Figure 4 ( A ) Representative images of control cells ( upper panel ) or cells treated with α - synucleinfibrilsATTO - 550 ( bottom panel ) for 16 hours ; in red : α - synucleinfibrils and in green : WGAAlexa - 488 ( plasma membrane dye marker ) , blue : DAPI . Scale bar represents 10 μm ( n = 3 independent experiments ) . ( B ) Relative percentage of TNT - connected cells after α - synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment . Data are mean ± s . e . m ( * * * , p < 0 . 001 by paired , two - tailed Student ' s t - test ) . ( C ) Confocal images showing one TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 ( in red ) 16 hours after co - culture and stained with WGAAlexa - 488 ( in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located ( top panel ) . Insets show 3D reconstructions of the TNT using ICY software ( middle panel : merge ; bottom panel : red channel ) . Several α - synucleinfibrils ( red ) are present in the lumen of the TNT ( WGAAlexa - 488 in white ) . Scale bars represent 10 μm ( confocal images ) and 4 μm ( insets ) . ( D ) Confocal image showing TNT connecting a donor cell containing α - synucleinfibrils and GFP transfected acceptor cell ( top panel ) strongly suggesting α - synucleinfibrils transfer from donor to acceptor cell . Insets show 3D reconstructions of TNT containing α - synucleinfibrils in the lumen of the TNT ; in red : α - synucleinfibrils , in green : GFP transfected acceptor cells in blue : WGAAlexa - 488 . Scale bars represent 10 μm and 3 μm for the insets . ( E ) Relative percentage of TNTs in cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs . ( F ) Quantification of the average number of ATTO - 550α - synucleinfibrils per acceptor cell after 24 hours co - culture with donor cells containing α - synucleinfibrils untransfected ( control ) or transfected with Myosin 10 ( Myosin 10 ) . Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α - synuclein puncta in acceptor cells ; n = 3 independent experiments ( * * , p < 0 . 01 by two - tailed Mann Whitney test ) . ( G ) Quantification of the relative proportion of TNTs in co - culture of donor cells containing α - synuclein puncta and GFP acceptor cells under sub - confluent or sparse culture conditions . Data are mean ± s . e . m ; n = 3 independent experiments ( * , p < 0 . 05 by paired , two - tailed Student ' s t - test ) . ( H ) Quantification of the percentage of acceptor cells containing α - synuclein puncta of the co - culture in G . n = 3 independent experiments ( * , p < 0 . 05 by two - tailed Mann Whitney test ) . Condition impairing TNT formation ( i . e . , sparse condition ) induces a drastic decrease of TNT number and α - synucleinfibrils transfer compared to condition allowing TNT formation ( sub - confluent condition ) .

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

Figure 4(A) Representative images of control cells (upper panel) or cells treated with α-synucleinfibrilsATTO-550 (bottom panel) for 16 hours; in red: α-synucleinfibrils and in green: WGAAlexa-488 (plasma membrane dye marker), blue: DAPI. Scale bar represents 10 μm (n = 3 independent experiments).(B) Relative percentage of TNT-connected cells after α-synucleinfibrils treatment as in A shows an increase in TNT number upon fibrils treatment. Data are mean ± s.e.m (***, p < 0.001 by paired, two-tailed Student's t-test).(C) Confocal images showing one TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 (in red) 16 hours after co-culture and stained with WGAAlexa-488 (in blue). The image represents a Z-projection of the several middle stacks where the TNT is located (top panel). Insets show 3D reconstructions of the TNT using ICY software (middle panel: merge; bottom panel: red channel). Several α-synucleinfibrils (red) are present in the lumen of the TNT (WGAAlexa-488 in white). Scale bars represent 10 μm (confocal images) and 4 μm (insets).(D) Confocal image showing TNT connecting a donor cell containing α-synucleinfibrils and GFP transfected acceptor cell (top panel) strongly suggesting α-synucleinfibrils transfer from donor to acceptor cell. Insets show 3D reconstructions of TNT containing α-synucleinfibrils in the lumen of the TNT; in red: α-synucleinfibrils, in green: GFP transfected acceptor cells in blue: WGAAlexa-488. Scale bars represent 10 μm and 3 μm for the insets.(E) Relative percentage of TNTs in cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Overexpression of Myosin 10 in the donor cell population led to an increase in the number of TNTs.(F) Quantification of the average number of ATTO-550α-synucleinfibrils per acceptor cell after 24 hours co-culture with donor cells containing α-synucleinfibrils untransfected (control) or transfected with Myosin 10 (Myosin 10). Note that Myosin 10 overexpression increases both the transfer of DiIvesicles and the number of transferred α-synuclein puncta in acceptor cells; n = 3 independent experiments (**, p < 0.01 by two-tailed Mann Whitney test).(G) Quantification of the relative proportion of TNTs in co-culture of donor cells containing α-synuclein puncta and GFP acceptor cells under sub-confluent or sparse culture conditions. Data are mean ± s.e.m; n = 3 independent experiments (*, p < 0.05 by paired, two-tailed Student's t-test).(H) Quantification of the percentage of acceptor cells containing α-synuclein puncta of the co-culture in G. n = 3 independent experiments (*, p < 0.05 by two-tailed Mann Whitney test). Condition impairing TNT formation (i.e., sparse condition) induces a drastic decrease of TNT number and α-synucleinfibrils transfer compared to condition allowing TNT formation (sub-confluent condition).


Figure 5 ( A ) Subcellular localization of α - synucleinfibrils in donor cells after 24 hours co - culture . The left panels show representative images of co - localization of α - synuclein puncta with early endosomes ( EEA1 , top panel ) , recycling endosomes ( Vamp3 , middle panel ) and lysosomal vesicles ( Lamp1 , bottom panel ) ( see arrows for co - localized spots ) ; in red : α - synucleinfibrils , in green : EEA1 , Vamp3 or Lamp1 and in blue : nuclei . Scale bar represents 10 μm . On the right , the bar graph represents the percentage of α - synucleinpuncta co - localized with endo - lysosomal vesicles in A . Data are mean ± s . e . m . from three independent experiments . ( B ) Confocal images showing a TNT connecting two cells loaded with α - synucleinfibrilsATTO - 550 immunostained with Lamp1 ( in green ) . Labelling of TNTs is performed using a plasma membrane dye ( WGAAlexa - 488 , in blue ) . The image represents a Z - projection of the several middle stacks where the TNT is located . The merged image with WGA - Alexa 633 ( blue ) , the orthogonal view and insets showing 3D reconstructions of the TNT ( with WGA in white ) are in the right panel . Several α - synuclein fibrillar foci ( in red ) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles ( in green ) . Of note , only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia . Scale bars represent 10 μm ( confocal images ) and 3 μm ( insets ) . The merge of α - synucleinfibrils ( red ) and Lamp1 ( green ) channels and the respective single channels are showed on the left panel . ( C ) The top panels show representative images of GFP - transfected acceptor cells after 24 h co - culture with donor cells containing α - synucleinfibrilsATTO - 550 . Immunofluorescence was performed with endo - lysosomal organelle markers ( top panel : EEA1 , middle panel : Vamp3 and Lamp1 : bottom panel ) and revealed that α - synucleinfibrils co - localized with lysosomal vesicles ( see arrow ) ; in red : α - synucleinfibrils , in green : organelle markers and in blue : nuclei . Scale bar represents 10 μm , arrows pinpoint assemblies co - localizing with organelle markers . The bar graph at the bottom shows the quantification of the percentage of co - localization of α - synucleinfibrils with organelle markers , revealing that α - synuclein puncta co - localized mostly with lysosomal vesicles . Data show mean ± s . e . m . from three independent experiments . ( D ) Representative confocal pictures of control cells loaded with ATTO - 550α - synucleinfibrils and LysoTracker Deep Red ( top panel ) , and of donor ( middle panel ) and acceptor GFP - transfected acceptor cells ( bottom panel , GFP - vector not shown ) cells , loaded with α - synucleinfibrilsATTO - 550 and LysoTracker Deep Red ( middle panel ) and co - cultured for 24 hours ; in red : α - synucleinfibrils , in green : LysoTracker Deep Red and in blue : nuclei . Scale bar represents 10 μm , arrows in inset pinpoint fibrils co - localizing with LysoTracker positive vesicles . ( E ) The percentage of co - localization of LysoTracker positive vesicles with α - synucleinfibrils revealed that 80 % of lysosomal vesicles that transferred from donor to acceptor cells contained α - synucleinfibrils thus demonstrating direct transfer of α - synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells . Data show mean ± s . e . m . from 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 5(A) Subcellular localization of α-synucleinfibrils in donor cells after 24 hours co-culture. The left panels show representative images of co-localization of α-synuclein puncta with early endosomes (EEA1, top panel), recycling endosomes (Vamp3, middle panel) and lysosomal vesicles (Lamp1, bottom panel) (see arrows for co-localized spots); in red: α-synucleinfibrils, in green: EEA1, Vamp3 or Lamp1 and in blue: nuclei. Scale bar represents 10 μm. On the right, the bar graph represents the percentage of α-synucleinpuncta co-localized with endo-lysosomal vesicles in A. Data are mean ± s.e.m. from three independent experiments.(B) Confocal images showing a TNT connecting two cells loaded with α-synucleinfibrilsATTO-550 immunostained with Lamp1 (in green). Labelling of TNTs is performed using a plasma membrane dye (WGAAlexa-488, in blue). The image represents a Z-projection of the several middle stacks where the TNT is located. The merged image with WGA-Alexa 633 (blue), the orthogonal view and insets showing 3D reconstructions of the TNT (with WGA in white) are in the right panel. Several α-synuclein fibrillar foci (in red) are present in the lumen of the TNT and embedded in Lamp1 positive vesicles (in green). Of note, only tubular structures physically connecting remote cells and not touching the substratum were identified as TNTs to distinguish them from filopodia. Scale bars represent 10 μm (confocal images) and 3 μm (insets). The merge of α-synucleinfibrils (red) and Lamp1 (green) channels and the respective single channels are showed on the left panel.(C) The top panels show representative images of GFP-transfected acceptor cells after 24 h co-culture with donor cells containing α-synucleinfibrilsATTO-550. Immunofluorescence was performed with endo-lysosomal organelle markers (top panel: EEA1, middle panel: Vamp3 and Lamp1: bottom panel) and revealed that α-synucleinfibrils co-localized with lysosomal vesicles (see arrow); in red: α-synucleinfibrils, in green: organelle markers and in blue: nuclei. Scale bar represents 10 μm, arrows pinpoint assemblies co-localizing with organelle markers. The bar graph at the bottom shows the quantification of the percentage of co-localization of α-synucleinfibrils with organelle markers, revealing that α-synuclein puncta co-localized mostly with lysosomal vesicles. Data show mean ± s.e.m. from three independent experiments.(D) Representative confocal pictures of control cells loaded with ATTO-550α-synucleinfibrils and LysoTracker Deep Red (top panel), and of donor (middle panel) and acceptor GFP-transfected acceptor cells (bottom panel, GFP-vector not shown) cells, loaded with α-synucleinfibrilsATTO-550 and LysoTracker Deep Red (middle panel) and co-cultured for 24 hours; in red: α-synucleinfibrils, in green: LysoTracker Deep Red and in blue: nuclei. Scale bar represents 10 μm, arrows in inset pinpoint fibrils co-localizing with LysoTracker positive vesicles.(E) The percentage of co-localization of LysoTracker positive vesicles with α-synucleinfibrils revealed that 80% of lysosomal vesicles that transferred from donor to acceptor cells contained α-synucleinfibrils thus demonstrating direct transfer of α-synucleinfibrils from donor to acceptor cells in majority inside lysosomal vesicles derived from donor cells. Data show mean ± s.e.m. from three independent experiments.


Figure 6 ( A ) Representative images of control ( upper panel ) and α - synuclein loaded neurons ( middle , 0 . 5µM , and bottom , 1µM , panels ) after 16 h . In red : α - synuclein fibrils , in green : MAP - 2 and in blue : nuclei . Scale bar represents 10 μm . ( B ) Representative images of donor ( upper panels ) and acceptor neurons ( bottom panels ) after 72 hours in co - culture . Donor neurons were loaded with α - synuclein fibrils prior to co - culture with CTG - labelled acceptor neurons and then cells were labelled either with MAP - 2 ( first and third panels ) or Lamp1 ( second and fourth panels ) . In red : α - synuclein fibrils , in green : CTG , in white : MAP - 2 / Lamp1 and in blue : nuclei . Scale bar represents 5 μm . ( C ) The graph bar shows the percentage of donor ( white bar ) and acceptor ( black bar ) neurons containing α - synuclein puncta . Data show mean ± s . e . m . ( D ) The box plot depicts the number of puncta in donor ( white ) and acceptor ( grey ) neurons . ( E ) Box plot showing the distribution of the average size of α - synuclein puncta in donor ( white ) and acceptor ( grey ) neurons . ( F ) The graph bar represents the percentage of α - synuclein puncta co - localized with lysosomes in donor ( white ) and acceptor ( black ) neurons . Data show mean ± s . e . m . Graphs in C , D , E and F correspond to data from 3 independent experiments analysed after 72 hours of co - culture ( * * * * , p < 0 . 0001 compared to donor by two - tailed Mann Whitney test ) .

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

Figure 6(A) Representative images of control (upper panel) and α-synuclein loaded neurons (middle, 0.5µM, and bottom, 1µM, panels) after 16 h. In red: α-synuclein fibrils, in green: MAP-2 and in blue: nuclei. Scale bar represents 10 μm.(B) Representative images of donor (upper panels) and acceptor neurons (bottom panels) after 72 hours in co-culture. Donor neurons were loaded with α-synuclein fibrils prior to co-culture with CTG-labelled acceptor neurons and then cells were labelled either with MAP-2 (first and third panels) or Lamp1 (second and fourth panels). In red: α-synuclein fibrils, in green: CTG, in white: MAP-2/ Lamp1 and in blue: nuclei. Scale bar represents 5 μm.(C) The graph bar shows the percentage of donor (white bar) and acceptor (black bar) neurons containing α-synuclein puncta. Data show mean ± s.e.m.(D) The box plot depicts the number of puncta in donor (white) and acceptor (grey) neurons.(E) Box plot showing the distribution of the average size of α-synuclein puncta in donor (white) and acceptor (grey) neurons.(F) The graph bar represents the percentage of α-synuclein puncta co-localized with lysosomes in donor (white) and acceptor (black) neurons. Data show mean ± s.e.m. Graphs in C, D, E and F correspond to data from 3 independent experiments analysed after 72 hours of co-culture (****, p < 0.0001 compared to donor by two-tailed Mann Whitney test).


Figure 7 ( B ) Representative images of acceptor neurons that were either ( i ) co - cultured with donor neurons ( upper panel , Co - culture ) , ( ii ) physically separated from donor cells ( middle panel , No contact ) or ( iii ) cultured with the conditioned medium of donor neurons ( bottom panel , CM ) . In red : α - synucleinfibrils , in white : acceptor neurons and in blue : nuclei . Scale bar represents 10 μm . ( C ) The graph bar shows the percentage of acceptor cells containing α - synuclein puncta from images such as those presented in B . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Student ' s t - test . Data show mean ± s . e . m from 3 independent experiments . ( D ) The box plot shows the number of α - synuclein puncta in acceptor neurons in co - culture ( white ) , in CM ( grey ) and in the No contact condition ( light grey ) . * * * * , p < 0 . 001 compared to the co - culture condition by two - tailed Mann Whitney test ( n = 3 independent experiments ) . . ( E ) Western blot analysis of α - synuclein indicates the presence of some α - synuclein in the CM of neurons loaded with the fibrils , after 72h compared to control medium and to lysate of cells directly exposed to the fibrils . α - tubulin was used as loading control .

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

Figure 7(B) Representative images of acceptor neurons that were either (i) co-cultured with donor neurons (upper panel, Co-culture), (ii) physically separated from donor cells (middle panel, No contact) or (iii) cultured with the conditioned medium of donor neurons (bottom panel, CM). In red: α-synucleinfibrils, in white: acceptor neurons and in blue: nuclei. Scale bar represents 10 μm.(C) The graph bar shows the percentage of acceptor cells containing α-synuclein puncta from images such as those presented in B. ****, p < 0.001 compared to the co-culture condition by two-tailed Student's t-test. Data show mean ± s.e.m from 3 independent experiments.(D) The box plot shows the number of α-synuclein puncta in acceptor neurons in co-culture (white), in CM (grey) and in the No contact condition (light grey). ****, p < 0.001 compared to the co-culture condition by two-tailed Mann Whitney test (n=3 independent experiments)..(E) Western blot analysis of α-synuclein indicates the presence of some α-synuclein in the CM of neurons loaded with the fibrils, after 72h compared to control medium and to lysate of cells directly exposed to the fibrils. α-tubulin was used as loading control.


Figure 8 ( A ) Representative images of donor ( upper panel ) and acceptor cells ( bottom panel ) after 24 hours in co - culture . Donor neurons were loaded with α - synucleinfibrils prior to co - culture with H2B - GFP - acceptor CAD cells . In red : α - synuclein puncta , in green : acceptor CADs , in white donor neurons and in blue : nuclei . The images of donor cells are representative Z - stack projections of the lower slices where these cells are located , and the images corresponding to acceptor CADs are Z - stack projections of the upper slices , covering the whole cell body . Scale bar represents 10 μm . ( B ) Box plots showing the number ( left side ) and average size ( right side ) of α - synuclein puncta in acceptor CAD cells after 24 hours in co - culture with donor neurons . ( n = 3 independent experiments ) . ( C ) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co - culture . In green : MAP - 2 , in white : WGA , in red : α - synuclein puncta and in blue nuclei . The merge image in the bottom panel shows in detail the TNT connecting the cells , and the insets and the 3D reconstruction of the neuron ( green ) and the CAD cell ( white ) show the presence of α - synuclein puncta inside the TNT .

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 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

Figure 8(A) Representative images of donor (upper panel) and acceptor cells (bottom panel) after 24 hours in co-culture. Donor neurons were loaded with α-synucleinfibrils prior to co-culture with H2B-GFP-acceptor CAD cells. In red: α-synuclein puncta, in green: acceptor CADs, in white donor neurons and in blue: nuclei. The images of donor cells are representative Z-stack projections of the lower slices where these cells are located, and the images corresponding to acceptor CADs are Z-stack projections of the upper slices, covering the whole cell body. Scale bar represents 10 μm.(B) Box plots showing the number (left side) and average size (right side) of α-synuclein puncta in acceptor CAD cells after 24 hours in co-culture with donor neurons. (n=3 independent experiments).(C) Representative images showing a TNT detected between CAD cells and neurons after 24 hours in co-culture. In green: MAP-2, in white: WGA, in red: α-synuclein puncta and in blue nuclei. The merge image in the bottom panel shows in detail the TNT connecting the cells, and the insets and the 3D reconstruction of the neuron (green) and the CAD cell (white) show the presence of α-synuclein puncta inside the TNT.


Figure 2 ( C ) Asynchronous exponentially growing cells were treated with 0 . 2M HU ( + HU ) , then HU was removed from the medium ( - HU ) . SDS - PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift ( Rad53 - P = * * ) . Red and green asterisk indicate the time where mec1 - S1991 phosphomutants show altered Rad53 recovery . A 5 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - the indicated dose of HU .

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Figure 2(C) Asynchronous exponentially growing cells were treated with 0.2M HU (+HU), then HU was removed from the medium (-HU). SDS-PAGE of total protein extracts taken at the indicated times were used to detect the kinetic of Rad53 phosphorylation upshift (Rad53-P=**). Red and green asterisk indicate the time where mec1-S1991 phosphomutants show altered Rad53 recovery. A 5-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- the indicated dose of HU.


Figure 3Analysis of replication fork progression at the single - molecule level by DNA combing . ( C ) Representative images of DNA fibers . Green : ssDNA , red : BrdU . Scale bar corresponds to 20 kb . ( D ) Graph depict the distribution of BrdU track length . WT 90 min ( n = 427 ) and 180 min ( n = 483 ) , mec1 - S1991A 90 min ( n = 309 ) and 180 min ( n = 477 ) .

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Figure 3Analysis of replication fork progression at the single-molecule level by DNA combing. (C) Representative images of DNA fibers. Green: ssDNA, red: BrdU. Scale bar corresponds to 20 kb. (D) Graph depict the distribution of BrdU track length. WT 90 min (n=427) and 180 min (n=483), mec1-S1991A 90 min (n=309) and 180 min (n=477).


Figure 4 ( A ) tRNA level measured by RT - qPCR in asynchronous ( async . ) culture and after 90 min on HU in the indicated strains . Expression is normalized to ACT1 . The repression is expressed as a ratio of HU treated / asynchronous cells in percent .

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Figure 4(A) tRNA level measured by RT-qPCR in asynchronous (async.) culture and after 90 min on HU in the indicated strains. Expression is normalized to ACT1. The repression is expressed as a ratio of HU treated/asynchronous cells in percent.


Figure 5 ( A - D ) Exponentially growing cells were treated with 0 . 2M HU and total protein extracts were collected at the indicated time points ( in min ) , samples were subjected to SDS - PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies . ( B , D ) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin . ( E , F ) Drop assay on HU showing a 10 - fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC + / - 200mM HU . ( F ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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Figure 5(A-D) Exponentially growing cells were treated with 0.2M HU and total protein extracts were collected at the indicated time points (in min), samples were subjected to SDS-PAGE followed by immunoblotting with Rpb1 and Tubulin antibodies. (B,D) Quantitation of total Rpb1 over time was done by normalizing Rpb1 levels to tubulin.(E,F) Drop assay on HU showing a 10-fold dilution series of cells from exponential SC cultures of the indicated strains that were spotted on SC +/- 200mM HU. (F) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


Figure 6 ( A ) Preparations of chromatin - bound proteins were prepared in triplicate and subjected to SDS - PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3 - TAP Quantification of chromatin - bound Rpb1 - S2P and Rpb1 - S5P is shown on the right . Mcm2 is used as a loading control . ( F , G ) A 10 - fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC + / - 200mM of HU . A high level of HU was used to be able to demonstrate robust suppression of the mec1 - S1991A phenotype . ( G ) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution .

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Figure 6(A) Preparations of chromatin-bound proteins were prepared in triplicate and subjected to SDS-PAGE and immunoblotting with the indicated antibodies in a strain that either does or does not express Rpb3-TAP Quantification of chromatin-bound Rpb1-S2P and Rpb1-S5P is shown on the right. Mcm2 is used as a loading control.(F,G) A 10-fold dilution series of cells from exponential SC cultures of the indicated strains were spotted on SC +/- 200mM of HU. A high level of HU was used to be able to demonstrate robust suppression of the mec1-S1991A phenotype. (G) Histogram presents quantification of 2 independent HU sensitivity assays with mean and individual data point values indicated for each yeast dilution.


Figure 7 ( B ) Amongst the Mec1 - S1991 - dependent phosphopeptides on HU , factors involved in transcription are highlighted . ( E ) Position relative to the nuclear envelope ( Zone 1 of the lacO - tagged GAL1 - GAL10 locus in the indicated strains grown either 12h on glucose , or 90 min galactose or galactose + 0 . 2M HU . The third panel shows the relative retention at the nuclear periphery ( Zone 1 ) on galactose + HU . The number of cells is > 300 for each condition .

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Figure 7(B) Amongst the Mec1-S1991-dependent phosphopeptides on HU, factors involved in transcription are highlighted.(E) Position relative to the nuclear envelope (Zone 1 of the lacO-tagged GAL1-GAL10 locus in the indicated strains grown either 12h on glucose, or 90 min galactose or galactose + 0.2M HU. The third panel shows the relative retention at the nuclear periphery (Zone 1) on galactose + HU. The number of cells is >300 for each condition.


Figure 8Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α - factor and released into S phase at 25 ° C . Actin was used as a loading control .

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Figure 8Total Rpb1 levels of exponentially growing cells that were synchronized in G1 with α-factor and released into S phase at 25°C. Actin was used as a loading control.


Figure 2A stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel A were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting A ) representative of 3 independent experimentsA stable inducible HEK - 293T cell line expressing myc - PP2AC ( panel B ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2AC by quantitative mass spectrometry ( B ) . B ) analysis of a biological triplicate . a stable inducible HeLa cell line expressing myc - PP2R1A ( C ) were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and myc - PP2R1A affinity purified , respectively . The binding of the indicated proteins was analysed by Western blotting C ) representative of 2 technical repeatsD ) HEK - 293T cells were treated with DMSO , 20 μM DT - 061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes . Bound complexes were analysed by mass spectrometry to identify PP2A components . Analysis conducted on a biological triplicate . E ) Doxycyclin - inducible CRISPR - Cas9 HeLa cell lines allow for depletion of specific B56 subunits . Growth assays in the presence of the indicated concentrations of DT - 061 or iHAP1 measured after 12 days of treatment . Mean and SD of three independent experiments are shown .

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Figure 2A stable inducible HEK-293T cell line expressing myc-PP2AC (panel A were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting A) representative of 3 independent experimentsA stable inducible HEK-293T cell line expressing myc-PP2AC (panel B) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2AC by quantitative mass spectrometry (B). B) analysis of a biological triplicate.a stable inducible HeLa cell line expressing myc- PP2R1A (C) were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and myc-PP2R1A affinity purified, respectively. The binding of the indicated proteins was analysed by Western blotting C) representative of 2 technical repeatsD) HEK-293T cells were treated with DMSO, 20 μM DT-061 or 2 μM iHAP1 for 2 hours and protein lysates were incubated with a microcystin affinity column to capture PPP complexes. Bound complexes were analysed by mass spectrometry to identify PP2A components. Analysis conducted on a biological triplicate.E) Doxycyclin-inducible CRISPR-Cas9 HeLa cell lines allow for depletion of specific B56 subunits. Growth assays in the presence of the indicated concentrations of DT-061 or iHAP1 measured after 12 days of treatment. Mean and SD of three independent experiments are shown.


Figure 3A ) Schematic of the experimental setup and results of CRISPR screen . Genes were ranked based on their normZ score . The top hits which were further analysed are highlighted . B ) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay . Cells were treated with DMSO ( Ctr ) or DT - 061 5 . 5 µM or iHAP1 0 . 5 µM . C ) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5 . 5 μM DT - 061 or 0 . 5 μM iHAP1 treatment . Data information : B - C ) Mean and SD of three independent experiments is shown .

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Figure 3A) Schematic of the experimental setup and results of CRISPR screen. Genes were ranked based on their normZ score. The top hits which were further analysed are highlighted.B) Validation of CRISPR screen results using RNAi depletion followed by SRB growth assay. Cells were treated with DMSO (Ctr) or DT-061 5.5 µM or iHAP1 0.5 µM. C) Incucyte growth assay after RNAi depletion of indicated proteins alone or in combination with DMSO or 5.5 μM DT-061 or 0.5 μM iHAP1 treatment. Data information: B-C) Mean and SD of three independent experiments is shown.


Figure 4A ) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics . Taxol and Nocodazole served as controls for the experimental setup . The assay was performed in the presence of the indicated drug concentrations . Mean and SEM are shown from three to six independent experiments . B ) Representative colour - coded temporal projection of U2OS EB1 - GFP cells following indicated treatment . Scale bar 10µm . C ) Quantification of microtubule dynamics acquired from manual tracking of EB1 - GFP comets as indicators for MT growth after drug treatment . The mean and SD are plotted from three independent experiments ( n = 10 cells per experiment ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 . D ) Representative temporal projection of mitotic spindles of U2OS EB1 - GFP cells following specified treatment . DNA counterstained with SiR - DNA shown in cyan in merged images . Scale bar 10µm . E ) Analysis of number of EB1 - GFP comets per mitotic spindle . The mean and SD are plotted from three independent experiments ( Total number of cells = 26 ( DMSO ) , 20 ( 30µM DT - 061 ) , 27 ( DMSO + APC / C in . ) and , 26 ( 2µM iHAP1 + APC / C in ) . Data information : P - values were calculated using Student ' s t - test or Mann - Whitney U - test ( unpaired , two - tailed ) . ns , not significant , * * * * p < 0 . 0001 .

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Figure 4A) In vitro tubulin polymerization assay showing concentration dependent effect of iHAP1 on polymerization dynamics. Taxol and Nocodazole served as controls for the experimental setup. The assay was performed in the presence of the indicated drug concentrations. Mean and SEM are shown from three to six independent experiments.B) Representative colour-coded temporal projection of U2OS EB1-GFP cells following indicated treatment. Scale bar 10µm.C) Quantification of microtubule dynamics acquired from manual tracking of EB1-GFP comets as indicators for MT growth after drug treatment. The mean and SD are plotted from three independent experiments (n = 10 cells per experiment). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.D) Representative temporal projection of mitotic spindles of U2OS EB1-GFP cells following specified treatment. DNA counterstained with SiR-DNA shown in cyan in merged images. Scale bar 10µm.E) Analysis of number of EB1-GFP comets per mitotic spindle. The mean and SD are plotted from three independent experiments (Total number of cells = 26 (DMSO), 20 (30µM DT-061), 27 (DMSO+APC/C in.) and, 26 (2µM iHAP1+APC/C in). Data information: P-values were calculated using Student's t-test or Mann-Whitney U-test (unpaired, two-tailed). ns, not significant, ****p<0.0001.


Figure 5A ) Measurement of time spent in mitosis by time - lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis . n indicates the number of cells analyzed from at least 2 independent experiments . Schematic of experimental protocol on the top . Cells were treated with iHAP1 when indicated at the concentration stated in the figure . B ) Representative stills from time - lapse imaging experiments depicting the two phenotypes . Scale bar represents 20 µm .

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Figure 5A) Measurement of time spent in mitosis by time-lapse imaging taking into consideration whether cells exited mitosis or underwent cell death during mitosis. n indicates the number of cells analyzed from at least 2 independent experiments. Schematic of experimental protocol on the top. Cells were treated with iHAP1 when indicated at the concentration stated in the figure. B) Representative stills from time-lapse imaging experiments depicting the two phenotypes. Scale bar represents 20 µm.


Figure 6A ) Time - lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus ( green ) ( n = 87 of cells from 2 experiments analysed all showing dispersal of marker ) . At time 0 , DT - 061 was added . On the right - hand side , the co - localisation of DT - 061 ( red ) and the Golgi marker is shown . B ) As in A ) but with a marker for ER and either with the addition of DT - 061 alone or DT - 061 + okadaic acid ( 50 nM ) Data information : Experiments were repeated at least twice and stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 20 µm . C ) Time required for ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top and median indicated by red line . Data information : Experiments were repeated at least twiceD ) Percentage of cells with ER signal translocation into the nucleus after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

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Figure 6A) Time-lapse imaging of HeLa cells expressing a marker staining the Golgi apparatus (green) (n= 87 of cells from 2 experiments analysed all showing dispersal of marker). At time 0, DT-061 was added. On the right-hand side, the co-localisation of DT-061 (red) and the Golgi marker is shown. B) As in A) but with a marker for ER and either with the addition of DT-061 alone or DT-061 + okadaic acid (50 nM) Data information: Experiments were repeated at least twice and stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.C) Time required for ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top and median indicated by red line. Data information: Experiments were repeated at least twiceD) Percentage of cells with ER signal translocation into the nucleus after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


Figure 7A - B ) Time - lapse imaging of H358 cells expressing a marker for the Golgi ( A ) or the ER ( B ) . Cells were treated with DT - 061 at time 0 . ( B , low panel ) . For A ) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker . Arrow indicates addition of compound . Data information : Experiments were repeated at least twice except filming of the Golgi marker which was done once . Stills show a representative cell for each treatment condition . Cells were treated with DT - 061 at the concentration stated in the figure . A - B ) Scale bar indicates 20 µm . C ) Time needed for ER translocation into the nucleus after drug treatment . Median ( red line ) : 160 min . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least twiceD ) Percentage of cells with ER nuclear translocation after drug treatment . Number of cells analysed per condition shown on top . Data information : Experiments were repeated at least

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Figure 7A-B) Time-lapse imaging of H358 cells expressing a marker for the Golgi (A) or the ER (B). Cells were treated with DT-061 at time 0. (B, low panel). For A) 177 cells were analysed from one experiment and all displayed dispersal of the Golgi marker. Arrow indicates addition of compound. Data information: Experiments were repeated at least twice except filming of the Golgi marker which was done once. Stills show a representative cell for each treatment condition. Cells were treated with DT-061 at the concentration stated in the figure. A-B) Scale bar indicates 20 µm.C) Time needed for ER translocation into the nucleus after drug treatment. Median (red line): 160 min. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twiceD) Percentage of cells with ER nuclear translocation after drug treatment. Number of cells analysed per condition shown on top. Data information: Experiments were repeated at least twice


Figure 8B ) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine . MCF7 cells were co - treated with 15 μM DT - 061 , 1 . 5 μM iHAP1 , 3 µg / mL Brefeldin A , or vehicle ( DMSO ) . The determined molar quantities of sphingolipid classes were normalized to that of PC . The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t - test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Cer : ceramide , HexCer : hexosylceramide , PC : phosphatidylcholine , and SM : sphingomyelin .

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Figure 8B) Increase in the molar quantity of sphingolipid classes assessed in MCF7 cells after feeding with the precursor sphingosine. MCF7 cells were co-treated with 15 μM DT-061, 1.5 μM iHAP1, 3 µg/mL Brefeldin A, or vehicle (DMSO). The determined molar quantities of sphingolipid classes were normalized to that of PC. The presented values represent averages of five independent experiments and statistical analysis was performed using an independent t-test. *p<0.05, **p<0.01, ***p<0.001. Cer: ceramide, HexCer: hexosylceramide, PC: phosphatidylcholine, and SM: sphingomyelin.


Figure 1 ( A ) Bone Marrow Derived Macrophages ( BMDMs ) were treated with either the AMPK activator 991 , Dex or a combination for 1 h before analysis by western blot . ( B , C ) Phospho - AMPK ( B ) and phospho - ACC ( C ) were quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 6 ( B , C ) , biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using student ' s t - test ( B , C ) ( D ) WT and AMPKα1 - / - BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 ( D ) biological replicates * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests ( D , ( E ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and analyzed by RNA - seq . Differentially expressed genes between any condition were clustered using c - means clustering . ( F ) WT and AMPKα1 - / - BMDMs were treated with Dex for 24 h and anti - / pro - inflammatory markers assessed using immunofluorescence . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 3 - 4 ( F ) biological replicates ( G WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . Proliferation ( Ki67 immunostaining ) ( G ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates ( G * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests Bar = 25 µm ( GH ) WT and AMPKα1 - / - BMDMs were treated with Dex for 72 h , washed and conditioned medium was recovered after 24 h used to treat muscle stem cells . differentiation ( desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more ) ( H ) were assessed in muscle stem cells . Data information : results are means ± SEM or z - score or displayed with boxes and whiskers in which the central band represents the median . N = 4 biological replicates H ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 statistical analysis using ANOVA tests , H ) . Bar = 25 µm H ) .

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Figure 1(A) Bone Marrow Derived Macrophages (BMDMs) were treated with either the AMPK activator 991, Dex or a combination for 1 h before analysis by western blot. (B, C) Phospho-AMPK (B) and phospho-ACC (C) were quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 6 (B,C), biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using student's t-test (B, C)(D) WT and AMPKα1-/- BMDMs were treated with Dex for 1 h before analysis by western blot and GR phosphorylation at S211 was quantified. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3 (D) biological replicates * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests (D,(E) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and analyzed by RNA-seq. Differentially expressed genes between any condition were clustered using c-means clustering.(F) WT and AMPKα1-/- BMDMs were treated with Dex for 24 h and anti-/pro-inflammatory markers assessed using immunofluorescence. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 3-4 (F) biological replicates(G WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. Proliferation (Ki67 immunostaining) (G) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates (G * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests Bar=25 µm (GH) WT and AMPKα1-/- BMDMs were treated with Dex for 72 h, washed and conditioned medium was recovered after 24 h used to treat muscle stem cells. differentiation (desmin immunostaining and counting the percentage of muscle cells with 2 nuclei or more) (H) were assessed in muscle stem cells. Data information: results are means ± SEM or z-score or displayed with boxes and whiskers in which the central band represents the median. N= 4 biological replicates H). * p<0.05, ** p<0.01, *** p<0.001 statistical analysis using ANOVA tests ,H). Bar=25 µm H).


Figure 2After cardiotoxin injection to damage the muscle , AMPKα1fl / fl ( WT ) and LysM - α1 - / - mice were treated with a single dose of Dexamethasone ( Dex ) intra - peritoneal ( i . p . ) ( 0 . 1 mg / kg ) at day 3 ( D3 ) and Tibialis Anterior ( TA ) muscles were harvested at day 8 ( D8 ) and 14 ( D14 ) after injury . ( A ) Representative embryonic myosin heavy chain ( EmbMHC ) immunostaining on whole TA muscle section at D8 and D14 after injury . ( B ) Quantification of ( A ) , results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section . Data information : results are displayed with box and whiskers in which the central band represents the median ( B N = 7 - 12 muscles ( B * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 500 µm ( A ) , ( F ) Macrophage polarization was determined by immunofluorescence in vehicle , DAMPs and DAMPs + Dex treated WT and AMPKα1 - / - BMDMs . Data information : results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 4 - 5 biological replicates ( F ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 using ANOVA tests . Bars = 25 µm ( F ) .

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Figure 2After cardiotoxin injection to damage the muscle, AMPKα1fl/fl (WT) and LysM-α1-/- mice were treated with a single dose of Dexamethasone (Dex) intra-peritoneal (i.p.) (0.1 mg/kg) at day 3 (D3) and Tibialis Anterior (TA) muscles were harvested at day 8 (D8) and 14 (D14) after injury. (A) Representative embryonic myosin heavy chain (EmbMHC) immunostaining on whole TA muscle section at D8 and D14 after injury. (B) Quantification of (A), results are expressed as percentage of positive myofibers expressing the EmbMHC on the whole muscle section. Data information: results are displayed with box and whiskers in which the central band represents the median (B N= 7-12 muscles (B * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 500 µm (A),(F) Macrophage polarization was determined by immunofluorescence in vehicle, DAMPs and DAMPs+Dex treated WT and AMPKα1-/- BMDMs. Data information: results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 4-5 biological replicates (F). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 using ANOVA tests. Bars = 25 µm (F).


Figure 3 ( F ) Macrophage polarization was determined by immunofluorescence in vehicle , LPS and LPS + Dex treated WT and AMPKα1 - / - BMDMs , data are expressed as percentage positive cells . Data information : Results are displayed with box and whiskers in which the central band represents the means ± SEM ( F ) . N = 3 ( F ) biological replicates . Statistical analysis by ANOVA F ) * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Bar = 25 µm ( F ) .

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Figure 3(F) Macrophage polarization was determined by immunofluorescence in vehicle, LPS and LPS+Dex treated WT and AMPKα1-/- BMDMs, data are expressed as percentage positive cells. Data information: Results are displayed with box and whiskers in which the central band represents the means ± SEM (F). N= 3 (F) biological replicates. Statistical analysis by ANOVA F) * p<0.05, ** p<0.01, *** p<0.001. Bar = 25 µm (F).


Figure 4 ( F ) WT and AMPKα1 - / - Bone Marrow Derived Macrophages ( BMDMs ) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated . Westen - blot for FOXO3 , AMPKα1 , actin and the phosphorylated form of FOXO3 were done . Quantification of the band intensity is provided normalized to those of actin and total FOXO3 , a representative example is shown . Data information : results are box and whiskers in which the central band represents the median F , N = 4 - 5 ( F biological replicates . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 * * * * p < 0 . 0001 using ANOVA and Kruskal - Wallis

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Figure 4(F) WT and AMPKα1-/- Bone Marrow Derived Macrophages (BMDMs) were treated with either Dex or the AMPK activator 991 and protein phosphorylated on serine were immunoprecipitated. Westen-blot for FOXO3, AMPKα1, actin and the phosphorylated form of FOXO3 were done. Quantification of the band intensity is provided normalized to those of actin and total FOXO3, a representative example is shown. Data information: results are box and whiskers in which the central band represents the median F, N= 4-5 (F biological replicates. * p<0.05, ** p<0.01, *** p<0.001 **** p<0.0001 using ANOVA and Kruskal-Wallis tests


Figure 1 , C . siRNA - mediated knockdown of EXOSC10 ( EXO10 ) , but not of DIS3 and DIS3L , significantly increased generation of poly - GA in HeLa cells expressing ( G4C2 ) 80 repeats . ctrl " indicates a control vector which lacks the G4C2 repeat but still contains the 5 ' flanking region and 3xTAG . . Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT - qPCR . E , F . Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing ( G4C2 ) 80 repeats visualized by in situ hybridization targeting sense RNA foci . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments . G . Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing ( G4C2 ) 80 repeats . Each dot represents average cellular RNA foci intensity ( Arbitrary unit ) of RNA foci - positive cells in each randomly taken image . In total 25 ( Ct ) and 24 ( EXO10 # 5 ) images were analyzed in 3 independent experiments .

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Figure 1, C. siRNA-mediated knockdown of EXOSC10 (EXO10), but not of DIS3 and DIS3L, significantly increased generation of poly-GA in HeLa cells expressing (G4C2) 80 repeats. ctrl" indicates a control vector which lacks the G4C2 repeat but still contains the 5' flanking region and 3xTAG.. Increased G4C2 repeat RNA expression upon EXOSC10 knockdown on RT-qPCR.E, F. Knockdown of EXOSC10 increased RNA foci in HeLa cells expressing (G4C2) 80 repeats visualized by in situ hybridization targeting sense RNA foci. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments. G. Knockdown of EXOSC10 increased RNA foci intensity in HeLa cells expressing (G4C2) 80 repeats. Each dot represents average cellular RNA foci intensity (Arbitrary unit) of RNA foci-positive cells in each randomly taken image. In total 25 (Ct) and 24 (EXO10#5) images were analyzed in 3 independent experiments.


Figure 2A , B . Dose - dependent suppression of poly - GA by exogenous EXOSC10 upon EXOSC10 knockdown . C . Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown . Concentration of transfected EXOSC10 plasmid was 0 . 2 ng / μl in 1 ml medium . D . Overexpression of EXOSC10 decreases G4C2 repeat RNA .

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Figure 2A, B. Dose-dependent suppression of poly-GA by exogenous EXOSC10 upon EXOSC10 knockdown.C. Suppression of G4C2 repeat RNA by exogenous EXOSC10 on EXOSC10 knockdown. Concentration of transfected EXOSC10 plasmid was 0.2 ng/μl in 1 ml medium.D. Overexpression of EXOSC10 decreases G4C2 repeat RNA.


Figure 3A , B . Under transcription inhibition with Actinomycin D , cells with EXOSC10 knockdown ( EXO10 KD ) showed more stable expression of the repeat RNA compared to cells with control knockdown with non - targeting siRNA ( Ct KD ) . C , D . Degradation of FAM - labeled synthetic ( G4C2 ) 8 repeat RNA with recombinant human EXOSC10 ( hEXO10 ) ( Upper left ) . 3 ' blocked FAM - labeled G4C2 repeat RNA resisting 3 ' exoribonuclease activity is not degraded by recombinant EXOSC10 ( Lower left ) . Lack of recombinant EXOSC10 ( Upper right ) or Mg2 + ion which is essential co - factor of EXOSC10 ( Lower right ) abolished the RNA degradation activity . 3 or 4 independent experiments . Quantification of the top bands are shown in ( D ) . E , F . Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 ( Left ) . Without recombinant EXOSC10 , no substrate degradation was observed ( Right ) . 5 or 6 independent experiments . Quantification is shown in ( F ) .

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Figure 3A, B. Under transcription inhibition with Actinomycin D, cells with EXOSC10 knockdown (EXO10 KD) showed more stable expression of the repeat RNA compared to cells with control knockdown with non-targeting siRNA (Ct KD).C, D. Degradation of FAM-labeled synthetic (G4C2)8 repeat RNA with recombinant human EXOSC10 (hEXO10) (Upper left). 3' blocked FAM-labeled G4C2 repeat RNA resisting 3' exoribonuclease activity is not degraded by recombinant EXOSC10 (Lower left). Lack of recombinant EXOSC10 (Upper right) or Mg2+ ion which is essential co-factor of EXOSC10 (Lower right) abolished the RNA degradation activity. 3 or 4 independent experiments. Quantification of the top bands are shown in (D).E, F. Degradation of antisense C4G2 repeat RNA substrate with recombinant human EXOSC10 (Left). Without recombinant EXOSC10, no substrate degradation was observed (Right). 5 or 6 independent experiments. Quantification is shown in (F).


Figure 4A . In situ hybridization . G4C2 RNA foci were detected in a C9 patient - derived fibroblast but not in a control - derived fibroblast with non - targeting siRNA treatment ( left panels , control / si Ct . middle panels , C9 / si Ct ) . Knockdown of EXSOC10 increased RNA foci ( right panels , C9 / siEXOSC10 # 5 ) . Nuclei were stained with DAPI ( blue ) . White arrows indicate nuclear RNA foci . The green arrows show cytoplasmic RNA foci . B . Quantification of % cell with G4C2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . C . Quantification of the number of G4C2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . D . Quantification of % cell with C4G2 RNA foci . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . E . Quantification of the number of C4G2 RNA foci / cell . 3 independent knockdown experiments were performed . 10 DAPI positive cells per experiment were randomly selected and z - stack images were obtained . In total , z - stack images from 90 cells per group ( control siRNA or EXOSC10 siRNA ) were counted for RNA foci . F , G . Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT - qPCR . 10 independent knockdown experiments ( Case1 N = 4 , Case2 N = 4 , Case3 N = 2 ) were performed .

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Figure 4A. In situ hybridization. G4C2 RNA foci were detected in a C9 patient-derived fibroblast but not in a control-derived fibroblast with non-targeting siRNA treatment (left panels, control/si Ct. middle panels, C9/si Ct). Knockdown of EXSOC10 increased RNA foci (right panels, C9/siEXOSC10#5). Nuclei were stained with DAPI (blue). White arrows indicate nuclear RNA foci. The green arrows show cytoplasmic RNA foci.B. Quantification of %cell with G4C2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. C. Quantification of the number of G4C2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. D. Quantification of %cell with C4G2 RNA foci. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. E. Quantification of the number of C4G2 RNA foci/cell. 3 independent knockdown experiments were performed. 10 DAPI positive cells per experiment were randomly selected and z-stack images were obtained. In total, z-stack images from 90 cells per group (control siRNA or EXOSC10 siRNA) were counted for RNA foci. F, G. Increased endogenous repeat RNA expression upon EXOSC10 knockdown in strand specific RT-qPCR. 10 independent knockdown experiments (Case1 N=4, Case2 N=4, Case3 N=2) were performed.


Figure 5A , B , C . Double knockdown of EXOSC10 and DIS3 significantly increased poly - GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing ( G4C2 ) 80 repeats . D . Overexpression of DIS3 compensated EXOSC10 knockdown - induced repeat RNA accumulation . E , F . Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown - induced GA increase .

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Figure 5A, B, C. Double knockdown of EXOSC10 and DIS3 significantly increased poly-GA and repeat RNA expression compared with single knockdown of EXOSC10 in cells expressing (G4C2) 80 repeats.D. Overexpression of DIS3 compensated EXOSC10 knockdown-induced repeat RNA accumulation.E, F. Catalytically dead mutant EXOSC10 rescued EXOSC10 knockdown-induced GA increase.


Figure 6C - F . HA - tagged poly - GA , poly - GP , poly - GR or poly - PR ( red ) and EXOSC10 ( green ) were immunostained with anti - HA and anti - EXOSC10 antibodies . Nuclei were stained with DAPI ( blue ) . G . EXOSC10 ( green ) were immunostained with anti - EXOSC10 antibody in cells transfected with control mock plasmid . Nuclei were stained with DAPI ( blue ) . H , I . Quantification of the frequency of cells with mislocalized EXOSC10 . Only DPR - positive cells were quantified ( except mock transfected cells expressing no DPR . ( Total cell counts : ( figure H ) mock 300 cells , GA 112 cells , GP 69 cells , GR 53 cells , ( figure I ) mock 445 cells , PR 159 cells ) .

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Figure 6C-F. HA-tagged poly-GA, poly-GP, poly-GR or poly-PR (red) and EXOSC10 (green) were immunostained with anti-HA and anti-EXOSC10 antibodies. Nuclei were stained with DAPI (blue).G. EXOSC10 (green) were immunostained with anti-EXOSC10 antibody in cells transfected with control mock plasmid. Nuclei were stained with DAPI (blue).H, I. Quantification of the frequency of cells with mislocalized EXOSC10. Only DPR-positive cells were quantified (except mock transfected cells expressing no DPR. (Total cell counts: (figure H) mock 300 cells, GA 112 cells, GP 69 cells, GR 53 cells, (figure I) mock 445 cells, PR 159 cells).


Figure 7B . Immunoblot validation of successful fractionation using antibodies against HDAC1 ( nuclear marker ) and GAPDH ( cytoplasmic marker ) . C . Co - immunoprecipitation of GFP - DPR interacting proteins in cytoplasmic fraction using GFP - trap beads . Immunoprecipitants were served for immunoblot with anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . D . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " . E . Co - immunoprecipitation of GFP - DPR interacting proteins in nuclear fraction using GFP - trap beads followed by immunoblot using anti - EXOSC10 antibody . The same membrane was reprobed with anti - GFP antibody . F . Quantification of the relative intensities of " EXOSC10 in co - IP fraction " / " EXOSC10 in input " .

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Figure 7B. Immunoblot validation of successful fractionation using antibodies against HDAC1 (nuclear marker) and GAPDH (cytoplasmic marker).C. Co-immunoprecipitation of GFP-DPR interacting proteins in cytoplasmic fraction using GFP-trap beads. Immunoprecipitants were served for immunoblot with anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. D. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input". E. Co-immunoprecipitation of GFP-DPR interacting proteins in nuclear fraction using GFP-trap beads followed by immunoblot using anti-EXOSC10 antibody. The same membrane was reprobed with anti-GFP antibody. F. Quantification of the relative intensities of "EXOSC10 in co-IP fraction" / "EXOSC10 in input".


Figure 8B , C . Increased expression of 3 ' extended snoRA48 precursor upon EXOSC10 knockdown ( B ) or GFP - poly - GR177 / GFP - poly - PR166 expression ( C ) on RT - qPCR . D , E . Increased expression of 3 ' extended snoRA68 precursor upon EXOSC10 knockdown ( D ) or GFP - poly - GR177 / GFP - poly - PR166 expressions ( E ) on RT - qPCR . F . RT - qPCR analysis showed increased cellular G4C2 repeat RNA expression from co - transfected EF1 ( G4C2 ) 80 repeat construct in the presence of GFP , GFP - poly - GR177 or GFP - poly - PR166 .

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Figure 8B, C. Increased expression of 3'extended snoRA48 precursor upon EXOSC10 knockdown (B) or GFP-poly-GR177/ GFP-poly-PR166 expression (C) on RT-qPCR. D, E. Increased expression of 3'extended snoRA68 precursor upon EXOSC10 knockdown (D) or GFP-poly-GR177 /GFP-poly-PR166 expressions (E) on RT-qPCR.F. RT-qPCR analysis showed increased cellular G4C2 repeat RNA expression from co-transfected EF1 (G4C2)80 repeat construct in the presence of GFP, GFP-poly-GR177 or GFP-poly-PR166.


Figure 1Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc - / - mice ( A ) . Quantification of Gadd45a protein expression , β - actin protein level was used as the loading control ( n = 7 mice per genotype ) , two independent experiments were carried out ( B ) . Quantification of Gadd45a - ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc - / - MEF cells ( n = 3 for each genotype ) . Dot - bot assay following Gadd45a - ChIP in WT and G3Terc - / - MEF cells . Kaplan - Meier survival curves of the mice with indicated genotypes ( WT , n = 44 ; Gadd45a - / - , n = 48 ; G3Terc - / - , n = 37 ; G3 - dKO , n = 41 ) . P value comparing the survival of G3Terc - / - with G3 - dKO mice is calculated with the log - rank test . The average body weight of mice with indicated genotypes at indicated time points ( G3 - dKO Female ( n = 19 ) vs G3Terc - / - Female ( n = 17 ) , P = 0 . 004 ; G3 - dKO Male ( n = 14 ) vs G3Terc - / - Male ( n = 14 ) , P = 0 . 0038 ) . P values are calculated with log - rank test . Whole mount images of colon samples from 7 - month old mice with indicated genotypes ( G ) . Quantification on density of colon crypts ( H ) ( n = 5 mice per group ) . H & E - stained small intestine sections from 7 - month old mice with indicated genotypes ( I ) . Quantification of the number of small intestine basal crypts ( J ) ( n = 5 - 6 mice per group ) .

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Figure 1Western blotting analysis of Gadd45a protein expression in mouse intestine crypt lysates from WT and G3Terc-/- mice (A). Quantification of Gadd45a protein expression, β-actin protein level was used as the loading control (n=7 mice per genotype), two independent experiments were carried out (B).Quantification of Gadd45a-ChIP values as percentage of total telomeric DNA and ALU elements in WT and G3Terc-/- MEF cells (n=3 for each genotype).Dot-bot assay following Gadd45a-ChIP in WT and G3Terc-/- MEF cells.Kaplan-Meier survival curves of the mice with indicated genotypes (WT, n=44; Gadd45a-/-, n=48; G3Terc-/-, n=37; G3-dKO, n=41). P value comparing the survival of G3Terc-/- with G3-dKO mice is calculated with the log-rank test.The average body weight of mice with indicated genotypes at indicated time points (G3-dKO Female (n=19) vs G3Terc-/- Female (n=17), P=0.004; G3-dKO Male (n=14) vs G3Terc-/- Male (n=14), P=0.0038). P values are calculated with log-rank test.Whole mount images of colon samples from 7-month old mice with indicated genotypes (G). Quantification on density of colon crypts (H) (n=5 mice per group).H&E-stained small intestine sections from 7-month old mice with indicated genotypes (I). Quantification of the number of small intestine basal crypts (J) (n=5-6 mice per group).


Figure 2Olfm4 - positive intestinal stem cells ( indicated by arrows ) in small intestine sections ( A ) . Quantification of intestinal stem cell ( Olfm4 + ) number per 100 μm ( n = 4 - 5 mice per group ) of small intestine sections ( B ) . Representative images of small intestine crypt culture ( day 2 ) . Arrows mark the surviving cysts ( C ) . Quantification of numbers of cysts per culture well ( D ) ( n = 4 - 5 mice / genotype ) . In vitro crypt culture ( day 8 ) from small intestine crypts isolated from mice with indicated genotypes . Arrows mark the crypt - like structure ( E ) . The number of organoids per culture wells ( n = 4 - 5 mice per group ) . Two hundred crypts are seeded per well ( F ) . Representative images of TUNEL staining in small intestine sections . Arrows indicate TUNEL positive cells ( G ) . Quantification of TUNEL positive cells per crypt ( n = 5 mice per group ) ( H ) . Representative images of phosphorylated histone 3 ( pH3 ) antibody staining in mouse small intestine . Arrows indicate pH3 positive cells ( I ) . Quantification of pH3 positive crypt cells per 100 μm of small intestines ( n = 4 - 6 mice per group ) ( J ) .

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Figure 2Olfm4-positive intestinal stem cells (indicated by arrows) in small intestine sections (A). Quantification of intestinal stem cell (Olfm4+) number per 100 μm (n=4-5 mice per group) of small intestine sections (B).Representative images of small intestine crypt culture (day 2). Arrows mark the surviving cysts (C). Quantification of numbers of cysts per culture well (D)(n=4-5 mice/genotype).In vitro crypt culture (day 8) from small intestine crypts isolated from mice with indicated genotypes. Arrows mark the crypt-like structure (E). The number of organoids per culture wells (n=4-5 mice per group). Two hundred crypts are seeded per well (F).Representative images of TUNEL staining in small intestine sections. Arrows indicate TUNEL positive cells (G). Quantification of TUNEL positive cells per crypt (n=5 mice per group) (H).Representative images of phosphorylated histone 3 (pH3) antibody staining in mouse small intestine. Arrows indicate pH3 positive cells (I). Quantification of pH3 positive crypt cells per 100 μm of small intestines (n=4-6 mice per group) (J).


Figure 3Representative images of immunostaining for p53 on small intestine sections . Arrows indicate p53 - positive cells ( A ) . Quantification of the percentage of p53 positive cells per crypt ( n = 5 - 6 mice per group ) ( B ) . Representative images of γH2AX antibody staining on small intestine paraffin sections from WT , Gadd45a - / - , G3Terc - / - , and G3 - dKO mice ( 7 months old ) . Arrows indicate γH2AX positive nuclei ( C ) . Quantification of the percentage of γH2AX positive cells per small intestine crypt ( n = 4 - 6 mice per group ) ( D ) . Representative images of small intestine crypt sections stained with antibody against pATM ( Ser - 1981 ) ( E ) . Quantification of frequencies of pATM ( Ser - 1981 ) positive crypt cells ( n = 4 - 6 of 7 - month old mice per genotype ) ( F ) . Representative image of the anaphase bridge ( indicated with arrows ) in the basal crypt of G3Terc - / - mice ( G ) . The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population ( n = 4 - 6 mice per genotype ) ( H ) . Heat map indicating the relative expression levels of DDR and cell cycle - related gene transcripts in MEFs from indicated genotypes ( Data are extracted from RNAseq datasets ) .

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Figure 3Representative images of immunostaining for p53 on small intestine sections. Arrows indicate p53-positive cells (A). Quantification of the percentage of p53 positive cells per crypt (n=5-6 mice per group) (B).Representative images of γH2AX antibody staining on small intestine paraffin sections from WT, Gadd45a-/-, G3Terc-/-, and G3-dKO mice (7 months old). Arrows indicate γH2AX positive nuclei (C). Quantification of the percentage of γH2AX positive cells per small intestine crypt (n=4-6 mice per group) (D).Representative images of small intestine crypt sections stained with antibody against pATM (Ser-1981)(E). Quantification of frequencies of pATM (Ser-1981) positive crypt cells (n=4-6 of 7-month old mice per genotype) (F).Representative image of the anaphase bridge (indicated with arrows) in the basal crypt of G3Terc-/- mice (G). The percentage of cells with anaphase bridges in pH3 positive basal crypt cell population (n=4-6 mice per genotype) (H).Heat map indicating the relative expression levels of DDR and cell cycle-related gene transcripts in MEFs from indicated genotypes (Data are extracted from RNAseq datasets).


Figure 4CpG island methylation status of subtelomeric regions on chromosome 1q . gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing . Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands , respectively , at each position ( A ) . Quantification of subtelomeric CpG island methylation ( B ) . Telomeric quantification and dot - bot assay of tri - methyl H3K9 ( H3K9me3 ) - ChIP in indicated genotypesTelomeric quantification and dot - bot assay of acetylated H3K9 ( H3K9ac ) - ChIP in indicated genotypesTelomeric quantification and dot - bot assay of HP1a - ChIP in indicated

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Figure 4CpG island methylation status of subtelomeric regions on chromosome 1q. gDNAs from MEF with indicated genotypes were PCR amplified and analysed by bisulfite sequencing. Yellow and blue bars denote the frequencies of methylated and unmethylated CpG islands, respectively, at each position (A). Quantification of subtelomeric CpG island methylation (B).Telomeric quantification and dot-bot assay of tri-methyl H3K9 (H3K9me3)-ChIP in indicated genotypesTelomeric quantification and dot-bot assay of acetylated H3K9 (H3K9ac)-ChIP in indicated genotypesTelomeric quantification and dot-bot assay of HP1a-ChIP in indicated genotypes


Figure 5Representative images of small intestine organoids from WT and G3Terc - / - mice incubated with or without APE1 inhibitor ( CRT0044876 , 10μM ) treatment . Arrows indicate the crypt - like structure in organoid cultures ( A ) . Quantification of organoid number ( per well ) and size ( in 104 μm2 ) ( n = 3 - 4 mice per genotype / treatment ) ( B - C ) . Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc - / - organoid cultures with or without APE1 inhibitor ( CRT0044876 , 10μM ) ( DNAs are stained with DAPI ) ( D ) . Quantification of γH2AX positive cells ( n = 3 - 4 mice per group ) ( E ) . Telomeric DNA quantification and dot - blot assay of H3K9me3 - ChIPTelomeric DNA quantification and dot - blot assay of H3K9ac - ChIPTelomeric DNA quantification and dot - blot assay of HP1a - ChIPRepresentative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor ( CRT0044876 , CRT ) . Arrows indicate Olfm4 positive intestine stem cells ( I ) . Quantification of Olfm4 positive intestine stem cells ( n = 4 mice per group ) ( J ) .

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Figure 5Representative images of small intestine organoids from WT and G3Terc-/- mice incubated with or without APE1 inhibitor (CRT0044876, 10μM) treatment. Arrows indicate the crypt-like structure in organoid cultures (A). Quantification of organoid number (per well) and size (in 104 μm2) (n=3-4 mice per genotype/treatment) (B-C).Representative images of γH2AX antibody staining on dissociated organoid cells from WT and G3Terc-/- organoid cultures with or without APE1 inhibitor (CRT0044876, 10μM) (DNAs are stained with DAPI) (D). Quantification of γH2AX positive cells (n=3-4 mice per group) (E).Telomeric DNA quantification and dot-blot assay of H3K9me3-ChIPTelomeric DNA quantification and dot-blot assay of H3K9ac-ChIPTelomeric DNA quantification and dot-blot assay of HP1a-ChIPRepresentative images of Olfm4 staining in small intestine sections from mice treated with or without APE1 inhibitor (CRT0044876, CRT). Arrows indicate Olfm4 positive intestine stem cells (I). Quantification of Olfm4 positive intestine stem cells (n=4 mice per group) (J).


Figure 6The knockdown efficiency of shRNA - GADD45A in human WI38 fibroblasts . The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A . Representative images of β - gal staining in human fibroblast cultures with control and shRNA - GADD45A treatments at passage 54 ( C ) . Quantification of the percentage of β - gal positive cells in human fibroblasts with control and shRNA - GADD45A treatments ( duplicates per group ) ( D ) . Representative images of GADD45A and γH2AX staining in colon sections from young ( 35 - 50 y / o ) and old ( 75 - 85 y / o ) people ( E ) . Quantification of the percentage of GADD45A positive cells in young and old human colon tissues ( n = 8 per age group ) ( F ) . Quantification of the percentage of γH2AX positive cells in young and old human colon tissues ( n = 6 per age group ) ( G ) .

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Figure 6The knockdown efficiency of shRNA-GADD45A in human WI38 fibroblasts.The proliferation curves of human WI38 fibroblasts treated with control shRNA or shRNA against GADD45A.Representative images of β-gal staining in human fibroblast cultures with control and shRNA-GADD45A treatments at passage 54 (C). Quantification of the percentage of β-gal positive cells in human fibroblasts with control and shRNA-GADD45A treatments (duplicates per group) (D).Representative images of GADD45A and γH2AX staining in colon sections from young (35-50 y/o) and old (75-85 y/o) people (E). Quantification of the percentage of GADD45A positive cells in young and old human colon tissues (n=8 per age group) (F). Quantification of the percentage of γH2AX positive cells in young and old human colon tissues (n=6 per age group) (G).


figf1 ( a ) Kinetics of the appearance of P14 cells in spleens of C57BL / 6 mice given adoptive transfer of 1 × 104 P14 cells , monitored after infection of recipients with LCMV Armstrong strain . ( b ) Flow cytometry of cell size at various stages of P14 differentiation , assessed by forward scatter ( FSC ) . ( c ) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation . Data are representative of two independent experiments with samples pooled from 5 - 20 mice for each time point .

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figf1(a) Kinetics of the appearance of P14 cells in spleens of C57BL/6 mice given adoptive transfer of 1 × 104 P14 cells, monitored after infection of recipients with LCMV Armstrong strain.(b) Flow cytometry of cell size at various stages of P14 differentiation, assessed by forward scatter (FSC).(c) Immunoblot analysis of LC3b and p62 at various stages of P14 differentiation. Data are representative of two independent experiments with samples pooled from 5-20 mice for each time point.


figf2 ( b ) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on days 5 , 8 and 30 after infection of recipients with LCMV . Numbers in blue outlined areas indicate percent GFP − cells among transduced ( Thy - 1 . 1 + ) P14 cells . ( c ) Summary of results in b , showing autophagy activity as percent GFP − cells . * P ≤ 0 . 0005 ( unpaired t - test ) . ( d ) Multispectral - imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a , assessed on day 8 after infection , showing GFP signals from GFP + transduced ( CD45 . 1 + Thy - 1 . 1 + ) P14 cells . Original magnification , × 60 . ( e ) Summary of results in d , presented as the frequency of cells with more than one GFP + punctum among vector transduced Thy - 1 . 1 + P14 cells . Each symbol represents an individual mouse ; small horizontal lines indicate the mean ( ± s . e . m . ) . Data are representative of three independent experiments ( b , d ) or three independent experiments with three or more mice per group ( e ; error bars , s . e . m . ) or six mice per group ( c ; error bars , s . e . m . ) .

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figf2(b) Flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on days 5, 8 and 30 after infection of recipients with LCMV. Numbers in blue outlined areas indicate percent GFP− cells among transduced (Thy-1.1+) P14 cells. (c) Summary of results in b, showing autophagy activity as percent GFP− cells. *P ≤ 0.0005 (unpaired t-test).(d) Multispectral-imaging flow cytometry of P14 cells from the spleens of recipient mice given adoptive transfer of P14 cells transduced with retrovirus as in a, assessed on day 8 after infection, showing GFP signals from GFP+ transduced (CD45.1+ Thy-1.1+) P14 cells. Original magnification, ×60. (e) Summary of results in d, presented as the frequency of cells with more than one GFP+ punctum among vector transduced Thy-1.1+ P14 cells. Each symbol represents an individual mouse; small horizontal lines indicate the mean (±s.e.m.). Data are representative of three independent experiments (b,d) or three independent experiments with three or more mice per group (e; error bars, s.e.m.) or six mice per group (c; error bars, s.e.m.).