( C ) HeLa cells were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub ( top ) or LC3 and p62 ( bottom ) . Bar , 5 µm . The percentages of LC3 - or p62 - positive beads per Ub - positive ( Ub + ) or Ub - negative ( Ub − ) beads were enumerated . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) HeLa cells were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for LC3 and Ub (top) or LC3 and p62 (bottom). Bar, 5 µm. The percentages of LC3- or p62-positive beads per Ub-positive (Ub+) or Ub-negative (Ub−) beads were enumerated. Statistical analysis was performed by Student's unpaired t test. *, P < 0.01.


( D and E ) HeLa cells stably expressing GFP - LC3 were transfected with Effectene - coated latex beads for 3 h . Bead - autophagosomes were fractionated as described in Materials and methods . The bead - autophagosome fraction was observed by confocal microscopy ( D ; bar , 10 µm ) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies ( E ) . In a control sample , scraped cells were mixed with Effectene - coated beads and immediately homogenized .

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(D and E) HeLa cells stably expressing GFP-LC3 were transfected with Effectene-coated latex beads for 3 h. Bead-autophagosomes were fractionated as described in Materials and methods. The bead-autophagosome fraction was observed by confocal microscopy (D; bar, 10 µm) or lysed with RIPA buffer and subjected to Western blot analysis using the indicated antibodies (E). In a control sample, scraped cells were mixed with Effectene-coated beads and immediately homogenized.


( F ) The bead - autophagosome fraction was lysed and subjected to immunoprecipitation with an anti - Ub IgG antibody ( FK2 ) or control IgG . Co - immunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

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(F) The bead-autophagosome fraction was lysed and subjected to immunoprecipitation with an anti-Ub IgG antibody (FK2) or control IgG. Co-immunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( G and H ) NIH3T3 cells stably expressing mStrawberry ( mStr ) - Gal3 and GFP - LC3 , mStr - Gal3 , and GFP - p62 , or mStr - Gal3 and GFP - Ub were transfected with Effectene - coated latex beads for 30 min and then washed . Live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after galectin3 localization was measured for at least 30 cases for each combination ( H ) . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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(G and H) NIH3T3 cells stably expressing mStrawberry (mStr)-Gal3 and GFP-LC3, mStr-Gal3, and GFP-p62, or mStr-Gal3 and GFP-Ub were transfected with Effectene-coated latex beads for 30 min and then washed. Live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after galectin3 localization was measured for at least 30 cases for each combination (H). Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( A and B ) NIH3T3 cells stably expressing mStr - Ub and GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , or ULK1 were transfected with Effectene - coated latex beads for 30 min . Then , live cells were observed at 1 - min intervals by fluorescence microscopy . Bar , 3 µm . The time after Ub localization was measured for at least 30 cases for each combination . Each value represents the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 ; NS , not significant .

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(A and B) NIH3T3 cells stably expressing mStr-Ub and GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, or ULK1 were transfected with Effectene-coated latex beads for 30 min. Then, live cells were observed at 1-min intervals by fluorescence microscopy. Bar, 3 µm. The time after Ub localization was measured for at least 30 cases for each combination. Each value represents the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05; NS, not significant.


( C ) NIH3T3 cells stably expressing GFP - tagged Ub , LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated beads for 3 h in the presence or absence ( mock ) of 30 µM UBEI - 41 ( a ubiquitin E1 - specific inhibitor ) and subjected to immunocytochemistry for galectin3 . The percentages of GFP - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD . Statistical analysis was performed by Student ' s unpaired t test . * , P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) NIH3T3 cells stably expressing GFP-tagged Ub, LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated beads for 3 h in the presence or absence (mock) of 30 µM UBEI-41 (a ubiquitin E1-specific inhibitor) and subjected to immunocytochemistry for galectin3. The percentages of GFP-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD. Statistical analysis was performed by Student's unpaired t test. *, P < 0.05.


( D and E ) Parent NIH3T3 cells , Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

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(D and E) Parent NIH3T3 cells, Atg4B mutant overexpressing NIH3T3 cells stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( E ) wild - type MEFs , and Atg5 - KO MEFs stably expressing GFP - tagged LC3 , Atg5 , WIPI - 1 , Atg14L1 , Atg9L1 , or ULK1 were transfected with Effectene - coated latex beads for 3 h and subjected to immunocytochemistry for galectin3 . The percentages of Atg - positive per galectin3 - positive beads were enumerated . At least 30 beads were counted ( n = 3 ) . The values are the mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) wild-type MEFs, and Atg5-KO MEFs stably expressing GFP-tagged LC3, Atg5, WIPI-1, Atg14L1, Atg9L1, or ULK1 were transfected with Effectene-coated latex beads for 3 h and subjected to immunocytochemistry for galectin3. The percentages of Atg-positive per galectin3-positive beads were enumerated. At least 30 beads were counted (n = 3). The values are the mean ± SD.


( A ) HeLa cells were lysed with 1 % Triton X - 100 lysis buffer and subjected to immunoprecipitation with FK2 - or control IgG - immobilized beads . The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies .

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(A) HeLa cells were lysed with 1% Triton X-100 lysis buffer and subjected to immunoprecipitation with FK2- or control IgG-immobilized beads. The coimmunoprecipitated molecules were examined by Western blotting using the indicated antibodies.


( C ) Purified recombinant GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG - tagged Atg16L1 constructs for 1 h at 25 ° C with gentle agitation . The beads were washed three times with ice - cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG .

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(C) Purified recombinant GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from HEK293T cells transiently expressing FLAG-tagged Atg16L1 constructs for 1 h at 25°C with gentle agitation. The beads were washed three times with ice-cold PBS and the bound complexes were eluted with 50 mM reduced glutathione and then subjected to Western blotting for FLAG.


( D ) Purified GST or GST - Ub - immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor ( TF ) or TF - FLAG - WD β - propellers for 1 h at 25 ° C with gentle agitation . Washing and elution were performed as described in C . The eluted samples were subjected to SDS - PAGE and Western blotting for FLAG .

(D) Purified GST or GST-Ub-immobilized glutathione Sepharose beads were incubated with lysates from bacteria expressing trigger factor (TF) or TF-FLAG-WD β-propellers for 1 h at 25°C with gentle agitation. Washing and elution were performed as described in C. The eluted samples were subjected to SDS-PAGE and Western blotting for FLAG.


( A ) Yeast two - hybrid interactions of the ULK1 complexes with other human Atg proteins . ULK1 , ULK2 , Atg13 , FIP200 , and Atg101 - AD fusions ( rows 2 - 6 ) or control AD constructs ( row 1 , Empty ) were coexpressed with control DBD constructs ( column 1 , Empty ) or Atg protein - DBD fusions ( columns 2 - 35 ) and tested for positive yeast two - hybrid interactions ( top ) or cotransformation ( control , bottom ) . White lines divide images derived from the same plate , and red lines divide images from different plates . Atg16L1 positively interacted with FIP200 in this assay .

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(A) Yeast two-hybrid interactions of the ULK1 complexes with other human Atg proteins. ULK1, ULK2, Atg13, FIP200, and Atg101-AD fusions (rows 2-6) or control AD constructs (row 1, Empty) were coexpressed with control DBD constructs (column 1, Empty) or Atg protein-DBD fusions (columns 2-35) and tested for positive yeast two-hybrid interactions (top) or cotransformation (control, bottom). White lines divide images derived from the same plate, and red lines divide images from different plates. Atg16L1 positively interacted with FIP200 in this assay.


( B ) FIP200 binds to exogenously expressed Atg16L1 . Myc - Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or One - STrEP - FLAG ( OSF ) - FIP200 ( lane 2 ) .

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(B) FIP200 binds to exogenously expressed Atg16L1. Myc-Atg16L1 coprecipitations with an empty vector control (lane 1) or One-STrEP-FLAG (OSF)-FIP200 (lane 2).


( C ) FIP200 binds endogenous Atg16L1 . Atg16L1 coprecipitations with an empty vector control ( lane 1 ) or OSF - FIP200 ( lane 2 ) .

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(C) FIP200 binds endogenous Atg16L1. Atg16L1 coprecipitations with an empty vector control (lane 1) or OSF-FIP200 (lane 2).


( D ) Wild - type or FIP200 - KO MEFs stably expressing the empty vector , full - length Atg16L1 , or ΔWD mutant were incubated in growth medium ( − ) or Earle ' s balanced salt solution ( EBSS ) ( + ) for 1 h and examined by Western blotting using the indicated antibodies . We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein ( Fujita et al . , 2009 ) because free Atg16L1 molecules not complexed with the Atg12 - Atg5 conjugate are preferentially degraded by the ubiquitin - proteasome system . In control cells ( Empty ) , two Atg16L1 splicing variants , the α - and β - forms , were detected . In full - length β - form - replaced cells ( Full ) , the α - form was not detected , whereas in ΔWD - replaced cells ( ΔWD ) both the α - and β - forms were minimally detected . Thus , we successfully obtained cells that express only endogenous levels of the full - length or ΔWD mutant .

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(D) Wild-type or FIP200-KO MEFs stably expressing the empty vector, full-length Atg16L1, or ΔWD mutant were incubated in growth medium (−) or Earle's balanced salt solution (EBSS) (+) for 1 h and examined by Western blotting using the indicated antibodies. We previously reported that stable expression of an exogenous Atg16L1 construct can be used to replace the endogenous Atg16L1 protein with an exogenous Atg16L1 protein (Fujita et al., 2009) because free Atg16L1 molecules not complexed with the Atg12-Atg5 conjugate are preferentially degraded by the ubiquitin-proteasome system. In control cells (Empty), two Atg16L1 splicing variants, the α- and β-forms, were detected. In full-length β-form-replaced cells (Full), the α-form was not detected, whereas in ΔWD-replaced cells (ΔWD) both the α- and β-forms were minimally detected. Thus, we successfully obtained cells that express only endogenous levels of the full-length or ΔWD mutant.


( E - L ) Atg16L1 - replaced wild - type or FIP200 - KO MEFs were infected with Salmonella ( MOI = 10 ) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub ( E and F ) or LC3 and Ub ( I and J ) . The percentages of Atg16L1 - positive ( G and H ) or LC3 - positive per Ub - positive bacteria were enumerated ( K and L ) . At least 50 Salmonella were counted . The average ± SD is shown for three independent experiments . Statistical analysis was performed by Student ' s t test . * , P < 0 . 05 ; NS , not significant . Bar , 5 µm .

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(E-L) Atg16L1-replaced wild-type or FIP200-KO MEFs were infected with Salmonella (MOI = 10) for 1 h and subjected to immunocytochemistry for Atg16L1 and Ub (E and F) or LC3 and Ub (I and J). The percentages of Atg16L1-positive (G and H) or LC3-positive per Ub-positive bacteria were enumerated (K and L). At least 50 Salmonella were counted. The average ± SD is shown for three independent experiments. Statistical analysis was performed by Student's t test. *, P < 0.05; NS, not significant. Bar, 5 µm.


( A ) Mapping of the Atg16L1 - binding site on FIP200 . Yeast two - hybrid mapping experiments showing that Atg16L1 binds to both the N - terminal and C - terminal coiled - coil regions of FIP200 .

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(A) Mapping of the Atg16L1-binding site on FIP200. Yeast two-hybrid mapping experiments showing that Atg16L1 binds to both the N-terminal and C-terminal coiled-coil regions of FIP200.


( B ) Mapping of the FIP200 - binding site within Atg16L1 . Yeast two - hybrid mapping experiments showing that the 1 - 840 fragment of FIP200 binds to the C - terminal 247 - 607 region , while the 1276 - 1591 fragment of FIP200 binds to the N - terminal 1 - 264 region of Atg16L1 .

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(B) Mapping of the FIP200-binding site within Atg16L1. Yeast two-hybrid mapping experiments showing that the 1-840 fragment of FIP200 binds to the C-terminal 247-607 region, while the 1276-1591 fragment of FIP200 binds to the N-terminal 1-264 region of Atg16L1.


( C and D ) Additional yeast two - hybrid mapping experiments showing that the binding site of the FIP200 1276 - 1591 fragment maps to residues 239 - 246 of Atg16L1 . White lines divide images derived from the same plate , and red lines divide images from different plates .

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(C and D) Additional yeast two-hybrid mapping experiments showing that the binding site of the FIP200 1276-1591 fragment maps to residues 239-246 of Atg16L1. White lines divide images derived from the same plate, and red lines divide images from different plates.


( E ) The N - terminal 1 - 249 fragment of Atg16L1 is sufficient to interact with FIP200 . Full - length ( lanes 1 and 2 ) or the N - terminal 1 - 249 fragments ( lanes 3 and 4 ) of Myc - Atg16L1 coprecipitations with empty vector controls ( lanes 1 and 3 ) or OSF - FIP200 1276 - 1591 ( lanes 2 and 4 ) .

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(E) The N-terminal 1-249 fragment of Atg16L1 is sufficient to interact with FIP200. Full-length (lanes 1 and 2) or the N-terminal 1-249 fragments (lanes 3 and 4) of Myc-Atg16L1 coprecipitations with empty vector controls (lanes 1 and 3) or OSF-FIP200 1276-1591 (lanes 2 and 4).


( F ) Co - precipitation experiments confirming that OSF - FIP200 1276 - 1591 coprecipitates with wild - type ( WT ) Myc - Atg16L1 ( 1 - 249 ) fragments , but not with analogous fragments containing a mutant FIP200 - binding site ( Myc - Atg16L1 ΔWD 239 - 242A ) .

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(F) Co-precipitation experiments confirming that OSF-FIP200 1276-1591 coprecipitates with wild-type (WT) Myc-Atg16L1 (1-249) fragments, but not with analogous fragments containing a mutant FIP200-binding site (Myc-Atg16L1 ΔWD 239-242A).


( B - E ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were infected with Salmonella ( MOI = 10 ) for 1 h and then analyzed by immunocytochemistry for LC3 ( B ) or Atg16L1 ( D ) . Bar , 5 µm . The percentage of LC3 - or Atg16L1 - positive Salmonella per Ub - positive Salmonella was enumerated by fluorescence microscopy ( C and E ) . At least 50 bacteria were counted . The average ± SD is shown for three independent experiments .

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(B-E) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were infected with Salmonella (MOI = 10) for 1 h and then analyzed by immunocytochemistry for LC3 (B) or Atg16L1 (D). Bar, 5 µm. The percentage of LC3- or Atg16L1-positive Salmonella per Ub-positive Salmonella was enumerated by fluorescence microscopy (C and E). At least 50 bacteria were counted. The average ± SD is shown for three independent experiments.


( F ) Atg16L1 - Δ / Δ MEFs stably expressing the indicated constructs were cultured in growth medium ( Fed ) or EBSS ( Starved ) for 1 h and then subjected to immunocytochemistry using an anti - LC3 antibody . The number of LC3 puncta in each cell was counted for more than 50 cells . The average ± SD is shown for three independent experiments .

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(F) Atg16L1-Δ/Δ MEFs stably expressing the indicated constructs were cultured in growth medium (Fed) or EBSS (Starved) for 1 h and then subjected to immunocytochemistry using an anti-LC3 antibody. The number of LC3 puncta in each cell was counted for more than 50 cells. The average ± SD is shown for three independent experiments.


( A ) ELISA titration curves ( OD at 450nm ) of Fab3 ( blue ; left panel ) and Fab71 ( blue ; right panel ) against mouse recPrP23 ‑ 231 compared to their best affinity - matured versions , Fab83 and Fab101 Curves were fitted by non - linear least - squares regression analysis for statistical analysis : log ( agonist ) vs . response .

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(A) ELISA titration curves (OD at 450nm) of Fab3 (blue; left panel) and Fab71 (blue; right panel) against mouse recPrP23‑231 compared to their best affinity-matured versions, Fab83 and Fab101 Curves were fitted by non-linear least-squares regression analysis for statistical analysis: log(agonist) vs. response.


( B - E ) FT - peptide competition ELISA to map the epitopes of the indicated Fabs . Peptides that strongly inhibit the binding of the Fabs to recPrP23 ‑ 231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes . Positive control : recPrP23 ‑ 231 ; negative controls : Neu , BSA , and the secondary antibody ( 2 ° Ab ) .

(B-E) FT-peptide competition ELISA to map the epitopes of the indicated Fabs. Peptides that strongly inhibit the binding of the Fabs to recPrP23‑231 are indicated by their residue numbers in the PrP sequence and reflect the respective binding epitopes. Positive control: recPrP23‑231; negative controls: Neu, BSA, and the secondary antibody (2° Ab).


( A ) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 ( CC123 ‑ 50 ) , Fab71 ( OR51 ‑ 91 ) or Fab1 ( GD ) for 45 days . Scale bar : 500 μm .

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(A) Fluorescence micrographs of tga20 slices chronically exposed to prions and cultured in the presence of either Fab3 (CC123‑50), Fab71 (OR51‑91) or Fab1 (GD) for 45 days. Scale bar: 500 μm.


( B ) Same as ( A ) , but in the presence of the affinity - maturated antibodies .

(B) Same as (A), but in the presence of the affinity-maturated antibodies.


( C ) NeuN immunofluorescence coverage of prion - infected slices cultured in the presence of the Fabs . Antibodies targeting the CC123 ‑ 50 and the OR51 ‑ 91 afforded protection against neurodegeneration . NBH : non - infectious brain homogenate . Each dot represents a cerebellar slice for all treatment groups .

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(C) NeuN immunofluorescence coverage of prion-infected slices cultured in the presence of the Fabs. Antibodies targeting the CC123‑50 and the OR51‑91 afforded protection against neurodegeneration. NBH: non-infectious brain homogenate. Each dot represents a cerebellar slice for all treatment groups.


( D ) Western blot analysis of COCS lysates . Prion - infected slices treated with Fab100 , but not with Fab83 and Fab25 showed reduced levels of PrPSc . n = 3 biological replicates .

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(D) Western blot analysis of COCS lysates. Prion-infected slices treated with Fab100, but not with Fab83 and Fab25 showed reduced levels of PrPSc. n = 3 biological replicates.


( C ) Left : Heat map showing the binding specificities ( Z - scores ) of selected Fab71 human analogous antibodies to human recPrP23 ‑ 230 compared to the negative controls ( BSA and Neu ) . Right : Heat map representing the reactivity ( - logEC50 ) obtained from dose - dependent ELISA binding curves of the analogous antibodies to human recPrP23 ‑ 230 . Red : low reactivity ; blue : high reactivity .

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(C) Left: Heat map showing the binding specificities (Z-scores) of selected Fab71 human analogous antibodies to human recPrP23‑230 compared to the negative controls (BSA and Neu). Right: Heat map representing the reactivity (-logEC50) obtained from dose-dependent ELISA binding curves of the analogous antibodies to human recPrP23‑230. Red: low reactivity; blue: high reactivity.


( C ) Boxplot with half - violin plot displaying the distribution of autoantibody reactivity in 48 , 718 plasma samples . Twenty - seven samples over the reactivity threshold of - log ( EC50 ) ≥ 2 and fitting error < 20 % were considered as hits in the primary screen . Black dots correspond to single samples . The boxplot divides the data set into three quartiles . The box extends from the first to the third quartile values of the data set , with a line at the median . Whiskers show the range of the data ( minimum and maximum ) .

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(C) Boxplot with half-violin plot displaying the distribution of autoantibody reactivity in 48,718 plasma samples. Twenty-seven samples over the reactivity threshold of -log(EC50) ≥ 2 and fitting error < 20% were considered as hits in the primary screen. Black dots correspond to single samples. The boxplot divides the data set into three quartiles. The box extends from the first to the third quartile values of the data set, with a line at the median. Whiskers show the range of the data (minimum and maximum).


( E ) Heat map representation for the validation of the hits from the primary screen ( 21 out of 27 hits ) . Confirmed hits displayed selective reactivity against human recPrP23 ‑ 230 . Controls : Patients # 11 - 13 , ApoE4 and BSA . Red : high reactivity ; blue : low reactivity .

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(E) Heat map representation for the validation of the hits from the primary screen (21 out of 27 hits). Confirmed hits displayed selective reactivity against human recPrP23‑230. Controls: Patients #11-13, ApoE4 and BSA. Red: high reactivity; blue: low reactivity.


( F ) Heat maps representing autoantibody clonality of hits analyzed by ELISA . Autoantibodies were mostly constituted by lambda light chains ( λ ) , while the kappa light chains ( κ ) showed prevalence in the total IgG fraction .

O O O O O O O O O O O O O O O O O O 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) Heat maps representing autoantibody clonality of hits analyzed by ELISA. Autoantibodies were mostly constituted by lambda light chains (λ), while the kappa light chains (κ) showed prevalence in the total IgG fraction.


( G ) Determination of antibody reactivity ( - log ( EC50 ) ) for three individuals at two different time points . Individuals were selected by displaying strong anti - PrP IgG reactivity in the first test ( Time 1 ) . Reactivity was maintained for even more than one year ( Time 2 ) .

(G) Determination of antibody reactivity (-log(EC50)) for three individuals at two different time points. Individuals were selected by displaying strong anti-PrP IgG reactivity in the first test (Time 1). Reactivity was maintained for even more than one year (Time 2).


( H ) Purified IgGs from patients with strong anti - PrP reactivity were tested against PrP variants and BSA ( negative control ) in dose - dependent ELISAs . Patient # 5 and # 6 showed binding mostly to the human recPrP121 ‑ 230 . Patient # 1 displayed a polyreactive pattern with binding to the human recPrP121 ‑ 230 and mouse recPrP23 - 110 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Purified IgGs from patients with strong anti-PrP reactivity were tested against PrP variants and BSA (negative control) in dose-dependent ELISAs. Patient #5 and #6 showed binding mostly to the human recPrP121‑230. Patient #1 displayed a polyreactive pattern with binding to the human recPrP121‑230 and mouse recPrP23-110.


( B , Performance in the multi - trail inverted screen test is impaired in ( B ) SUR2 - STOP1149 ( WT n = 9 [ 3 male ; 6 female ] ; STOP n = 9 [ 3 male ; 6 female ] ; p = 0 . 011 according to Mann Whitney U test ) Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Performance in the multi-trail inverted screen test is impaired in (B) SUR2-STOP1149 (WT n = 9 [3 male; 6 female]; STOP n = 9 [3 male; 6 female]; p = 0.011 according to Mann Whitney U test) Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.


C ) Performance in the multi - trail inverted screen test is impaired in ( C ) SUR2 - STOP475 mice ( WT n = 9 [ 4 male ; 5 female ] ; STOP n = 15 [ 5 male ; 10 female ] ; p = 2 . 9x10 - 4 according to Mann Whitney U test ) . Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C) Performance in the multi-trail inverted screen test is impaired in (C) SUR2-STOP475 mice (WT n = 9 [4 male; 5 female]; STOP n = 15 [5 male; 10 female]; p = 2.9x10-4 according to Mann Whitney U test). Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.


( D ) Treadmill exercise tolerance is also reduced in SUR2 - STOP475 mice ( WT n = 4 [ 2 male ; 2 female ] ; STOP n = 8 [ 4 male ; 4 female ] ; p = 0 . 008 according to Mann Whitney U test ) . Treadmill workload tolerated was also reduced ( WT 18 . 1 ± 2 . 2 vs STOP475 9 . 2 ± 0 . 9 J ; p = 0 . 008 from Man Whitney U test ) , mean mouse body weight was WT 22 . 1 ± 0 . 9 vs STOP475 21 . 9 ± 0 . 6 g . Data information : Bars show mean ( ± SEM ) with individual biological replicates shown as circles / squares . * denotes p value < α = 0 . 05 for all panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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)Treadmill exercise tolerance is also reduced in SUR2-STOP475 mice (WT n = 4 [2 male; 2 female]; STOP n = 8 [4 male; 4 female]; p = 0.008 according to Mann Whitney U test). Treadmill workload tolerated was also reduced (WT 18.1 ± 2.2 vs STOP475 9.2 ± 0.9 J; p = 0.008 from Man Whitney U test), mean mouse body weight was WT 22.1 ± 0.9 vs STOP475 21.9 ± 0.6 g. Data information: Bars show mean (± SEM) with individual biological replicates shown as circles/squares. * denotes p value < α = 0.05 for all panels.


( A ) Example traces of inside - out patch clamp recordings of acutely isolated FDB myofibers of WT ( black , top left ) , SUR2 - STOP475 ( purple , top right ) , and skeletal muscle dominant negative ( SkM - DN ; magenta , bottom left ) mice . Patches were voltage clamped at - 50 mV and excised at time - points indicated by arrows . ATP ( 5 mM ) was administered to the cytoplasmic face of membrane patches as indicated by horizontal bars . Dashed lines denote the zero KATP current level .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O 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) Example traces of inside-out patch clamp recordings of acutely isolated FDB myofibers of WT (black, top left), SUR2-STOP475 (purple, top right), and skeletal muscle dominant negative (SkM-DN; magenta, bottom left) mice. Patches were voltage clamped at -50 mV and excised at time-points indicated by arrows. ATP (5 mM) was administered to the cytoplasmic face of membrane patches as indicated by horizontal bars. Dashed lines denote the zero KATP current level.


( B ) Maximal ATP - sensitive current levels from excised patches . Bars show mean ( ± SEM ) with individual recordings from each myofiber shown as dots / squares ( cells from ≥ 3 mice ; WT n = 10 recordings ; STOP n = 8 ; SkM - DN n = 11 ; Dunn ' s test following Kruskal - Wallis omnibus test p = 1 . 0 x 10 - 4 for WT vs STOP and 0 . 014 for WT vs SkM - DN ; * denotes p value < adjusted α = 0 . 0167 ) .

O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Maximal ATP-sensitive current levels from excised patches. Bars show mean (± SEM) with individual recordings from each myofiber shown as dots/squares (cells from ≥ 3 mice; WT n = 10 recordings; STOP n = 8; SkM-DN n = 11; Dunn's test following Kruskal-Wallis omnibus test p = 1.0 x 10-4 for WT vs STOP and 0.014 for WT vs SkM-DN ; * denotes p value < adjusted α = 0.0167).


( D ) Distance travelled in treadmill exercise tolerance tests ( all male ; WT n = 6 ; AAA + n = 8 ; Myf5 - cre n = 8 ; SkM - DN n = 7 ; Dunn ' s test following Kruskal - Wallis omnibus test p = 0 . 005 for WT vs SkM - DN , 0 . 037 for AAA + vs SkM - DN , and 0 . 014 for Myf5 - cre vs SkM - DN ; * denotes p value < adjusted α = 0 . 0083 ) . Treadmill workload tolerated was also reduced ( WT 23 . 6 ± 2 . 3 vs SkMDN 10 . 1 ± 1 . 8 J ; p = 0 . 0017 from Dunn ' s test ) , mean mouse body weight was WT 28 . 9 ± 1 . 6 , AAA + 26 . 6 ± 0 . 9 , Myf5 - cre 25 . 3 ± 0 . 8 , SkMDN 24 . 2 ± 0 . 8 g ; no significant differences in body weights were observed , p from Dunn ' s test following Kruskal - Wallis omnibus test > adjusted α = 0 . 0083 for all comparisons . Bars show mean ( ± SEM ) with individual mouse performance shown as dots / diamonds / triangles / squares .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Distance travelled in treadmill exercise tolerance tests (all male; WT n = 6; AAA+ n = 8; Myf5-cre n = 8; SkM-DN n = 7; Dunn's test following Kruskal-Wallis omnibus test p = 0.005 for WT vs SkM-DN, 0.037 for AAA+ vs SkM-DN, and 0.014 for Myf5-cre vs SkM-DN; * denotes p value < adjusted α = 0.0083). Treadmill workload tolerated was also reduced (WT 23.6 ± 2.3 vs SkMDN 10.1 ± 1.8 J; p = 0.0017 from Dunn's test), mean mouse body weight was WT 28.9 ± 1.6, AAA+ 26.6 ± 0.9, Myf5-cre 25.3 ± 0.8, SkMDN 24.2 ± 0.8 g; no significant differences in body weights were observed, p from Dunn's test following Kruskal-Wallis omnibus test > adjusted α = 0.0083 for all comparisons. Bars show mean (± SEM) with individual mouse performance shown as dots/diamonds/triangles/squares.


( A ) Left - example HE stained tibialis anterior ( TA ) muscles from WT and SUR2 - STOP475 mice . Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers . Right - quantification of centrally nucleated fibers . Bars show mean ( ± SEM ) with individual biological replicates shown as dots / squares ( WT n = 5 ; STOP n = 4 ; p = 0 . 016 according to Mann Whitney U test ) ( B ) Left - example HE stained TA muscles from control and SkM - DN mice . Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers . Right - quantification of centrally nucleated fibers . Bars show mean ( ± SEM ) with individual biological replicates shown as dots / squares ( Control n = 7 [ comprising 2 x WT , 3 x Myf5 + - cre , and 2 x AAA + littermate controls ] ; SkM - DN n = 5 ; p = 0 . 002 according to Mann Whitney U test ) . Data information : * denotes p value < α = 0 . 05 for all panels . Scale bars show 50 µm in each panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left - example HE stained tibialis anterior (TA) muscles from WT and SUR2-STOP475 mice. Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers. Right - quantification of centrally nucleated fibers. Bars show mean (± SEM) with individual biological replicates shown as dots/squares (WT n = 5; STOP n = 4; p = 0.016 according to Mann Whitney U test) (B) Left - example HE stained TA muscles from control and SkM-DN mice. Black arrows identify peripheral nuclei and yellow arrows indicate centrally nucleated fibers. Right - quantification of centrally nucleated fibers. Bars show mean (± SEM) with individual biological replicates shown as dots/squares (Control n = 7 [comprising 2 x WT, 3 x Myf5+-cre, and 2 x AAA+ littermate controls]; SkM-DN n = 5; p = 0.002 according to Mann Whitney U test). Data information: * denotes p value < α = 0.05 for all panels. Scale bars show 50 µm in each panel.


( C ) Example of HE stained WT TA muscle ( left ) and two examples of HE stained SkM - DN TA muscle alongside immunolabelling showing presence of CD68 + / CD206 + ( macrophage marker ) cells indicated by green arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Example of HE stained WT TA muscle (left) and two examples of HE stained SkM-DN TA muscle alongside immunolabelling showing presence of CD68+/CD206+ (macrophage marker) cells indicated by green arrows.


( D ) Left - Picrosirius red stained TA muscle from WT and SkM - DN mice . Right - quantification of red stained area ( WT n = 4 ; SkM - DN n = 4 ; p = 0 . 028 according to Mann Whitney U test ) . Bars show mean ( ± SEM ) with individual measurements shown as dots / squares . ( E ) Left - TA muscle for WT and SkM - DN mice showing Pax7 positive immunolabelled satellite cells ( red arrows ) . Right - quantification of Pax7 + cells ( WT n = 4 ; SkM - DN n = 4 ; p = 0 . 028 according to Mann Whitney U test ) . Bars show mean ( ± SEM ) with individual measurements shown as dots / squares . Data information : * denotes p value < α = 0 . 05 for all panels . Scale bars show 50 µm in each panel .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left - Picrosirius red stained TA muscle from WT and SkM-DN mice. Right - quantification of red stained area (WT n = 4; SkM-DN n = 4; p = 0.028 according to Mann Whitney U test). Bars show mean (± SEM) with individual measurements shown as dots/squares. (E) Left - TA muscle for WT and SkM-DN mice showing Pax7 positive immunolabelled satellite cells (red arrows). Right - quantification of Pax7+ cells (WT n = 4; SkM-DN n = 4; p = 0.028 according to Mann Whitney U test). Bars show mean (± SEM) with individual measurements shown as dots/squares. Data information: * denotes p value < α = 0.05 for all panels. Scale bars show 50 µm in each panel.


( A ) Example traces showing force generation in isometric contractility studies of isolated EDL muscle from WT ( left , black ) and SUR2 - STOP475 ( right , purple ) mice . Muscle was stimulated every 2 s . Scale bars show 20 mN ( y - axis ) and 2 s ( x - axis ) . ( B ) Enlarged representations of the 1st ( at 0 seconds ) , 10th ( 20 s ) and 30th ( 60 s ) contractions , identified in concatenated traces in ( A ) by arrows . Scale bars show 20 mN ( y - axis ) and 0 . 4 s ( x - axis ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example traces showing force generation in isometric contractility studies of isolated EDL muscle from WT (left, black) and SUR2-STOP475 (right, purple) mice. Muscle was stimulated every 2 s. Scale bars show 20 mN (y-axis) and 2 s (x-axis). (B) Enlarged representations of the 1st (at 0 seconds), 10th (20 s) and 30th (60 s) contractions, identified in concatenated traces in (A) by arrows. Scale bars show 20 mN (y-axis) and 0.4 s (x-axis).


( A ) Survival of WT and SUR2 - STOP475 mice administered with verapamil in drinking water ( n = 7 [ 4 male , 3 female ] for WT administered 0 . 9 g / l verapamil ; n = 14 [ 8 male , 6 female ] for SUR2 - STOP475 administered 0 . 9 g / l verapamil ; n = 6 [ 3 male , 3 female ] for SUR2 - STOP475 administered 0 . 3 g / l verapamil ) . Verapamil induced deaths were observed in both male and female mice ( 7 of 8 male and 4 of 6 female SUR2 - STOP475 mice died within 28 days of 0 . 9 g / l verapamil ) . ( B ) Survival of WT and SUR2 - STOP1149 mice administered with verapamil in drinking water ( n = 10 [ 3 male , 7 female ] for SUR2 - STOP1149 mice administered 0 . 9 g / l verapamil ) .

O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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-CONTROLLED_VAR O O

(A) Survival of WT and SUR2-STOP475 mice administered with verapamil in drinking water (n = 7 [4 male, 3 female] for WT administered 0.9 g/l verapamil; n = 14 [8 male, 6 female] for SUR2-STOP475 administered 0.9 g/l verapamil; n = 6 [3 male, 3 female] for SUR2-STOP475 administered 0.3 g/l verapamil). Verapamil induced deaths were observed in both male and female mice (7 of 8 male and 4 of 6 female SUR2-STOP475 mice died within 28 days of 0.9 g/l verapamil). (B) Survival of WT and SUR2-STOP1149 mice administered with verapamil in drinking water (n = 10 [3 male, 7 female] for SUR2-STOP1149 mice administered 0.9 g/l verapamil).


( D ) Heart rates calculated from ECG recordings from WT and SUR2 - STOP mice administered with no verapamil ( 0 Verap ) or 0 . 9 g / l verapamil in drinking water .

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(D) Heart rates calculated from ECG recordings from WT and SUR2-STOP mice administered with no verapamil (0 Verap) or 0.9 g/l verapamil in drinking water.


( F ) Cumulative durations of inversion in multi - trial inverted screen test for WT mice ( black circles ) and SkM - DN mice without ( magenta squares ) or with 0 . 9 g / l verapamil ( pink triangles ) administration . Data from WT and SkM - DN without verapamil Bars show mean ± SEM ( WT 0 verapamil n = 9 [ 6 male ; 3 female ] ; SkM - DN 0 verapamil n = 16 [ 12 male ; 4 female ] ; SkM - DN with 0 . 9 g / l verapamil n = 6 [ 4 male ; 2 female ] ; Dunn ' s test p = 0 . 006 for WT 0 verapamil vs SkM - DN 0 verapamil and 0 . 027 for WT 0 verapamil vs SkM - DN 0 . 9 g / l verapamil following Kruskal - Wallis omnibus test ; * denotes p value < adjusted α = 0 . 017 ) .

O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

(F) Cumulative durations of inversion in multi-trial inverted screen test for WT mice (black circles) and SkM-DN mice without (magenta squares) or with 0.9 g/l verapamil (pink triangles) administration. Data from WT and SkM-DN without verapamil Bars show mean ± SEM (WT 0 verapamil n = 9 [6 male; 3 female]; SkM-DN 0 verapamil n = 16 [12 male; 4 female]; SkM-DN with 0.9 g/l verapamil n = 6 [4 male; 2 female]; Dunn's test p = 0.006 for WT 0 verapamil vs SkM-DN 0 verapamil and 0.027 for WT 0 verapamil vs SkM-DN 0.9 g/l verapamil following Kruskal-Wallis omnibus test; * denotes p value < adjusted α = 0.017).


( A ) Left - Average performance in the inverted screen test of mice homozygous for the ncDHPR allele alone ( ncDHPR ) or homozygous for both the ncDHPR and SUR2 - STOP475 alleles ( STOP475 / ncDHPR ) . Right - Summary of inverted screen test data . Data shown as mean ± SEM ( WT n = 9 [ 4 male ; 5 female ] ; ncDHPR n = 15 [ male 6 ; female 9 ] ; STOP475 n = 15 [ 5 male ; 10 female ] ; STOP475 / ncDHPR n = 12 [ 4 male ; 12 female ] ; Dunn ' s test p = 4 . 0 x 10 - 4 for WT vs STOP475 , 6 . 9 x 10 - 4 for WT vs STOP475 / ncDHPR , 0 . 001 for ncDHPR vs STOP475 / ncDHPR verapamil , following Kruskal - Wallis omnibus test ) . * denotes p value < adjusted α = 0 . 0083 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(A) Left - Average performance in the inverted screen test of mice homozygous for the ncDHPR allele alone (ncDHPR) or homozygous for both the ncDHPR and SUR2-STOP475 alleles (STOP475/ncDHPR). Right - Summary of inverted screen test data. Data shown as mean ± SEM (WT n = 9 [4 male; 5 female]; ncDHPR n = 15 [male 6; female 9]; STOP475 n = 15 [5 male; 10 female]; STOP475/ncDHPR n = 12 [4 male; 12 female]; Dunn's test p = 4.0 x 10-4 for WT vs STOP475, 6.9 x 10-4 for WT vs STOP475/ncDHPR, 0.001 for ncDHPR vs STOP475/ncDHPR verapamil, following Kruskal-Wallis omnibus test). * denotes p value < adjusted α = 0.0083.


( B ) Left - Example of HE stained tibialis anterior muscle from STOP475 / ncDHPR mouse , black arrow shows peripheral nuclei and yellow arrows show central nuclei . Right - Quantification of centrally nucleated fibers , data from WT and SUR2 - STOP475 mice ( WT n = 5 ; STOP475 n = 4 ; STOP475 / ncDHPR n = 8 ; Dunn ' s test p = 0 . 007 for WT vs STOP475 and 0 . 011 for WT vs STOP475 / ncDHPR , following Kruskal - Wallis test , * denotes p value < adjusted α = 0 . 067 ) . Scale bar shows 50 µm . Bars show mean ( ± SEM ) with individual measurements shown as dots / squares / triangles .

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(B) Left - Example of HE stained tibialis anterior muscle from STOP475/ncDHPR mouse, black arrow shows peripheral nuclei and yellow arrows show central nuclei. Right - Quantification of centrally nucleated fibers, data from WT and SUR2-STOP475 mice (WT n = 5; STOP475 n = 4; STOP475/ncDHPR n = 8; Dunn's test p = 0.007 for WT vs STOP475 and 0.011 for WT vs STOP475/ncDHPR, following Kruskal-Wallis test, * denotes p value < adjusted α = 0.067). Scale bar shows 50 µm. Bars show mean (± SEM) with individual measurements shown as dots/squares/triangles.


( F ) Summary of unstimulated force ( normalised by physiological cross - sectional area ) at 60 s for each experiment . Bars show mean ± SEM with individual biological replicates shown as dots / squares / triangles ( WT without glibenclamide n = 6 [ 3 male ; 3 female ] ; WT with glibenclamide n = 10 [ 5 male ; 5 female ] ; ncDHPR without glibenclamide n = 6 [ 3 male ; 3 female ] ; ncDHPR with glibenclamide n = 6 [ 3 male ; 3 female ] ; Dunn ' s test p = 7 . 0 x 10 - 4 for WT without glibenclamide vs WT with glibenclamide and 0 . 002 for ncDHPR without glibenclamide vs ncDHPR with glibenclamide , following Kruskal - Wallis omnibus test ; * denotes p value < adjusted α = 0 . 0083 ) .

O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O 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-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O

(F) Summary of unstimulated force (normalised by physiological cross-sectional area) at 60 s for each experiment. Bars show mean ± SEM with individual biological replicates shown as dots/squares/triangles (WT without glibenclamide n = 6 [3 male; 3 female]; WT with glibenclamide n = 10 [5 male; 5 female]; ncDHPR without glibenclamide n = 6 [3 male; 3 female]; ncDHPR with glibenclamide n = 6 [3 male; 3 female]; Dunn's test p = 7.0 x 10-4 for WT without glibenclamide vs WT with glibenclamide and 0.002 for ncDHPR without glibenclamide vs ncDHPR with glibenclamide, following Kruskal-Wallis omnibus test; * denotes p value < adjusted α = 0.0083).


G The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses in HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . H Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

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G The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses in HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. H Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


A Flow cytometry characterization of binding between bat - ACE2 orthologs and the SARS - CoV - 2 or RshSTT182 / 200 RBDs . BHK cells expressing eGFP - fused ACE2s were stained with the indicated His - tagged proteins . The SARS - CoV - 2 NTD was used as a negative control .

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A Flow cytometry characterization of binding between bat-ACE2 orthologs and the SARS-CoV-2 or RshSTT182/200 RBDs. BHK cells expressing eGFP-fused ACE2s were stained with the indicated His-tagged proteins. The SARS-CoV-2 NTD was used as a negative control.


A Flow cytometry characterization of binding between ACE2s and the RshSTT182 / 200 or SARS - CoV - 2 RBDs . Cells stained with the RshSTT182 / 200 RBD , the SARS - CoV - 2 RBD and the SARS - CoV - 2 NTD proteins are shown in orange , marine and red , respectively . The SARS - CoV - 2 NTD was used as a negative control .

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A Flow cytometry characterization of binding between ACE2s and the RshSTT182/200 or SARS-CoV-2 RBDs. Cells stained with the RshSTT182/200 RBD, the SARS-CoV-2 RBD and the SARS-CoV-2 NTD proteins are shown in orange, marine and red, respectively. The SARS-CoV-2 NTD was used as a negative control.


D The entry efficiency of SARS - CoV - 2 and RshSTT182 pseudoviruses into HeLa - hACE2 cells . Green fluorescence indicates the entry of the pseudoviruses into the Hela - hACE2 cells . Unaltered HeLa cells were used as a negative control . E Statistics for transduction of pseudoviruses . Data represents the results of five replicates in one representative assay . All data are presented as mean ± SD . Pseudovirus infection assays were performed at least twice .

D The entry efficiency of SARS-CoV-2 and RshSTT182 pseudoviruses into HeLa-hACE2 cells. Green fluorescence indicates the entry of the pseudoviruses into the Hela-hACE2 cells. Unaltered HeLa cells were used as a negative control. E Statistics for transduction of pseudoviruses. Data represents the results of five replicates in one representative assay. All data are presented as mean ± SD. Pseudovirus infection assays were performed at least twice.


B The neutralization curve of serum samples in COVID - 19 convalescents and vaccinees against RshSTT182 and SARS - CoV - 2 are shown . Bar represents the SD of two replicates .

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B The neutralization curve of serum samples in COVID-19 convalescents and vaccinees against RshSTT182 and SARS-CoV-2 are shown. Bar represents the SD of two replicates.


( A ) Representative micrographs of optic nerve sections collected from mice 8 days after ONC , showing the accumulation of IB - 4 + activated myeloid cells in the injury site , which is demarcated by GFAP staining . Scale bar , 50 µm .

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(A) Representative micrographs of optic nerve sections collected from mice 8 days after ONC, showing the accumulation of IB-4+ activated myeloid cells in the injury site, which is demarcated by GFAP staining. Scale bar, 50 µm.


( B ) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [ Cx3cr1gfp / + WT ] BM chimeric mice , immunostained for GFP ( red ) , to depict monocyte - derived macrophages , and IB - 4 ( green ) to label activated myeloid cells . Scale bar , 100 µm . Inset scale , 20 µm .

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(B) Representative micrograph of the lesion site in optic nerve sections 7 days after crush injury in [Cx3cr1gfp/+ WT] BM chimeric mice, immunostained for GFP (red), to depict monocyte-derived macrophages, and IB-4 (green) to label activated myeloid cells. Scale bar, 100 µm. Inset scale, 20 µm.


( C ) Representative micrograph of optic nerve section 7 days after crush injury , immunostained for CD3 ( red , arrows ) . Scale bar , 100 µm .

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(C) Representative micrograph of optic nerve section 7 days after crush injury, immunostained for CD3 (red, arrows). Scale bar, 100 µm.


( D ) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina . Leukocytes were identified by gating on CD45 + cells , and were further gated on CD11b + or TCRβ + to detect monocytes / macrophages and T cells , respectively . Of the CD11b + cells , infiltrating monocytes were regarded as CD45hi .

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(D) Representative dotplots showing the general gating strategy used in flow cytometry experiments for the analysis of immune cells in the retina. Leukocytes were identified by gating on CD45+ cells, and were further gated on CD11b+ or TCRβ+ to detect monocytes/macrophages and T cells, respectively. Of the CD11b+ cells, infiltrating monocytes were regarded as CD45hi.


( E ) Representative contour plot from [ Cx3cr1gfp / + WT ] BM chimeric mouse , verifying that infiltrating blood - derived macrophages ( CX3CR1 - GFP + ) are indeed CD45hi , and showing that they are detectable only in retinas from eyes that underwent ONC , while absent in noninjured controls ( red vs . blue line ) . Cells were pre - gated on CD11b . n = 3 - 4 per group .

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(E) Representative contour plot from [Cx3cr1gfp/+ WT] BM chimeric mouse, verifying that infiltrating blood-derived macrophages (CX3CR1-GFP+) are indeed CD45hi, and showing that they are detectable only in retinas from eyes that underwent ONC, while absent in noninjured controls (red vs. blue line). Cells were pre-gated on CD11b. n = 3-4 per group.


( F ) Quantitative analysis of flow cytometry data , showing the induction of an immune response in the retina after ONC , evident by a general increase in leukocytes ( CD45 + ) , including CD11b + CD45hi cells ( representing infiltrating macrophages ) and T cells ( TCRβ + ) . n = 4 per group . Data shown are representative of six independent experiments . Graphs show mean ± SE of each group . * * * P < 0 . 001 . FSC - forward scatter ; noninj - noninjured control ; ONC - optic nerve crush .

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(F) Quantitative analysis of flow cytometry data, showing the induction of an immune response in the retina after ONC, evident by a general increase in leukocytes (CD45+), including CD11b+CD45hi cells (representing infiltrating macrophages) and T cells (TCRβ+). n = 4 per group. Data shown are representative of six independent experiments. Graphs show mean ± SE of each group. *** P < 0.001. FSC - forward scatter; noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of adhesion molecules ( A ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of adhesion molecules (A) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC. n = 6-13 per group. Data shown are representative of three independent experiments.


( B ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 12 hours after ONC . Scale bar , 20 µm . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(B) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 12 hours after ONC. Scale bar, 20 µm. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A , C - E ) Changes in mRNA transcript levels of an array of chemokines ( C , D ) and cytokine receptors ( E ) in retinas from ONC eyes compared with contralateral noninjured retinas , 8 hours , and 1 , 3 and 7 days after ONC ( optic nerve crush ) . n = 6 - 13 per group . Data shown are representative of three independent experiments .

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(A, C-E) Changes in mRNA transcript levels of an array of chemokines (C, D) and cytokine receptors (E) in retinas from ONC eyes compared with contralateral noninjured retinas, 8 hours, and 1, 3 and 7 days after ONC (optic nerve crush). n = 6-13 per group. Data shown are representative of three independent experiments.


( A , B ) Quantitative flow cytometry analysis of myeloid cells in the RPE complex ( A ) and retinas ( B ) from injured ( filled bars ) and contralateral noninjured eyes ( empty bars ) , on days 1 , 3 and 7 after ONC . Cells were pre - gated on total leukocytes ( CD45 + ) , and analyzed for the expression of CD11b . CD11b + cells were further discriminated based on their expression levels of Ly6C . n = 6 - 7 per group . Bar graphs throughout the figure show mean ± SE of each group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different time points are indicated by asterisks between bars . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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(A, B) Quantitative flow cytometry analysis of myeloid cells in the RPE complex (A) and retinas (B) from injured (filled bars) and contralateral noninjured eyes (empty bars), on days 1, 3 and 7 after ONC. Cells were pre-gated on total leukocytes (CD45+), and analyzed for the expression of CD11b. CD11b+ cells were further discriminated based on their expression levels of Ly6C. n = 6-7 per group. Bar graphs throughout the figure show mean ± SE of each group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different time points are indicated by asterisks between bars. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for flow cytometry . ( A ) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti - VCAM - 1 antibody , as compared to isotype - treated controls . n = 3 per group . Asterisks above bars indicate significant differences compared with noninjured tissue ; significant differences between different antibody treatments are indicated by asterisks between bars .

Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for flow cytometry. (A) Flow cytometry quantification of myeloid cells in retinas from mice that were treated with anti-VCAM-1 antibody, as compared to isotype-treated controls. n = 3 per group. Asterisks above bars indicate significant differences compared with noninjured tissue; significant differences between different antibody treatments are indicated by asterisks between bars.


Mice were subjected to ONC and i . v . - injected with either an anti - VCAM - 1 blocking antibody or an isotype control antibody . Eyes were collected after 2 or 3 days for Real - Time PCR analysis , respectively . ( B ) Expression ratios of Tnf , Il12a and Il1b , compared to Tgfb2 , showing that blocking VCAM - 1 results in skewing the retina towards a pro - inflammatory milieu after ONC . n = 11 - 13 per group . Data shown are representative of two independent experiments . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . noninj - noninjured control ; ONC - optic nerve crush .

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Mice were subjected to ONC and i.v.-injected with either an anti-VCAM-1 blocking antibody or an isotype control antibody. Eyes were collected after 2 or 3 days for Real-Time PCR analysis, respectively. (B) Expression ratios of Tnf, Il12a and Il1b, compared to Tgfb2, showing that blocking VCAM-1 results in skewing the retina towards a pro-inflammatory milieu after ONC. n = 11-13 per group. Data shown are representative of two independent experiments. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. noninj - noninjured control; ONC - optic nerve crush.


( A ) Live cell microscopy imaging of noninjured and ONC eyes , showing the presence of DiR + monocyte clusters in injured eyes only . Some of the cell clusters are co - localized with GFP , indicating monocytes that have crossed the RPE and infiltrated the retina . n = 2 - 3 per group . Data shown are representative of two independent experiments .

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(A) Live cell microscopy imaging of noninjured and ONC eyes, showing the presence of DiR+monocyte clusters in injured eyes only. Some of the cell clusters are co-localized with GFP, indicating monocytes that have crossed the RPE and infiltrated the retina. n = 2-3 per group. Data shown are representative of two independent experiments.


( B ) Representative micrographs of eye sections collected from mice 1 day after ONC , or 29 days after the induction of EAU , stained for β - dystroglycan ( red , arrows ) and laminin ( green ) , showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls , which is different from the appearance of blood vessels in EAU retina . Scale bar , 50 µm .

(B) Representative micrographs of eye sections collected from mice 1 day after ONC, or 29 days after the induction of EAU, stained for β-dystroglycan (red, arrows) and laminin (green), showing a comparable structure of blood vessels in the inner retina of ONC eyes and noninjured controls, which is different from the appearance of blood vessels in EAU retina. Scale bar, 50 µm.


( C - E ) [ Actbegfp / + WT ] BM chimeric mice were generated and subjected to ONC ( optic nerve crush ) . ( C ) Representative micrographs showing the abundance of GFP + cells ( green ) in the RPE ( RPE65 , red ) and retina of the ONC eye compared to noninjured contralateral eye . Retinal layers can be distinguished by Hoechst staining ( blue ; scale bar , 100 µm ) . ( D ) Higher magnification micrographs of ONC eyes , showing the association of blood - derived GFP + cells ( green , arrows ) with the RPE ( red ) ; Note processes extending from the cells to the RPE ( scale bar , 20 µm ) . ( E ) Representative micrograph of ONC eye showing GFP + cells ( green ) in proximity to the GCL , the site of dying retinal ganglion cells ( scale bar , 20 µm ) . n = 4 per group .

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(C-E) [Actbegfp/+ WT] BM chimeric mice were generated and subjected to ONC (optic nerve crush). (C) Representative micrographs showing the abundance of GFP+ cells (green) in the RPE (RPE65, red) and retina of the ONC eye compared to noninjured contralateral eye. Retinal layers can be distinguished by Hoechst staining (blue; scale bar, 100 µm). (D) Higher magnification micrographs of ONC eyes, showing the association of blood-derived GFP+ cells (green, arrows) with the RPE (red); Note processes extending from the cells to the RPE (scale bar, 20 µm). (E) Representative micrograph of ONC eye showing GFP+ cells (green) in proximity to the GCL, the site of dying retinal ganglion cells (scale bar, 20 µm). n = 4 per group.


( A - D ) Changes in mRNA transcript levels of a series of adhesion molecules ( A ) , chemokines ( B , C ) and cytokine receptors ( D ) in injured retinas compared with contralateral noninjured retinas , assessed 1 day after GT intoxication . n = 6 per group .

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(A-D) Changes in mRNA transcript levels of a series of adhesion molecules (A), chemokines (B, C) and cytokine receptors (D) in injured retinas compared with contralateral noninjured retinas, assessed 1 day after GT intoxication. n = 6 per group.


( E ) Representative micrographs showing the expression of ICAM - 1 and VCAM - 1 ( red ) by the RPE ( green ) 1 day after GT intoxication . Bar graphs throughout the figure show mean ± SE of each group . * , P < 0 . 05 ; * * , P < 0 . 01 ; * * * P < 0 . 001 . GT - glutamate ; noninj - noninjured control .

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(E) Representative micrographs showing the expression of ICAM-1 and VCAM-1 (red) by the RPE (green) 1 day after GT intoxication. Bar graphs throughout the figure show mean ± SE of each group. *, P < 0.05; **, P < 0.01; *** P < 0.001. GT - glutamate; noninj - noninjured control.


( A ) Representative micrograph of an eye from day 7 after glutamate intoxication , from a mouse that was i . v . - injected with monocytes , showing monocyte localization to the SRS after this mode of application , and indicating a potential homing route for the cells ( IL - 10 , red ; CX3CR1 - GFP , green ) .

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(A) Representative micrograph of an eye from day 7 after glutamate intoxication, from a mouse that was i.v.-injected with monocytes, showing monocyte localization to the SRS after this mode of application, and indicating a potential homing route for the cells (IL-10, red; CX3CR1-GFP, green).


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( B , C ) Representative micrographs of noninjured and injured retinas stained for the RGC marker , Brn3a ( red ; scale bar , 100 µm ) , and quantification of RGC survival . Arrows indicate Brn3a + cells . Bar graph shows mean ± SE of each group . * , P < 0 . 05 .

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(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (B, C) Representative micrographs of noninjured and injured retinas stained for the RGC marker, Brn3a (red; scale bar, 100 µm), and quantification of RGC survival. Arrows indicate Brn3a+ cells. Bar graph shows mean ± SE of each group. *, P < 0.05.


( B - E ) Mice were injected i . v . t . with either monocytes or PBS 1 day after GT intoxication , and ocular sections from 7 days after the injury were analyzed . ( D , E ) Representative micrographs of GT - intoxicated eyes that were i . v . t . - injected with monocytes , showing the expression of an array of cytokines and neurotrophic factors ( red ) by the injected monocytes ( CX3CR1 - GFP , green ) . The injected cells were found in the vitreous and in proximity to injured RGCs ( left panels ) , as well as in the SRS , adjacent to the RPE ( right panels ) . Insets show higher magnification of representative cells ( scale bar , 100 µm ; inset scale , 20 µm ) . n = 4 - 5 per group . GCL - ganglion cell layer ; GT - glutamate ; mon - monocytes ; RGC - retinal ganglion cell ; RPE - retinal pigment epithelium ; SRS - subretinal space ; vit - vitreous .

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(B-E) Mice were injected i.v.t. with either monocytes or PBS 1 day after GT intoxication, and ocular sections from 7 days after the injury were analyzed. (D, E) Representative micrographs of GT-intoxicated eyes that were i.v.t.-injected with monocytes, showing the expression of an array of cytokines and neurotrophic factors (red) by the injected monocytes (CX3CR1-GFP, green). The injected cells were found in the vitreous and in proximity to injured RGCs (left panels), as well as in the SRS, adjacent to the RPE (right panels). Insets show higher magnification of representative cells (scale bar, 100 µm; inset scale, 20 µm). n = 4-5 per group. GCL - ganglion cell layer; GT - glutamate; mon - monocytes; RGC - retinal ganglion cell; RPE - retinal pigment epithelium; SRS - subretinal space; vit - vitreous.


A LC - ESI - MS / MS spectrum of Dnmt3a pulled down by Biotin - As in NIH3T3 cells . NIH3T3 cells were treated with 10 μg / mL Biotin - As for 2 hours , followed by pull - down assay and determination using LC - ESI - MS / MS .

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A LC-ESI-MS/MS spectrum of Dnmt3a pulled down by Biotin-As in NIH3T3 cells. NIH3T3 cells were treated with 10 μg/mL Biotin-As for 2 hours, followed by pull-down assay and determination using LC-ESI-MS/MS.


B - D Validation of the binding between DNMT3A and Biotin - As at 5 μg / mL in NIH3T3 ( B ) , MEFs ( C ) and CCRF - CEM ( D ) cells in the Biotin - As pull - down assay .

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B-D Validation of the binding between DNMT3A and Biotin-As at 5 μg/mL in NIH3T3 (B), MEFs (C) and CCRF-CEM (D) cells in the Biotin-As pull-down assay.


F Measurement of DNMT3A - ADD thermodynamic stability upon ATO treatment . Purified DNMT3A - ADD was mixed with ATO at the indicated ratios in 20 mM HEPES ( pH 7 . 5 ) for 1 hour . Melting curves were measured by DSF ( left panel ) , and the calculated ΔTm values are shown ( right panel ) .

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F Measurement of DNMT3A-ADD thermodynamic stability upon ATO treatment. Purified DNMT3A-ADD was mixed with ATO at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Melting curves were measured by DSF (left panel), and the calculated ΔTm values are shown (right panel).


G LC - MS spectra of purified DNMT3A - ADD before and after ATO incubation at 1 : 5 molar ratio for 16 hours .

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G LC-MS spectra of purified DNMT3A-ADD before and after ATO incubation at 1:5 molar ratio for 16 hours.


H , I Biotin - As pull - down assays for the transfected DNMTs ( H ) and DNMT3A - C520A / C524A mutation ( I ) in 293T cells treated with 5 μg / mL Biotin - As .

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H, I Biotin-As pull-down assays for the transfected DNMTs (H) and DNMT3A-C520A/C524A mutation (I) in 293T cells treated with 5 μg/mL Biotin-As.


A Immunoblotting of DNMT3A protein levels in MEFs ( upper panel ) and CCRF - CEM cells ( lower panel ) treated with ATO in the indicated conditions .

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A Immunoblotting of DNMT3A protein levels in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO in the indicated conditions.


B MEFs ( upper panel ) and CCRF - CEM cells ( lower panel ) were pretreated with 20 μM DMSO , 1 μM MG132 , 0 . 02 μM Concanamycin A ( Conc A ) , 20 μM Calpeptin ( Calp ) , or 20 μM Z - VAD - FMK ( Z - VAD ) for 2 hours , followed by 16 - hour ATO co - treatment at 1 μg / mL for MEFs or 0 . 3 μg / mL for CCRF - CEM cells . Immunoblotting of DNMT3A in whole cell lysate is shown .

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B MEFs (upper panel) and CCRF-CEM cells (lower panel) were pretreated with 20 μM DMSO, 1 μM MG132, 0.02 μM Concanamycin A (Conc A), 20 μM Calpeptin (Calp), or 20 μM Z-VAD-FMK (Z-VAD) for 2 hours, followed by 16-hour ATO co-treatment at 1 μg/mL for MEFs or 0.3 μg/mL for CCRF-CEM cells. Immunoblotting of DNMT3A in whole cell lysate is shown.


C HCT116 cells were pretreated with 3 μM MG132 for 2 hours , followed by ATO co - treatment for 16 hours at the indicated concentrations . Immunoblotting of DNMT3A in whole cell lysate is shown .

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C HCT116 cells were pretreated with 3 μM MG132 for 2 hours, followed by ATO co-treatment for 16 hours at the indicated concentrations. Immunoblotting of DNMT3A in whole cell lysate is shown.


D In vivo ubiquitination assay . 293T cells were transiently transfected with HA - ubiquitin and Myc - DNMT3A . After 24 hours , cells were treated with or without MG132 for 2 hours at the indicated concentrations , followed by ATO co - treatment for 6 hours at the indicated concentrations . Myc - DNMT3A was immunoprecipitated ( IP ) with anti - Myc antibody , followed by immunoblotting ( IB ) as indicated .

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D In vivo ubiquitination assay. 293T cells were transiently transfected with HA-ubiquitin and Myc-DNMT3A. After 24 hours, cells were treated with or without MG132 for 2 hours at the indicated concentrations, followed by ATO co-treatment for 6 hours at the indicated concentrations. Myc-DNMT3A was immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) as indicated.


E Immunoblotting of the transfected wild - type DNMT3A , C520A / C524A and C541A mutants in 293T cells upon 1 μg / mL ATO treatment for the indicated durations .

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E Immunoblotting of the transfected wild-type DNMT3A, C520A/C524A and C541A mutants in 293T cells upon 1 μg/mL ATO treatment for the indicated durations.


F Measurement of DNMT3A - ADD thermodynamic stability upon ATO or AsPh3 treatment . Purified DNMT3A - ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES ( pH 7 . 5 ) for 1 hour . Chemical structure of AsPh3 is shown . Melting curves were measured by DSF ( upper panel ) , and the calculated ΔTm values are shown ( lower panel ) .

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F Measurement of DNMT3A-ADD thermodynamic stability upon ATO or AsPh3 treatment. Purified DNMT3A-ADD was mixed with ATO or AsPh3 at the indicated ratios in 20 mM HEPES (pH 7.5) for 1 hour. Chemical structure of AsPh3 is shown. Melting curves were measured by DSF (upper panel), and the calculated ΔTm values are shown (lower panel).


G Immunoblotting of DNMT3A in MEFs ( upper panel ) and CCRF - CEM cells ( lower panel ) treated with ATO or AsPh3 in the indicated conditions .

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G Immunoblotting of DNMT3A in MEFs (upper panel) and CCRF-CEM cells (lower panel) treated with ATO or AsPh3 in the indicated conditions.


H , I Immunoblotting of endogenous DNMT3A , DNMT3B and DNMT1 protein levels in CCRF - CEM ( H ) and THP - 1 ( I ) cells treated with DAC or ATO in the indicated conditions . DAC : + , 0 . 2 μM . ATO : + , 0 . 06 μg / mL ; + + , 0 . 2 μg / mL ; + + + , 0 . 6 μg / mL . Red asterisk indicates the specific band .

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H, I Immunoblotting of endogenous DNMT3A, DNMT3B and DNMT1 protein levels in CCRF-CEM (H) and THP-1 (I) cells treated with DAC or ATO in the indicated conditions. DAC: +, 0.2 μM. ATO: +, 0.06 μg/mL; ++, 0.2 μg/mL; +++, 0.6 μg/mL. Red asterisk indicates the specific band.


A Dot blot analysis of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days . The relative 5meC levels were quantified by ImageJ . The full blotting image of Fig 4A is shown in Fig EV4D , the treatment is the same .

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A Dot blot analysis of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ. The full blotting image of Fig 4A is shown in Fig EV4D, the treatment is the same.


B COBRA - IAP of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days . The signal density of unmethylated and methylated DNA were quantified by ImageJ .

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B COBRA-IAP of global DNA methylation levels in MEFs upon ATO or DAC treatment at the indicated concentrations for 7 days. The signal density of unmethylated and methylated DNA were quantified by ImageJ.


C 5meC enrichment analysis of the IAP , Xist , H19 loci in MEFs upon ATO treatment at 0 . 3 μg / mL for 4 or 7 days as measured by MeDIP - qPCR .

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C 5meC enrichment analysis of the IAP, Xist, H19 loci in MEFs upon ATO treatment at 0.3 μg/mL for 4 or 7 days as measured by MeDIP-qPCR.


D Dot blot analysis of global DNA methylation levels in CCRF - CEM cells upon ATO or DAC treatment at the indicated concentrations for 7 days . The relative 5meC levels were quantified by ImageJ .

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D Dot blot analysis of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment at the indicated concentrations for 7 days. The relative 5meC levels were quantified by ImageJ.


E COBRA - LINE - 1 of global DNA methylation levels in CCRF - CEM cells upon ATO or DAC treatment in the indicated conditions . The signal density of unmethylated and methylated DNA were quantified by ImageJ .

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E COBRA-LINE-1 of global DNA methylation levels in CCRF-CEM cells upon ATO or DAC treatment in the indicated conditions. The signal density of unmethylated and methylated DNA were quantified by ImageJ.


F 5meC enrichment analysis of the XIST , H19 loci in CCRF - CEM cells upon ATO treatment at the indicated concentrations for 63 days as measured by MeDIP - qPCR .

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F 5meC enrichment analysis of the XIST, H19 loci in CCRF-CEM cells upon ATO treatment at the indicated concentrations for 63 days as measured by MeDIP-qPCR.


B ) Electrophoretic mobility shift assay ( EMSA ) examination of the binding affinity of CENPC - CT and CM peptide to a CENP - A nucleosome . Binding to a canonical H3 nucleosome was also examined using EMSA .

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B) Electrophoretic mobility shift assay (EMSA) examination of the binding affinity of CENPC-CT and CM peptide to a CENP-A nucleosome. Binding to a canonical H3 nucleosome was also examined using EMSA.


C ) EMSA results of the binding affinities of CENPC - CT WT , CENPC - CT 3A679 - 681 , and CENPC - CT ∆ 678 - 683 to the CENP - A nucleosome .

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C) EMSA results of the binding affinities of CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 to the CENP-A nucleosome.


D ) Stable expression of GFP - fused CENPC - CT and its mutants ( shown in ( B ) ) in CENP - C knockout chicken DT40 cells . α - Tubulin ( Tub ) was probed as a loading control . Parental CENP - C knockout cells ( parental ) were also examined .

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D) Stable expression of GFP-fused CENPC-CT and its mutants (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control. Parental CENP-C knockout cells (parental) were also examined.


E ) Localization analysis of GFP - fused CENPC - CT WT , CENPC - CT 3A679 - 681 , and CENPC - CT ∆ 678 - 683 ( green ) on the mitotic chromosomes in chicken DT40 cells . CENP - T ( red ) was used as a centromere marker . DNA was stained using 4 ' , 6 - diamidino - 2 - phenylindole ( DAPI ; blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT WT, CENPC-CT 3A679-681, and CENPC-CT ∆678-683 (green) on the mitotic chromosomes in chicken DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar indicates 10 μm.


F ) Localization of CENP - C in CENP - A knockout DT40 cells stably expressing GFP - fused gCENP - A WT , RG - AA , or ∆ RG . Scale bar indicates 10 μm . CENP - C was stained by an anti - CENP - C antibody ( red ) and DNA was stained by DAPI ( blue ) . CENP - C signals on kinetochores in mitotic cells were quantified in each cell . Bar graph indicates mean ± SD ( n = 7 ; * * * * , P < 0 . 0001 , unpaired t test , two tailed ) .

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F) Localization of CENP-C in CENP-A knockout DT40 cells stably expressing GFP-fused gCENP-A WT, RG-AA, or ∆RG. Scale bar indicates 10 μm. CENP-C was stained by an anti-CENP-C antibody (red) and DNA was stained by DAPI (blue). CENP-C signals on kinetochores in mitotic cells were quantified in each cell. Bar graph indicates mean ± SD (n = 7; ****, P < 0.0001, unpaired t test, two tailed).


C ) EMSA results of the binding affinities of CENP - C fragments indicated in ( B ) to the CENP - A nucleosome . All fragments were phosphorylated by CDK1 .

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C) EMSA results of the binding affinities of CENP-C fragments indicated in (B) to the CENP-A nucleosome. All fragments were phosphorylated by CDK1.


D ) Stable expression of GFP - fused CENPC - CT and CENPC643 - 864 ( shown in ( B ) ) in CENP - C knockout chicken DT40 cells . α - Tubulin ( Tub ) was probed as a loading control .

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D) Stable expression of GFP-fused CENPC-CT and CENPC643-864 (shown in (B)) in CENP-C knockout chicken DT40 cells. α-Tubulin (Tub) was probed as a loading control.


E ) Localization analysis of GFP - fused CENPC - CT and CENPC643 - 864 lacking the CM upstream region ( green ) on mitotic chromosomes in CENP - C knockout DT40 cells . CENP - T ( red ) was used as a centromere marker . DNA was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT and CENPC643-864 lacking the CM upstream region (green) on mitotic chromosomes in CENP-C knockout DT40 cells. CENP-T (red) was used as a centromere marker. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) The position of a CDK1 phosphorylation site , T651CENP - C , is indicated in the diagram of chicken CENPC - CT . EMSA was performed to examine the binding affinities of phosphorylated or non - phosphorylated CENPC - CT ( left panel ) and CM peptide ( right panel ) to the CENP - A nucleosome .

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A) The position of a CDK1 phosphorylation site, T651CENP-C, is indicated in the diagram of chicken CENPC-CT. EMSA was performed to examine the binding affinities of phosphorylated or non-phosphorylated CENPC-CT (left panel) and CM peptide (right panel) to the CENP-A nucleosome.


D ) All constructs analyzed in ( E ) were stably expressed in CENP - C knockout cells .

D) All constructs analyzed in (E) were stably expressed in CENP-C knockout cells.


E ) Localization analysis of GFP - fused CENPC - CT , CENPC - CTR656E , and CENPC - CTR655E _ R656E ( green ) on mitotic chromosomes in chicken DT40 cells . DNA was stained using DAPI ( blue ) . Scale bar indicates 10 μm .

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E) Localization analysis of GFP-fused CENPC-CT, CENPC-CTR656E, and CENPC-CTR655E_R656E (green) on mitotic chromosomes in chicken DT40 cells. DNA was stained using DAPI (blue). Scale bar indicates 10 μm.


A ) Competitive pull - down assays for the CENP - A nucleosome binding of CENPC - CT and CENPN - NT . The left panel displays a complex of MBP - CENPN - NT ( MBP - CN - NT ) with the CENP - A nucleosome ( CA - nuc ) incubated in the absence or presence of phosphorylated CENPC - CT ( CC - CT ) and pulled down by MBP affinity . The right panel displays a complex of phosphorylated MBP - CENPC - CT ( MBP - CC - CT ) with the CENP - A nucleosome ( CA - nuc ) incubated in the absence or presence of CENPN - NT ( CN - NT ) and pulled down by MBP affinity . The signal intensities of all histones in CENP - A nucleosomes , which were precipitated with MBP proteins in indicated lanes ( * ) , were quantified . The signal intensities were normalized to MBP - CN - NT or MBP - CC - CT signals . Bar graph indicates mean with SD ( n = 3 ) .

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A) Competitive pull-down assays for the CENP-A nucleosome binding of CENPC-CT and CENPN-NT. The left panel displays a complex of MBP-CENPN-NT (MBP-CN-NT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of phosphorylated CENPC-CT (CC-CT) and pulled down by MBP affinity. The right panel displays a complex of phosphorylated MBP-CENPC-CT (MBP-CC-CT) with the CENP-A nucleosome (CA-nuc) incubated in the absence or presence of CENPN-NT (CN-NT) and pulled down by MBP affinity. The signal intensities of all histones in CENP-A nucleosomes, which were precipitated with MBP proteins in indicated lanes (*), were quantified. The signal intensities were normalized to MBP-CN-NT or MBP-CC-CT signals. Bar graph indicates mean with SD (n = 3).


PIP strip lipid binding assay with recombinant PTPIP51 _ ΔTM , PTPIP51 _ TPR , and PTPIP51 _ ΔTPR .

PIP strip lipid binding assay with recombinant PTPIP51_ΔTM, PTPIP51_TPR, and PTPIP51_ΔTPR.


PA binding of PTPIP51 ( PTPIP51 _ ΔTM and PTPIP51 _ TPR ) was monitored by a PA precipitation assay and a liposome flotation assay . The POPC : POPE : POPA ratios in each liposome were 50 : 50 : 0 , 50 : 40 : 10 , 50 : 30 : 20 , and 50 : 20 : 30 . Diagrams of the liposome precipitation and flotation assays are illustrated separately .

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PA binding of PTPIP51 (PTPIP51_ΔTM and PTPIP51_TPR) was monitored by a PA precipitation assay and a liposome flotation assay. The POPC:POPE:POPA ratios in each liposome were 50:50:0, 50:40:10, 50:30:20, and 50:20:30. Diagrams of the liposome precipitation and flotation assays are illustrated separately.


Results of fluorescence polarization ( FP ) experiments to monitor the interaction between the FFAT - like motif - containing peptide of PTPIP51 and VAPB _ ΔTM . The Kd value of the interaction determined in the FP experiment was ~ 53 µM . Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested . The data are presented as the mean ± SD of technical quadruplicate experiments .

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Results of fluorescence polarization (FP) experiments to monitor the interaction between the FFAT-like motif-containing peptide of PTPIP51 and VAPB_ΔTM. The Kd value of the interaction determined in the FP experiment was ~53 µM. Peptides with mutation of conserved phenylalanine and tyrosine residues in the FFAT motif were also tested. The data are presented as the mean ± SD of technical quadruplicate experiments.


CL levels after PTPIP51 depletion and reconstitution were monitored . A doxycycline - induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells . Left , western blot analysis of HeLa cells in which PTPIP51 was depleted and restored . β - ACTIN was used as the loading control . Right , the mitochondrial CL level ( nmol / mg ) according to PTPIP51 expression . To reconstitute PTPIP51 , the full - length PTPIP51 gene was transiently expressed . The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment ( n = 3 ) , and p - values calculated using Student ' s t - test are shown .

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CL levels after PTPIP51 depletion and reconstitution were monitored. A doxycycline-induced shRNA expression system was used to deplete the PTPIP51 protein in HeLa cells. Left, western blot analysis of HeLa cells in which PTPIP51 was depleted and restored. β-ACTIN was used as the loading control. Right, the mitochondrial CL level (nmol/mg) according to PTPIP51 expression. To reconstitute PTPIP51, the full-length PTPIP51 gene was transiently expressed. The data are presented as the mean ± SD of technical triplicate experiments from one representative experiment (n = 3), and p-values calculated using Student's t-test are shown.