore intensities of Cyclin B1 and MAD1 in nocodazole-arrested cells (noco) treated with the MPS1 inhibitors, AZ-3146 (5μM) or Reversine (500nM), either before (D) or after (E) mitotic entry.F. Immunofluorescence images of LacI-MAD1 and Cyclin B1 in U2OS cells containing a LacO arrays on chromosome 1G. Live imaging of Cyclin B1-mCherry (CycB1-mCh) in LacO-U2OS cells transfected with LacI-MAD1-FL (full length: aa1-718) or various LacI-MAD1 truncations (amino acid numbers indicated).H,I. Immunofluorescence images (H) and quantifications (I) of Cyclin B1 and MAD1 kinetochore levels in control (MAD1-WT) or MAD1β HeLa cells (two independent clones: C13 or C24) treated with nocodazole.J,K. Immunofluorescence images (J) and quantification (K) of Cyclin B1 and MAD1 kinetochore localisation in doxycycline-inducible MAD1α and β knockouts treated with or without dox for 10 days and then arrested in nocodazole. Cells were selected that had full MAD1 knockout in the doxycycline treatment (this constituted approximately 30% of cells).L. Relative kinetochore volumes occupied by Cyclin B1 and MAD1 (relative to CenpC) in nocodazole-arrested MAD1α and MAD1β cells (calculated from experiments shown in (H,I).


Figure 2A . Elution profiles and SDS - PAGE for SEC runs on the indicated column of the Cyclin B1 : CDK1 complex ( blue profile ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . Note that the same Cyclin B1 : CDK1 elution profile and SDS - PAGE is also displayed as reference in panels D and F to improve clarity . For the same reason , the MBP - MAD1 : MAD2 elution profile and SDS - PAGE is also displayed in panels 2A - C . B . Elution profiles and SDS - PAGE for SEC runs on the indicated column of CDK1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . C . Elution profiles and SDS - PAGE for SEC runs on the indicated column of Cyclin B1 ( blue ) , MBP - MAD1 : MAD2 ( red ) , and their combination ( green ) . D . Elution profiles and SDS - PAGE for SEC runs of the Cyclin B1 : CDK1 complex ( blue ) , MBP - MAD1 ∆ 93 : MAD2 ( green ) , and their combination ( red ) . F . Elution profiles and SDS - PAGE for SEC runs of Cyclin B1 : CDK1 ( blue ) , MBP - MAD13EK : MAD2 ( E52K , E53K , and E56K mutations ; red ) , and their combination ( green ) . In A - D , individual potential binding partners were combined at a concentration of 5 µM .

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

Figure 2A. Elution profiles and SDS-PAGE for SEC runs on the indicated column of the Cyclin B1:CDK1 complex (blue profile), MBP-MAD1:MAD2 (red), and their combination (green). Note that the same Cyclin B1:CDK1 elution profile and SDS-PAGE is also displayed as reference in panels D and F to improve clarity. For the same reason, the MBP-MAD1:MAD2 elution profile and SDS-PAGE is also displayed in panels 2A-C. B. Elution profiles and SDS-PAGE for SEC runs on the indicated column of CDK1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). C. Elution profiles and SDS-PAGE for SEC runs on the indicated column of Cyclin B1 (blue), MBP-MAD1:MAD2 (red), and their combination (green). D. Elution profiles and SDS-PAGE for SEC runs of the Cyclin B1:CDK1 complex (blue), MBP-MAD1∆93:MAD2 (green), and their combination (red). F. Elution profiles and SDS-PAGE for SEC runs of Cyclin B1:CDK1 (blue), MBP-MAD13EK:MAD2 (E52K, E53K, and E56K mutations; red), and their combination (green). In A-D, individual potential binding partners were combined at a concentration of 5 µM.


Figure 3A . Western blot analysis of indicated vsv - MAD1 - WT or 3EK HeLa clones treated with or without doxycycline for 10 days . B . Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole - arrested MAD1 - WT - C13 AND 3EK - C14 just after nuclear envelope breakdown ( early prometaphase ) or later in mitosis when the chromatin is condensed ( late prometaphase ) . Note , that early and late prometaphase was defined based on the level of chromatin condensation . C , D . Relative kinetochore volumes occupied by MAD1 ( C ) and ZW10 ( D ) ( relative to CenpC ) in MAD1 - WT and - 3EK cells in early and late prometaphase . E . Quantification of MAD1 kinetochore intensity from indicated MAD1 - WT and 3EK clones treated as in ( B ) .

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

Figure 3A. Western blot analysis of indicated vsv-MAD1-WT or 3EK HeLa clones treated with or without doxycycline for 10 days.B. Immunofluorescence images showing MAD1 and ZW10 kinetochore levels in nocodazole-arrested MAD1-WT-C13 AND 3EK-C14 just after nuclear envelope breakdown (early prometaphase) or later in mitosis when the chromatin is condensed (late prometaphase). Note, that early and late prometaphase was defined based on the level of chromatin condensation.C,D. Relative kinetochore volumes occupied by MAD1 (C) and ZW10 (D) (relative to CenpC) in MAD1-WT and -3EK cells in early and late prometaphase.E. Quantification of MAD1 kinetochore intensity from indicated MAD1-WT and 3EK clones treated as in (B).