Figure 2 ( 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 ) . ( 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 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 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 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

Figure 2(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).(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.


Figure 3 ( 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 . ( 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 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 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 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

Figure 3(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.(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.


Figure 4 ( 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 ) .

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Figure 4(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).


Figure 5 ( 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 . ( 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 ) .

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Figure 5(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.(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).


Figure 6 ( 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 . ( 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 ) .

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Figure 6(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.(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).


Figure 2G 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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Figure 2G 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.


Figure 3A 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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Figure 3A 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.


Figure 4A 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 .

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Figure 4A 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.


Figure 5B 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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Figure 5B 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.


Figure 1 ( 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 . ( 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 . ( 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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Figure 1(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.(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.(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.


Figure 2 ( 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 . ( 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 .

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 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

Figure 2(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.(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.


Figure 3 ( 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 .

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

Figure 3(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.


Figure 4Mice 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 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Mice 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.


Figure 5 ( 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 . ( 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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Figure 5(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.(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.


Figure 6 ( 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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Figure 6(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.


Figure 7 ( 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 . ( 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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Figure 7(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.(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.


Figure 2A 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 . 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 . 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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Figure 2A 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.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.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.


Figure 3A 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 . 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 . 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 ) . 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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Figure 3A 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.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.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).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.


Figure 4A 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 . 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 . 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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Figure 4A 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.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.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.


Figure 1B ) 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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Figure 1B) 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.


Figure 2C ) 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 . 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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Figure 2C) 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.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).


Figure 3C ) 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 . 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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Figure 3C) 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.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.


Figure 4A ) 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 . 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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Figure 4A) 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.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.


Figure 6A ) 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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Figure 6A) 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).


Figure 3PIP 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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Figure 3PIP 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.


Figure 4Results 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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Figure 4Results 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.


Figure 5CL 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 . Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution . Color codes for each sample were presented in the lower right subpanel . Experiments were technically repeated three times , and the results are represented as the means ± SD of technical replicates ( n = 3 ) . * p < 0 . 05 ; * * p < 0 . 01 ( Student ' s t - test ) . Quality control ( QC ) samples , which were pooled identical aliquots of the mitochondria samples , were measured four times throughout the run for data reproducibility . Upper right , western blot analysis of HeLa cells in which PTPIP51 WT or ∆ FFAT mutant were depleted and restored . β - Actin was used as a loading control .

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Figure 5CL 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.Quantification of CL in mitochondria using lipidomic analysis after PTPIP51 depletion and reconstitution. Color codes for each sample were presented in the lower right subpanel. Experiments were technically repeated three times, and the results are represented as the means ± SD of technical replicates (n = 3). *p < 0.05; **p < 0.01 (Student's t-test). Quality control (QC) samples, which were pooled identical aliquots of the mitochondria samples, were measured four times throughout the run for data reproducibility. Upper right, western blot analysis of HeLa cells in which PTPIP51 WT or ∆FFAT mutant were depleted and restored. β-Actin was used as a loading control.


Figure 1Representative micrographs of a time - lapse microscopy of K and KM non - regressed tumour cells ( H2B - GFP green ) . Top : Mitotic cell with a chromatin bridge ( yellow arrow ) . Bottom : Mitotic cell with misalignment and cytokinesis failure resulting in binucleation ( yellow arrow ) . Percentage of mitotic errors in K and KM primary tumours and in K and KM non - regressed tumours . Percentage of cells in K and KM primary tumours and in K and KM non - regressed tumours with the indicated mitotic errors . Scale bar 20 µm .

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Figure 1Representative micrographs of a time-lapse microscopy of K and KM non-regressed tumour cells (H2B-GFP green). Top: Mitotic cell with a chromatin bridge (yellow arrow). Bottom: Mitotic cell with misalignment and cytokinesis failure resulting in binucleation (yellow arrow).Percentage of mitotic errors in K and KM primary tumours and in K and KM non-regressed tumours. Percentage of cells in K and KM primary tumours and in K and KM non-regressed tumours with the indicated mitotic errors. Scale bar 20 µm.


Figure 2Somatic copy number alterations in each individual Kras and Kras / Mad2 non - regressed tumours . 7 K non - regressed and 16 KM non - regressed tumours were sequenced . Focal deletion ( DEL , green ) , Focal amplification ( AMP , light green ) , whole chromosome gain ( WCG , dark grey ) and loss ( WCL , light grey ) , partial chromosome gain ( PCG , dark blue ) and loss ( PCL , light blue ) and gross chromosomal rearrangement ( GCR , red ) . Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene . Colour of the block corresponds to the segment mean ( the degree to which a genome segment is lost ( blue ) or gained ( red ) . Immunostaining of phospho - cMet in non - regressed tumours carrying or not the amplification in chromosome 6 . Yellow numbers indicate the total number of cMet positive cells / total number of cells counted . Scale bar 100 µm .

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Figure 2Somatic copy number alterations in each individual Kras and Kras/Mad2 non-regressed tumours. 7 K non-regressed and 16 KM non-regressed tumours were sequenced. Focal deletion (DEL, green), Focal amplification (AMP, light green), whole chromosome gain (WCG, dark grey) and loss (WCL, light grey), partial chromosome gain (PCG, dark blue) and loss (PCL, light blue) and gross chromosomal rearrangement (GCR, red).Overlaying of the amplifications in chromosome 6 across multiple regions showing that all amplicons contained the cMet oncogene. Colour of the block corresponds to the segment mean (the degree to which a genome segment is lost (blue) or gained (red).Immunostaining of phospho-cMet in non-regressed tumours carrying or not the amplification in chromosome 6. Yellow numbers indicate the total number of cMet positive cells/total number of cells counted. Scale bar 100 µm.


Figure 3Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification . 0 indicates when doxycycline was removed . Each colour represents one tumour . Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth . Genome - wide log2 - ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non - regressed tumour showing no amplification in the primary tumour ( upper panels ) and a small amplification in the non - regressed tumours ( bottom panels , yellow arrow ) . Immunostaining of phospho - cMet in 3 biopsied primary tumours ( PT ) and their corresponding non - regressed tumours ( KM5 , KM7 and KM8 , which are also shown in Fig 2C ) . Scale bar 100 µm . Representative two - dimensional scatter plots constructed with overlaid dPCR data of the reference ( VIC ) and cMet ( FAM ) from one tumour without cMet amplification and one with cMet amplified . Dots represent results of independent PCR reactions in the wells of a digital PCR chip . Reactions in the bottom left corner ( yellow ) are negative for both targets , while the ones in the top right corner ( green ) are double positives . Reactions in the top left ( blue ) and bottom right ( red ) corners are positive for cMet and the reference targets , respectively . Representative photographs of FISH staining with a probe for Met ( red signal ) and a probe for a reference gene EML4 ( green signals ) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots ( white arrows ) . The lower panel shows an example of a tumour with cMet amplification ( several red dots ) and 2 green dots ( yellow arrow ) . Scale bar 10 µm .

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Figure 3Tumour diameter before and after doxycycline withdrawal in 4 K and 5 KM breast tumours with cMet amplification. 0 indicates when doxycycline was removed. Each colour represents one tumour. Blue and green squares indicate the timeframe between doxycycline withdrawal and the moment in which tumours resumed growth.Genome-wide log2-ratio plots of chromosome 6 of two primary tumour biopsies and their corresponding non-regressed tumour showing no amplification in the primary tumour (upper panels) and a small amplification in the non-regressed tumours (bottom panels, yellow arrow).Immunostaining of phospho-cMet in 3 biopsied primary tumours (PT) and their corresponding non-regressed tumours (KM5, KM7 and KM8, which are also shown in Fig 2C). Scale bar 100 µm.Representative two-dimensional scatter plots constructed with overlaid dPCR data of the reference (VIC) and cMet (FAM) from one tumour without cMet amplification and one with cMet amplified. Dots represent results of independent PCR reactions in the wells of a digital PCR chip. Reactions in the bottom left corner (yellow) are negative for both targets, while the ones in the top right corner (green) are double positives. Reactions in the top left (blue) and bottom right (red) corners are positive for cMet and the reference targets, respectively.Representative photographs of FISH staining with a probe for Met (red signal) and a probe for a reference gene EML4 (green signals) The upper panel is a negative example for cMet amplification containing 2 red and 2 green dots (white arrows). The lower panel shows an example of a tumour with cMet amplification (several red dots) and 2 green dots (yellow arrow). Scale bar 10 µm.


Figure 4Relative volume of tumours grown in 12 Rag2 - / - animals after treatment with Tepotinib or vehicle control ( 3K and 3KM tumours for each condition ) . No statistical significance was found by One - way ANOVA . Mean + / - SEM .

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Figure 4Relative volume of tumours grown in 12 Rag2-/- animals after treatment with Tepotinib or vehicle control (3K and 3KM tumours for each condition). No statistical significance was found by One-way ANOVA. Mean +/- SEM.


Figure 5Relative volume of tumours grown in Rag2 - / - animals after treatment with Tepotinib or vehicle control . One - way ANOVA , Tukey ' s multiple comparison . Ns : not significant ; * * p < 0 . 024 . Mean + / - SEM . A total of 19 Rag2 - / - mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls . For tumours with no cMet , 13 Rag2 - / - mice were treated with Tepotinib and 9 were used as control . Immunostaining of phospho - cMet in the same tumours grown in Rag2 - / - mice after treatment with Tepotinib or vehicle control . Scale bar 200 µm . Quantification of pH3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control . Quantification of caspase 3 in tumours grown in Rag2 - / - animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control .

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Figure 5Relative volume of tumours grown in Rag2-/- animals after treatment with Tepotinib or vehicle control. One-way ANOVA, Tukey's multiple comparison. Ns: not significant; ** p<0.024. Mean +/- SEM. A total of 19 Rag2-/- mice injected with cMet expressing tumours were treated with Tepotinib and 14 used as controls. For tumours with no cMet, 13 Rag2-/- mice were treated with Tepotinib and 9 were used as control.Immunostaining of phospho-cMet in the same tumours grown in Rag2-/- mice after treatment with Tepotinib or vehicle control. Scale bar 200 µm.Quantification of pH3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.Quantification of caspase 3 in tumours grown in Rag2-/- animals after 5 days treatment with the cMet inhibitor Tepotinib or vehicle control.


Figure 1 ( B - D ) Flow cytometric quantification of DHE > Eth . positive wild type yeast cells ( allowing the detection of both dead cells and cells exhibiting sub - lethal oxidative stress ) after 42 h ( B ) or 66 h ( C , D ) expressing Abeta constructs . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 5 - 12 biologically independent cultures . * * * , p < 0 . 001 , * * , p < 0 . 01 , * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test . ( E ) Annexin V ( AV ) / PI co - staining to assess cell death in yeast expressing A42 after 48 h . Data represent means ± s . d . n = 6 biologically independent cultures . * * * , p < 0 . 001 ( comparing PI positive populations ) . Unpaired , two - tailed t - test . ( F ) Immunoblot of whole cell extract ( WCE ) of wild type yeast cells after 16 h expression of Abeta constructs . For western blot ( WB ) Abeta - specific antibody ( Abeta ) 6E10 was used showing long ( Long exp . ) and short time exposure ( Short exp . ) . Ssc1 was used as a loading control . ( G ) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression . Colors indicate fluorescence intensity .

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Figure 1(B-D) Flow cytometric quantification of DHE>Eth. positive wild type yeast cells (allowing the detection of both dead cells and cells exhibiting sub-lethal oxidative stress) after 42 h (B) or 66 h (C, D) expressing Abeta constructs. Dot plots show all data points along with the mean (bar) ± s.d.. n = 5-12 biologically independent cultures. ***, p < 0.001, **, p < 0.01, *, p < 0.05. ANOVA with Tukey's post-hoc test.(E) Annexin V (AV)/PI co-staining to assess cell death in yeast expressing A42 after 48 h. Data represent means ± s.d. n = 6 biologically independent cultures. ***, p < 0.001 (comparing PI positive populations). Unpaired, two-tailed t-test.(F) Immunoblot of whole cell extract (WCE) of wild type yeast cells after 16 h expression of Abeta constructs. For western blot (WB) Abeta-specific antibody (Abeta) 6E10 was used showing long (Long exp.) and short time exposure (Short exp.). Ssc1 was used as a loading control.(G) Confocal microscopy of wild type yeast cells after 18 h of Abeta expression. Colors indicate fluorescence intensity.


Figure 2 ( A ) Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial , microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP - Abeta42 ( A42 ) , EGFP - Abeta40 ( A40 ) and EGFP empty vector ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Purity of fractions was tested with antibodies against Cyc1 ( mitochondria ) , Sec61 ( microsomes ) and Pgk1 ( cytosol ) . ( B ) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 16 biologically independent cultures . * * , p < 0 . 01 . Unpaired , two - tailed t - test . ( C ) Relative protein abundance of EGFP - Abeta42 ( A42 ) versus empty vector ( ev ) expressing wild type yeast cells . The significant subset of cytosolic heat shock proteins ( HSP ) detected in a proteomics analysis of isolated mitochondria is depicted . Log2 SILAC ratios of A42 / ev of two independent proteome measurements are shown . Dot plots show all data points along with the mean ( line ) . Significance was determined using an outlier test ( Significance A , p < 0 . 003 ) . ( D ) Screen of 123 deletion strains assessing Abeta42 ( A42 ) - induced toxicity ( assessed by DHE > Eth . positive cells indicative of the sum of sub - lethal oxidative stress and cell death ) in yeast . Relative A42 - toxicity normalized to wild type ( WT ) cells is depicted . Potential hits ( mutants that reduce A42 toxicity ) are shown in green . Data represent means ± s . d . . n ≥ 3 biologically independent cultures .

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Figure 2(A) Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial, microsomal and cytosolic fractions of wild type yeast cells after 18 h expression of EGFP-Abeta42 (A42), EGFP-Abeta40 (A40) and EGFP empty vector (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Purity of fractions was tested with antibodies against Cyc1 (mitochondria), Sec61 (microsomes) and Pgk1 (cytosol).(B) Cellular ATP content of wild type yeast cells after 42 h expression of EGFP-A42 (A42) or EGFP empty vector (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 16 biologically independent cultures. **, p < 0.01. Unpaired, two-tailed t-test.(C) Relative protein abundance of EGFP-Abeta42 (A42) versus empty vector (ev) expressing wild type yeast cells. The significant subset of cytosolic heat shock proteins (HSP) detected in a proteomics analysis of isolated mitochondria is depicted. Log2 SILAC ratios of A42/ev of two independent proteome measurements are shown. Dot plots show all data points along with the mean (line). Significance was determined using an outlier test (Significance A, p <0.003).(D) Screen of 123 deletion strains assessing Abeta42 (A42)-induced toxicity (assessed by DHE>Eth. positive cells indicative of the sum of sub-lethal oxidative stress and cell death) in yeast. Relative A42-toxicity normalized to wild type (WT) cells is depicted. Potential hits (mutants that reduce A42 toxicity) are shown in green. Data represent means ± s.d.. n ≥ 3 biologically independent cultures.


Figure 3Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) of wild type ( WT ) ( A ) and YDJ1 deleted ( Δydj1 ) cells ( B ) . Mean ± s . d . . n = 5 biologically independent cultures . Comparisons by two - way ANOVA ( mixed - design ) followed by simple main effects ( * * * , p < 0 . 001 , vs . control ) . Immunoblot of whole cell extract ( Input ) and eluate of FLAG - tagged Ydj1 . Immunoprecipitation ( IP : FLAG ) of YDJ1 deletion strain ( Δydj1 ) cells expressing EGFP - A42 ( A42 ) or EGFP empty vector ( ev ) and co - overexpressing Ydj1 - FLAG using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) . Confocal microscopy of wild type ( WT ) yeast cells expressing EGFP - A42 ( A42 ) only and co - overexpressing Ydj1 - FLAG or with the corresponding vector control after 18 h of expression . Colors indicate fluorescence intensity . Confocal microscopy of wild type ( WT ) yeast cells expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG after 18 h of expression . Mitochondria were visualized with MitoTracker Red ( magenta ) . See also Fig EV2J , EV1C . Immunoblot of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) and cytosolic ( Cyt . ) fractions of wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time exposure ( Short exp . ) . Tom22 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction . Ratio of full length EGFP - A42 in the mitochondrial fraction / cytosolic fraction , using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three / 4 independent experiments . Dot plots show all data points along with the mean ( line / bar ) ± s . d . . n = 3 - 4 biologically independent cultures . * , p < 0 . 05 . Unpaired , two - tailed t - test . Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity was measured at two time points ( t0 and t2 , 2 h after CH administration ) and normalized to t0 in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) cells after 18 h of expression of EGFP - A42 ( A42 ) and co - overexpressing Ydj1 - FLAG or corresponding empty vector controls ( ev ) . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc . See also Appendix Fig S2D . Quantification of the ratio between EGFP - A42 tetramer and monomer in wild type ( WT ) and YDJ1 deletion strain ( Δydj1 ) expressing EGFP - A42 ( J ) as well as between WT expressing EGFP - A42 only and co - overexpressing Ydj1 - FLAG ( K ) at indicated time points . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 8 ( J ) n = 12 ( K ) biologically independent cultures . * * * , p < 0 . 001 ; * * , p < 0 . 01 ; * , p < 0 . 05 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2A - B .

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Figure 3Quantification of cells positive for PI indicating cell death at indicated time points after start of expression of EGFP-A42 (A42) or EGFP empty vector (ev) of wild type (WT) (A) and YDJ1 deleted (Δydj1) cells (B). Mean ± s.d.. n = 5 biologically independent cultures. Comparisons by two-way ANOVA (mixed-design) followed by simple main effects (***, p < 0.001, vs. control).Immunoblot of whole cell extract (Input) and eluate of FLAG-tagged Ydj1. Immunoprecipitation (IP: FLAG) of YDJ1 deletion strain (Δydj1) cells expressing EGFP-A42 (A42) or EGFP empty vector (ev) and co-overexpressing Ydj1-FLAG using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).Confocal microscopy of wild type (WT) yeast cells expressing EGFP-A42 (A42) only and co-overexpressing Ydj1-FLAG or with the corresponding vector control after 18 h of expression. Colors indicate fluorescence intensity.Confocal microscopy of wild type (WT) yeast cells expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG after 18 h of expression. Mitochondria were visualized with MitoTracker Red (magenta). See also Fig EV2J, EV1C.Immunoblot of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.) and cytosolic (Cyt.) fractions of wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time exposure (Short exp.). Tom22-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.Ratio of full length EGFP-A42 in the mitochondrial fraction / cytosolic fraction, using densitometry quantification of the immunoblot representatively shown in Fig 3F and Appendix Fig S1C from three/4 independent experiments. Dot plots show all data points along with the mean (line/bar) ± s.d.. n = 3-4 biologically independent cultures. *, p < 0.05. Unpaired, two-tailed t-test.Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity was measured at two time points (t0 and t2, 2 h after CH administration) and normalized to t0 in wild type (WT) and YDJ1 deletion strain (Δydj1) cells after 18 h of expression of EGFP-A42 (A42) and co-overexpressing Ydj1-FLAG or corresponding empty vector controls (ev). Dot plots show all data points along with the mean (bar) ± s.d.. n = 8 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc. See also Appendix Fig S2D.Quantification of the ratio between EGFP-A42 tetramer and monomer in wild type (WT) and YDJ1 deletion strain (Δydj1) expressing EGFP-A42 (J) as well as between WT expressing EGFP-A42 only and co-overexpressing Ydj1-FLAG (K) at indicated time points. Dot plots show all data points along with the mean (bar) ± s.d.. n = 8(J) n=12(K) biologically independent cultures. ***, p < 0.001; **, p < 0.01; *, p < 0.05. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2A-B.


Figure 4Quantification of propidium iodide ( PI ) - staining positive cells indicating cell death at indicated time points of expression of EGFP - A42 ( A42 ) Δydj1 cells with co - expression of DnaJA1 - FLAG ( DnaJA1 ) ( B ) or corresponding empty vector controls ( ev ) ( A ) . Mean ± SD n = 4 - 6 biologically independent cultures . Comparisons by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus control ) . ( C ) Immunoblot ( WB ) of whole - cell extract ( Input ) and eluate of FLAG - tagged DnaJA1 immunoprecipitation ( IP : FLAG ) of wild - type strain ( WT ) expressing EGFP - A42 ( A42 ) and co - expressing DnaJA1 - FLAG ( A1 ) using Abeta - specific antibody ( 6E10 ) and FLAG antibody ( FLAG ) . Assay for protein degradation using cycloheximide ( CH ) to stall protein translation . EGFP fluorescence intensity of YDJ1 deletion strain ( Δydj1 ) and wild type ( WT ) cells expressing EGFP - A42 ( A42 ) only or co - expressing DnaJA1 was measured at two time points ( t0 and t2 ) and normalized to t0 . Dot plots show all data points along with the mean ( bar ) ± s . d . . n = 6 biologically independent cultures . * * * , p < 0 . 001 . ANOVA with Tukey ' s post - hoc test . See also Appendix Fig S2C . Immunoblot ( WB ) of total cytoplasmic post - nuclear supernatant ( PNS ) , mitochondrial ( Mito . ) , microsomal ( Micro . ) , and cytosolic ( Cyt . ) fractions of YDJ1 - deleted cells ( Δydj1 ) after 18 h expression of EGFP - A42 ( A42 ) only or co - expressing DnaJA1 - FLAG ( DnaJA1 ) using Abeta - specific antibody ( Abeta ) 6E10 with long ( Long exp . ) and short time ( Short exp . ) exposure ( two sections from one immunoblot ) . Cox4 - specific antibody is a marker for mitochondria , Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction . Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 ( A1 ) and quantified by densitometry in ( G ) . Quantification of the oligomer - to - monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in ( F ) . Dot plots show all data points along with the mean ( line ) ± SD n = 3 biologically independent cultures . P - values by two - way ANOVA ( mixed design ) followed by simple main effects ( * P < 0 . 05 , versus A42 control ) . See also Appendix Fig S2D . A42 beta - sheet - rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ . Data represent means of at least eight measurements . See also Appendix Fig S1E .

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

Figure 4Quantification of propidium iodide (PI)-staining positive cells indicating cell death at indicated time points of expression of EGFP-A42 (A42) Δydj1 cells with co-expression of DnaJA1-FLAG (DnaJA1) (B) or corresponding empty vector controls (ev) (A). Mean ± SD n = 4-6 biologically independent cultures. Comparisons by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus control).(C)Immunoblot (WB) of whole-cell extract (Input) and eluate of FLAG-tagged DnaJA1 immunoprecipitation (IP: FLAG) of wild-type strain (WT) expressing EGFP-A42 (A42) and co-expressing DnaJA1-FLAG (A1) using Abeta-specific antibody (6E10) and FLAG antibody (FLAG).Assay for protein degradation using cycloheximide (CH) to stall protein translation. EGFP fluorescence intensity of YDJ1 deletion strain (Δydj1) and wild type (WT) cells expressing EGFP-A42 (A42) only or co-expressing DnaJA1 was measured at two time points (t0 and t2) and normalized to t0. Dot plots show all data points along with the mean (bar) ± s.d.. n = 6 biologically independent cultures. ***, p < 0.001. ANOVA with Tukey's post-hoc test. See also Appendix Fig S2C.Immunoblot (WB) of total cytoplasmic post-nuclear supernatant (PNS), mitochondrial (Mito.), microsomal (Micro.), and cytosolic (Cyt.) fractions of YDJ1-deleted cells (Δydj1) after 18 h expression of EGFP-A42 (A42) only or co-expressing DnaJA1-FLAG (DnaJA1) using Abeta-specific antibody (Abeta) 6E10 with long (Long exp.) and short time (Short exp.) exposure (two sections from one immunoblot). Cox4-specific antibody is a marker for mitochondria, Sec61 for microsomes and Ssa1 was used to verify cytosolic fraction.Representative immunoblot of synthetic Abeta42 oligomer formation monitored in vitro over the indicated time course with or without DnaJA1 (A1) and quantified by densitometry in (G).Quantification of the oligomer-to-monomer ratio of synthetic Abeta42 with or without DnaJA1 obtained from immunoblots representatively shown in (F). Dot plots show all data points along with the mean (line) ± SD n = 3 biologically independent cultures. P-values by two-way ANOVA (mixed design) followed by simple main effects (*P < 0.05, versus A42 control). See also Appendix Fig S2D.A42 beta-sheet-rich assembly formation monitored by increase in ThT fluorescence over time with BSA or with DnaJ. Data represent means of at least eight measurements. See also Appendix Fig S1E.