c , Heat map illustrating differentially expressed genes ( DEGs ) encoding heat shock proteins between WT and Apc - / - organoids .

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

c, Heat map illustrating differentially expressed genes (DEGs) encoding heat shock proteins between WT and Apc-/- organoids.


d , Volcano plot of DEGs between normal mouse intestinal tissue and adenomas ( GSE65461 ) .

d, Volcano plot of DEGs between normal mouse intestinal tissue and adenomas (GSE65461).


e , f , Relative gene expression of Hspb1 ( * * * P = 0 . 0004 , n = 6 ) ( e ) and protein expression of HSP25 ( f ) in unrecombined versus recombined murine organoids .

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

e, f, Relative gene expression of Hspb1 (***P=0.0004, n=6)(e) and protein expression of HSP25 (f) in unrecombined versus recombined murine organoids.


g , Gene expression of Hspb1 in normal ( N ) versus adenoma ( A ) tissue ( * * * P = 0 . 0002 , n = 4 ) .

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

g, Gene expression of Hspb1 in normal (N) versus adenoma (A) tissue (***P=0.0002, n=4).


H , RNA - ISH for Hspb1 in murine intestinal adenoma . Scalebar , 100 μm . Zoom panel , 25

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

H, RNA-ISH for Hspb1 in murine intestinal adenoma. Scalebar, 100 μm. Zoom panel, 25 μm


i , Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue , ( * * * * P < 0 . 0001 , paired t - test , n = 59 pairs ) .

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

i, Relative gene expression of human HSPB1 in paired sets of normal versus adenoma tissue, (****P<0.0001, paired t-test, n=59 pairs).


Relative gene expression of AXIN2 ( * P = 0 . 0129 ) , P21 ( * P = 0 . 0103 ) , and HSPB1 ( * P = 0 . 0236 ) ( b ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline .

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

Relative gene expression of AXIN2 (*P=0.0129), P21(*P=0.0103), and HSPB1(*P=0.0236) (b) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


protein expression of HSP27 ( c ) in Ls174t - dnTCF4 cells the presence or absence of doxycycline .

protein expression of HSP27 (c) in Ls174t-dnTCF4 cells the presence or absence of doxycycline.


Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR - 99021 ( * * P = 0 . 0016 , one way ANOVA , n = 4 ) ( d )

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

Relative gene expression of Hspb1 in murine WT organoids treated with different concentrations of CHIR-99021 (**P=0.0016, one way ANOVA, n=4) (d)


protein expression of HSP25 in WT and Apc - / - organoids ( e ) .

protein expression of HSP25 in WT and Apc-/- organoids (e).


g , h , Representative images of single cell Hspb1 KO clones passaged after loss of Apc ( g ) , and quantification of their clonogenic potential ( * P = 0 . 0147 ( # 1 ) , * * P = 0 . 0035 ( # 2 ) , * * P = 0 . 0032 ( # 3 ) ) ( h ) .

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

g, h, Representative images of single cell Hspb1 KO clones passaged after loss of Apc (g), and quantification of their clonogenic potential (*P=0.0147(#1), **P=0.0035(#2), **P=0.0032(#3)) (h).


i , j , Representative images of Apc - / - organoids cultured in the absence or presence of 60 μM BVDU ( i ) , and quantification of their clonogenic potential ( * * P = 0 . 0039 ) ( j ) .

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

i, j, Representative images of Apc-/- organoids cultured in the absence or presence of 60 μM BVDU (i), and quantification of their clonogenic potential (**P=0.0039) (j).


k , l , Relative gene expression of Wnt target genes Axin2 ( * * P = 0 . 0080 ) and Lgr5 ( * P = 0 . 0142 ) in Apc - / - organoids cultured in the absence or presence of 60 μM BVDU .

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

k, l, Relative gene expression of Wnt target genes Axin2 (**P=0.0080) and Lgr5 (*P=0.0142) in Apc-/- organoids cultured in the absence or presence of 60 μM BVDU.


m , RNA - ISH for Lgr5 in control or BVDU - treated Apc - / - organoids . Scale bar , 50 μm , zoom panel 20 μm .

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

m, RNA-ISH for Lgr5 in control or BVDU-treated Apc-/- organoids. Scale bar, 50 μm, zoom panel 20 μm.


b , c , Visualization of proliferating cells in tissues incubated with EdU for 2 - hours ( b ) , and quantification of the hyperproliferative zone ( * * P = 0 . 0057 , n = 3 mice ) ( c ) .

O O O O O O O O O O O O O O O O O O O O O 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, c, Visualization of proliferating cells in tissues incubated with EdU for 2-hours (b), and quantification of the hyperproliferative zone (**P=0.0057, n=3 mice) (c).


e , f , RNA - ISH for Notum reveals Apc - mutant clones in crypt bottoms of control or BVDU - treated mice ( e ) , and quantification of the abundance of mutant crypts ( n = 2 mice per condition , n = 5 technical replicates per mouse ) ( f ) .

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

e, f, RNA-ISH for Notum reveals Apc-mutant clones in crypt bottoms of control or BVDU-treated mice (e), and quantification of the abundance of mutant crypts (n=2 mice per condition, n=5 technical replicates per mouse) (f).


g , h , Clone size distributions of Notum + crypts in control ( n = 265 crypts ) or BVDU - treated ( n = 260 crypts ) mice , the average clone size and number of fixed clones are included in the figure .

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

g, h, Clone size distributions of Notum+ crypts in control (n=265 crypts) or BVDU-treated (n=260 crypts) mice, the average clone size and number of fixed clones are included in the figure.


Survival curves for control and BVDU - treated mice ( P = 0 . 0351 , Mantel - Cox test ) ( j )

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

Survival curves for control and BVDU-treated mice (P=0.0351, Mantel-Cox test) (j)


macroscopic images of adenoma development in the distal small intestine ( k )

macroscopic images of adenoma development in the distal small intestine (k)


boxplots for the number of adenomas per intestinal region ( * * P = 0 . 0013 ) ( l ) , n = 8 control , n = 11 treated .

boxplots for the number of adenomas per intestinal region(**P=0.0013) (l), n=8 control, n=11 treated.


( a ) [ 3H ] thymidine incorporation at 72 h by wild - type ( WT ) and Irgm1 - / - CD4 + T cells activated with soluble mAb to CD3 and irradiated syngenic wild - type splenocytes .

(a) [3H]thymidine incorporation at 72 h by wild-type (WT) and Irgm1-/- CD4+ T cells activated with soluble mAb to CD3 and irradiated syngenic wild-type splenocytes.


( b ) [ 3H ] thymidine incorporation at 72 h by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA protein ( OVA ) or OVA peptide of amino acids 323 - 339 ( OVA ( 323 - 339 ) ) in the presence of syngenic splenic DCs .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) [3H]thymidine incorporation at 72 h by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA protein (OVA) or OVA peptide of amino acids 323-339 (OVA(323-339)) in the presence of syngenic splenic DCs.


( c , d ) Kinetic analysis of [ 3H ] thymidine incorporation ( c ) and cell numbers ( d ) in cultures of OT - II and Irgm1 − / − OT - II spleen cells stimulated with OVA peptide .

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

(c,d) Kinetic analysis of [3H]thymidine incorporation (c) and cell numbers (d) in cultures of OT-II and Irgm1−/− OT-II spleen cells stimulated with OVA peptide.


( e ) Flow cytometric analysis of the activation and proliferation of OVA peptide - stimulated OT - II and Irgm1 - / - OT - II CD4 + T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU . Numbers in quadrants indicate percent viable CD4 + cells .

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

(e) Flow cytometric analysis of the activation and proliferation of OVA peptide-stimulated OT-II and Irgm1-/- OT-II CD4+ T cells pulsed with BrdU during the final 4 h of 72 h of culture and then stained with mAb to CD25 and BrdU. Numbers in quadrants indicate percent viable CD4+ cells.


( f ) Enzyme - linked immunosorbent assay of IL - 2 produced by OT - II and Irgm1 - / - OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DCs , measured at 72 h . Data are representative of more than five ( a , b ) or three ( c - f ) independent experiments with similar results ( mean and s . d . of triplicate cultures , a - d , f ) .

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

(f) Enzyme-linked immunosorbent assay of IL-2 produced by OT-II and Irgm1-/- OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DCs, measured at 72 h. Data are representative of more than five (a,b) or three (c-f) independent experiments with similar results (mean and s.d. of triplicate cultures, a-d,f).


( a ) Immunoblot analysis of Irgm1 in lysates of flow cytometry - sorted naive wild - type CD4 + lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 ( 10 μg / ml ) alone or with mAb to CD28 ( 2 μg / ml ) in the presence or absence of IFN - γ - neutralizing mAb . Actin , loading control .

(a) Immunoblot analysis of Irgm1 in lysates of flow cytometry-sorted naive wild-type CD4+ lymphocytes activated for 48 h with immobilized agonistic mAb to CD3 (10 μg/ml) alone or with mAb to CD28 (2 μg/ml) in the presence or absence of IFN-γ-neutralizing mAb. Actin, loading control.


( b ) Enzyme - linked immunosorbent assay of IFN - γ produced by OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions ( TH1 or TH2 ) or nonpolarized conditions ( N ) , assessed in 72 - hour cultures . ND , not detected .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Enzyme-linked immunosorbent assay of IFN-γ produced by OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with OVA peptide in the presence of splenic DC in polarized conditions (TH1 or TH2) or nonpolarized conditions (N), assessed in 72-hour cultures. ND, not detected.


( c ) Accumulation of polarized and nonpolarized OT - II ( open symbols ) or Irgm1 − / − OT - II ( filled symbols ) CD4 + T cells after peptide stimulation , quantified by counting of viable cells .

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

(c) Accumulation of polarized and nonpolarized OT-II (open symbols) or Irgm1−/− OT-II (filled symbols) CD4+ T cells after peptide stimulation, quantified by counting of viable cells.


( d ) Viable OT - II and Irgm1 − / − OT - II CD4 + T cells 72 h after restimulation with OVA peptide in the presence of T cell - depleted APC samples .

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

(d) Viable OT-II and Irgm1−/− OT-II CD4+ T cells 72 h after restimulation with OVA peptide in the presence of T cell-depleted APC samples.


( e ) Flow cytometry of the viability and division of OT - II and Irgm1 − / − OT - II CD4 + T cells stimulated with peptide , allowed to ' rest ' for 7 d in IL - 2 , then labeled with CFSE and restimulated with OVA peptide in the presence of T cell - depleted APC samples and assessed 72 h later .

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

(e) Flow cytometry of the viability and division of OT-II and Irgm1−/− OT-II CD4+ T cells stimulated with peptide, allowed to 'rest' for 7 d in IL-2, then labeled with CFSE and restimulated with OVA peptide in the presence of T cell-depleted APC samples and assessed 72 h later.


( f ) Flow cytometric analysis of the population expansion of CFSE - labeled OT - II and Irgm1 − / − OT - II CD4 + T cells activated with OVA peptide in the presence or absence of IFN - γ - neutralizing mAb . Results in e , f obtained after gating on CD4 + T cells ; numbers in outlined areas ( e ) and in top left corners ( f ) indicate percent CD4 + cells . PI , propidium iodide .

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

(f) Flow cytometric analysis of the population expansion of CFSE-labeled OT-II and Irgm1−/− OT-II CD4+ T cells activated with OVA peptide in the presence or absence of IFN-γ-neutralizing mAb. Results in e,f obtained after gating on CD4+ T cells; numbers in outlined areas (e) and in top left corners (f) indicate percent CD4+ cells. PI, propidium iodide.


( g ) [ 3H ] thymidine incorporation by wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng − / − splenocytes . Data are representative of two independent experiments with similar results ( mean and s . d . of triplicate cultures , b - d , g ) .

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

(g) [3H]thymidine incorporation by wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells stimulated for 72 h with soluble agonistic mAb to CD3 and irradiated syngenic Ifng−/− splenocytes. Data are representative of two independent experiments with similar results (mean and s.d. of triplicate cultures, b-d,g).


( a ) Flow cytometric analysis of the survival of CD45 . 2 + Ifng − / − ( open symbols ) or Irgm1 − / − Ifng − / − ( filled symbols ) CD4 + T lymphocytes preactivated with agonistic mAb to CD3 in vitro , then transferred intraperitoneally into congenic CD45 . 1 + C57BL / 6 . SJL recipients left untreated or inoculated intraperitoneally with T . gondii or M . avium 3 d earlier , assessed in peritoneal exudate cell samples collected at day 4 after transfer .

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

(a) Flow cytometric analysis of the survival of CD45.2+ Ifng−/− (open symbols) or Irgm1−/−Ifng−/− (filled symbols) CD4+ T lymphocytes preactivated with agonistic mAb to CD3 in vitro, then transferred intraperitoneally into congenic CD45.1+ C57BL/6.SJL recipients left untreated or inoculated intraperitoneally with T. gondii or M. avium 3 d earlier, assessed in peritoneal exudate cell samples collected at day 4 after transfer.


( b ) Survival of M . avium - infected wild - type , Irgm1 − / − , Ifng − / − and Irgm1 − / − Ifng − / − mice ( n = 5 mice per genotype ) .

(b) Survival of M. avium-infected wild-type, Irgm1−/−, Ifng−/− and Irgm1−/−Ifng−/− mice (n = 5 mice per genotype).


( c - e ) Splenic bacterial loads ( c ) , circulating lymphocytes ( d ) and splenic lymphocytes ( e ) , assessed at week 4 after M . avium infection ( n = 5 mice per genotype ) . Each symbol in a , c - e represents an individual mouse . Data are representative of two independent experiments .

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

(c-e) Splenic bacterial loads (c), circulating lymphocytes (d) and splenic lymphocytes (e), assessed at week 4 after M. avium infection (n = 5 mice per genotype). Each symbol in a,c-e represents an individual mouse. Data are representative of two independent experiments.


( a ) [ 3H ] thymidine incorporation by naive CD4 + lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN - γ - deficient irradiated APCs , with IL - 2 ( 10 U / ml ) added 24 h after the initiation of culture .

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

(a) [3H]thymidine incorporation by naive CD4+ lymphocytes stimulated for 72 h with agonistic mAb to CD3 and IFN-γ-deficient irradiated APCs, with IL-2 (10 U/ml) added 24 h after the initiation of culture.


( b ) Accumulation of viable CD4 + T cells at 72 h in the presence of medium alone ( Med ) or IFN - γ ( 5 U / ml ) .

O O O O O O O O O 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) Accumulation of viable CD4+ T cells at 72 h in the presence of medium alone (Med) or IFN-γ (5 U/ml).


( c ) Flow cytometric analysis of the viability and division of CD4 + T cells labeled with CFSE and activated as described in a with T cell - depleted Ifng − / − APC samples in the presence or absence of IFN - γ ( 5 U / ml ) . Numbers in plots indicate percent CFSE + CD4 + T cell populations .

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

(c) Flow cytometric analysis of the viability and division of CD4+ T cells labeled with CFSE and activated as described in a with T cell-depleted Ifng−/− APC samples in the presence or absence of IFN-γ (5 U/ml). Numbers in plots indicate percent CFSE+CD4+ T cell populations.


( d - f ) Death of Ifng − / − and Irgm1 − / − Ifng − / − T cell populations activated with mAb to CD3 and expanded in IL - 2 , then treated with IFN - γ ( d ) , agonistic mAb to CD3 ( e ) or Fas ligand ( FasL ; f ) , assessed by propidium iodide staining at 48 h after treatment . Open bars , Ifng − / − ; filled bars , Irgm1 − / − Ifng − / − ( a , b , d - f ) . Data are representative of three independent experiments with similar results ( mean and s . d . of triplicate cultures a , b , d - f ) .

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

(d-f) Death of Ifng−/− and Irgm1−/−Ifng−/− T cell populations activated with mAb to CD3 and expanded in IL-2, then treated with IFN-γ (d), agonistic mAb to CD3 (e) or Fas ligand (FasL; f), assessed by propidium iodide staining at 48 h after treatment. Open bars, Ifng−/−; filled bars, Irgm1−/−Ifng−/− (a,b,d-f). Data are representative of three independent experiments with similar results (mean and s.d. of triplicate cultures a,b,d-f).


( a - f ) Electron microscopy analysis of Ifng − / − CD4 + lymphocytes ( a ) and Irgm1 − / − Ifng − / − CD4 + lymphocytes ( b - f ) exposed for 30 h to IFN - γ ( 5 U / ml ) . ( c ) Enlargement of the area outlined in b ; arrowheads indicate two autophagic vacuoles . ( d ) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells . Each symbol represents one section . ( e ) Irgm1 − / − CD4 + T cell with many vacuoles containing dense materials . ( f ) Two Irgm1 − / − CD4 + cells with vacuolated cytoplasma . Original magnification ( a - c , e , f ) , × 1 , 000 . Data are representative of two experiments .

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

(a-f) Electron microscopy analysis of Ifng−/− CD4+ lymphocytes (a) and Irgm1−/−Ifng−/− CD4+ lymphocytes (b-f) exposed for 30 h to IFN-γ (5 U/ml). (c) Enlargement of the area outlined in b; arrowheads indicate two autophagic vacuoles. (d) Quantification of vacuoles in 50 randomly selected electron microscopy sections of more than 400 cells. Each symbol represents one section. (e) Irgm1−/− CD4+ T cell with many vacuoles containing dense materials. (f) Two Irgm1−/− CD4+ cells with vacuolated cytoplasma. Original magnification (a-c,e,f), × 1,000. Data are representative of two experiments.


( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells in the presence or absence of wortmannin or Ly294002 ( g ) , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) .

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

(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells in the presence or absence of wortmannin or Ly294002 (g), assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


( g , h ) Death of Ifng − / − and Irgm1 − / − Ifng − / − CD4 + T cells transfected with control or beclin1 - specific small interfering RNA , assessed by flow cytometry with propidium iodide staining at 48 h after IFN - γ exposure . Right ( h ) , immunoblot analysis of beclin1 expression . C , control small interfering RNA ; B , beclin1 - specific small interfering RNA . Data are representative of three independent experiments ( mean and s . d . of triplicate cultures ) .

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

(g,h) Death of Ifng−/− and Irgm1−/−Ifng−/− CD4+ T cells transfected with control or beclin1-specific small interfering RNA, assessed by flow cytometry with propidium iodide staining at 48 h after IFN-γ exposure. Right (h), immunoblot analysis of beclin1 expression. C, control small interfering RNA; B, beclin1-specific small interfering RNA. Data are representative of three independent experiments (mean and s.d. of triplicate cultures).


B Vero E6 cells were inoculated with SARS - CoV - 2 , fixed with paraformaldehyde after two days , stained with 30 nM of the indicated Alexa Fluor 488 ( AF488 ) - labeled anti - RBD VHH antibodies , and imaged by confocal laser scanning microscopy ( CLSM ) . C Immunofluorescence ( IF ) staining as in B , but with the indicated AF568 - labeled anti - S1ΔRBD VHH antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Vero E6 cells were inoculated with SARS-CoV-2, fixed with paraformaldehyde after two days, stained with 30 nM of the indicated Alexa Fluor 488 (AF488)-labeled anti-RBD VHH antibodies, and imaged by confocal laser scanning microscopy (CLSM). C Immunofluorescence (IF) staining as in B, but with the indicated AF568-labeled anti-S1ΔRBD VHH antibodies.


BLI sensorgrams of intermediate VHHs A SARS - CoV - 2 RBD - loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s , followed by dipping in assay buffer for 600 s . The response curves ( black ) were fitted using either a 1 : 1 ( for VHH - 72 ) or 2 : 1 ( for Re6B06 and Re9F06 ) binding models to obtain KDs . Fitted curves are shown in red . For Re6B06 and Re9F06 , some secondary binding with weaker affinity at high VHH concentrations was observed ( denoted as KD2 ) .

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

BLI sensorgrams of intermediate VHHs A SARS-CoV-2 RBD-loaded sensors were incubated with nanobodies at the indicated concentrations for 300 s, followed by dipping in assay buffer for 600 s. The response curves (black) were fitted using either a 1:1 (for VHH-72) or 2:1 (for Re6B06 and Re9F06) binding models to obtain KDs. Fitted curves are shown in red. For Re6B06 and Re9F06, some secondary binding with weaker affinity at high VHH concentrations was observed (denoted as KD2).


A Vero E6 cells were infected with SARS - CoV - 2 , pre - incubated with indicated concentrations of the neutralizing VHH - 72 Cells were fixed two days after inoculation , stained with sets of anti - RBD ( green ) and anti - S1ΔRBD ( red ) nanobodies and analyzed by CLSM . We used a cocktail of such fluorophore - labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody .

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A Vero E6 cells were infected with SARS-CoV-2, pre-incubated with indicated concentrations of the neutralizing VHH-72 Cells were fixed two days after inoculation, stained with sets of anti-RBD (green) and anti-S1ΔRBD (red) nanobodies and analyzed by CLSM. We used a cocktail of such fluorophore-labeled VHH antibodies to ensure that negative fluorescence readings truly indicated neutralization and thus absence of viral infection and not just masking of the IF epitope by the tested nanobody.


B Vero E6 cells were infected as in A . Viral RNA in the culture supernatants was quantified by reverse transcription ( RT ) - qPCR . RNA signals are normalized to the respective minus - VHH controls ( 100 % ) . Note the log10 scale of the y - axis . " Inoculum " ( red dotted line ) marks the RNA level detected at the time of infection .

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B Vero E6 cells were infected as in A. Viral RNA in the culture supernatants was quantified by reverse transcription (RT)-qPCR. RNA signals are normalized to the respective minus-VHH controls (100%). Note the log10 scale of the y-axis. "Inoculum" (red dotted line) marks the RNA level detected at the time of infection.


Neutralization of SARS - CoV - 2 by the indicated VHH antibodies at nanomolar ( A and B ) or picomolar ( C and D ) concentrations . The neutralization experiment was performed ; n " indicates the number of independent biological

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

Neutralization of SARS-CoV-2 by the indicated VHH antibodies at nanomolar (A and B) or picomolar (C and D) concentrations. The neutralization experiment was performed ;n" indicates the number of independent biological replicates


A HeLa cells were transiently transfected to express the SARS - CoV - 2 Spike protein . Following fixation , cells were stained for 1 hour with fluorophore - labeled Re10B10 ( 5 nM , green ) and Re7E02 ( 15 nM , red ) in the presence of the indicated unlabeled VHH competitors . Competitor ( 150 nM ) was added 20 min prior to the labeled nanobodies . The weakly binding competitors Re5A08 , Re9F06 , Re6B06 , and , Re6D06 were added as trimers ( see below ) . Cells were imaged by CLSM . For each competitor , the sequence class and the binding site on the RBD ( epitope 1 or 2 ) are indicated .

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A HeLa cells were transiently transfected to express the SARS-CoV-2 Spike protein. Following fixation, cells were stained for 1 hour with fluorophore-labeled Re10B10 (5 nM, green) and Re7E02 (15 nM, red) in the presence of the indicated unlabeled VHH competitors. Competitor (150 nM) was added 20 min prior to the labeled nanobodies. The weakly binding competitors Re5A08, Re9F06, Re6B06, and, Re6D06 were added as trimers (see below). Cells were imaged by CLSM. For each competitor, the sequence class and the binding site on the RBD (epitope 1 or 2) are indicated.


F Neutralization of SARS - CoV - 2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations . The neutralization experiment was

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F Neutralization of SARS-CoV-2 by the thermostable Re5D06 variants R15 and R28 at the indicated concentrations. The neutralization experiment was performed


B Re6B06 monomer and trimer variants ( 20 nM ) were analyzed by BLI for RBD - binding , with 900 s association and 1500 s dissociation steps . Note the avidity effect of Re6B06 trimerization .

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B Re6B06 monomer and trimer variants (20 nM) were analyzed by BLI for RBD-binding, with 900 s association and 1500 s dissociation steps. Note the avidity effect of Re6B06 trimerization.


C Neutralization of SARS - CoV - 2 by the indicated VHH antibodies . The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized . Re6B06 , for example , is 30 000 - fold more potent when symmetry - matched with the virus Spike .

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C Neutralization of SARS-CoV-2 by the indicated VHH antibodies. The neutralization experiment was performed Note the gain in neutralization efficacy when a VHH is trimerized. Re6B06, for example, is 30 000-fold more potent when symmetry-matched with the virus Spike.


C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI . Immobilized RBD was incubated with 100 nM of nanobodies for 450 s , followed by assay buffer for 900 s .

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C Binding of nanobodies to the quadruple RBD mutant was monitored by BLI. Immobilized RBD was incubated with 100 nM of nanobodies for 450 s, followed by assay buffer for 900 s.


A and B Neutralization of SARS - CoV - 2 B . 1 . 351 / Beta by the indicated VHH antibody constructs . The neutralization experiment was performed as described in Fig 3A , with the difference being that a mutant - optimized anti - RBD / S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown , corresponding to the standard ( upper row ) or a five - fold higher virus inoculum ( lower row ) that was used for even more stringent neutralization conditions .

A and B Neutralization of SARS-CoV-2 B.1.351/Beta by the indicated VHH antibody constructs. The neutralization experiment was performed as described in Fig 3A, with the difference being that a mutant-optimized anti-RBD/S1 VHH cocktail was used for immunofluorescence staining Two titrations for the Re9F06 trimer are shown, corresponding to the standard (upper row) or a five-fold higher virus inoculum (lower row) that was used for even more stringent neutralization conditions.


( B ) A fragment of genomic DNA ( 2 . 2 kb ) was amplified by PCR and in vitro sgRNA - guided Cas9 - mediated cleavage assay was performed with each of the sgRNAs . sgRNA - targeting sites are indicated by arrows , genomic DNA size is indicated by asterisk .

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(B) A fragment of genomic DNA (2.2 kb) was amplified by PCR and in vitro sgRNA-guided Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA size is indicated by asterisk.


( C ) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45 . 2 B cells from CLK09 ( left ) and CLK19 ( right ) KI mouse . Green , B220 ; Blue , CD3 ; White , CD45 . 2 ; Red , GL7 . White arrows indicate the CD45 . 2 B cells .

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(C) Immunohistochemistry of day 8 spleen sections for host mouse receiving 5x105 isolated CD45.2 B cells from CLK09 (left) and CLK19 (right) KI mouse. Green, B220; Blue, CD3; White, CD45.2; Red, GL7.White arrows indicate the CD45.2 B cells.


( A ) CD45 . 2 frequency among GCs induced by eOD - GT8 60 mer at different time points . Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD - GT8 60mer . On days 8 , 15 and 36 , the splenic B cells were isolated to measure the CD45 . 2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS . Gated plots represent the CD45 . 2 frequency among GC B cells . ( B ) Quantification of CD45 . 2 frequency among GCs at three time points . X - axis represents adoptively transferred mice groups with different precursor frequency ( 1 in 104 , 2 in 105 , 3 in 106 ) at three time point , y axis represents CD45 . 2 frequency among GC . Each circle represents one mouse . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . Bars indicate mean ± SD from mice in each group .

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(A) CD45.2 frequency among GCs induced by eOD-GT8 60 mer at different time points. Adoptively transferred mice with 1 in 104 of precursor frequency were immunized with eOD-GT8 60mer. On days 8, 15 and 36, the splenic B cells were isolated to measure the CD45.2 cell frequency in GC B cells by FACS using the markers mentioned above by FACS. Gated plots represent the CD45.2 frequency among GC B cells. (B) Quantification of CD45.2 frequency among GCs at three time points. X-axis represents adoptively transferred mice groups with different precursor frequency (1 in 104, 2 in 105, 3 in 106) at three time point, y axis represents CD45.2 frequency among GC. Each circle represents one mouse. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. Bars indicate mean ± SD from mice in each group.


( C ) IgG titers detected by ELISA . eOD - GT8 or eOD - GT8 KO soluble proteins were used to coat ELISA plates . Sera from adoptively transferred mice at different time points were used to detect by ELISA . Bars indicate geometric mean and geometric SD from mice in each group . For CLK21 and CLK09 panel , n = 5 mice in each group . For CLK19 panel , n = 3 mice in each group . X - axis represents precursor frequency , and Y - axis represents the change of area under curve ( AUC coated eOD - GT8 - AUC coated eOD - GT8 KO ) . Each circle represents one mouse .

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(C) IgG titers detected by ELISA. eOD-GT8 or eOD-GT8 KO soluble proteins were used to coat ELISA plates. Sera from adoptively transferred mice at different time points were used to detect by ELISA. Bars indicate geometric mean and geometric SD from mice in each group. For CLK21 and CLK09 panel, n=5 mice in each group. For CLK19 panel, n=3 mice in each group. X-axis represents precursor frequency, and Y-axis represents the change of area under curve (AUC coated eOD-GT8 -AUC coated eOD-GT8 KO). Each circle represents one mouse.


B . BrdU - sensitized U - 2 - OS cells stably expressing GFP - NLS - CDKL5 were pre - incubated with DMSO ( mock ) , olaparib ( 5 μM ) , talazoparib ( 50 nM ) or PDD00017273 ( 0 . 3 μM ) for 1 h prior to micro - irradiation and live - imaging at the indicated times post - irradiation . One of three independent experiments is shown . Scale bar is 10

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B. BrdU-sensitized U-2-OS cells stably expressing GFP-NLS-CDKL5 were pre-incubated with DMSO (mock), olaparib (5 μM), talazoparib (50 nM) or PDD00017273 (0.3 μM) for 1 h prior to micro-irradiation and live-imaging at the indicated times post-irradiation. One of three independent experiments is shown. Scale bar is 10 μm


E . BrdU - sensitized parental or PARP1Δ / Δ , PARP2Δ / Δ , PARP1 / 2Δ / Δ U - 2 - OS cells transiently expressing GFP NLS - CDKL5 were subjected to 355 nm line micro - irradiation followed by time lapse imaging . One of two independent experiments is shown . Scale bar is 10

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E. BrdU-sensitized parental or PARP1Δ/Δ, PARP2Δ/Δ, PARP1/2Δ/Δ U-2-OS cells transiently expressing GFP NLS-CDKL5 were subjected to 355 nm line micro-irradiation followed by time lapse imaging. One of two independent experiments is shown. Scale bar is 10 μm


G . Cells subjected to the workflow in F . were detergent - extracted and fixed before staining with anti - GFP or fibrillarin ( nucleoli ) . Scale bar is 10 μm H . Quantification of the detergent - insoluble GFP - NLS - CDKL5 signal ( minus nucleolar signal ) . The mean ± SD from three biological experiments is shown . Statistical significance was assessed by one - way - ANOVA - test . Asterisks * * indicate P - value of < 0 . 01 ; ns - not significant .

G. Cells subjected to the workflow in F. were detergent-extracted and fixed before staining with anti-GFP or fibrillarin (nucleoli). Scale bar is 10 μm H. Quantification of the detergent-insoluble GFP-NLS-CDKL5 signal (minus nucleolar signal). The mean ± SD from three biological experiments is shown. Statistical significance was assessed by one-way-ANOVA-test. Asterisks ** indicate P-value of <0.01; ns - not significant.


B . BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS , the GFP - NLS - CDKL5 deletion mutants shown in A , or full length ( FL ) GFP - NLS - CDKL5 were subjected to line micro - irradiation ( 355 nm ) and time lapse imaging . Three independent experiments were performed , and one representative experiment is shown . Scale bar is 10

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B. BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS, the GFP-NLS-CDKL5 deletion mutants shown in A, or full length (FL) GFP-NLS-CDKL5 were subjected to line micro-irradiation (355 nm) and time lapse imaging. Three independent experiments were performed, and one representative experiment is shown. Scale bar is 10 μm


D . Same as in B except that the GFP - NLS tagged CDKL5 fragments indicated were examined . Scale bar is 10

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D. Same as in B except that the GFP-NLS tagged CDKL5 fragments indicated were examined. Scale bar is 10 μm


F . Recombinant fragments of CDKL5 fused to GST ( 1 . 2 , 2 . 5 , 5 , 10 µg ) , or GST , were dot - blotted on nitrocellulose membrane and then incubated with synthetic PAR . PAR binding was detected by far western blotting . APLF was used as positive control . One of three independent experiments is

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F. Recombinant fragments of CDKL5 fused to GST (1.2, 2.5, 5, 10 µg), or GST, were dot-blotted on nitrocellulose membrane and then incubated with synthetic PAR. PAR binding was detected by far western blotting. APLF was used as positive control. One of three independent experiments is shown


( G - H ) . U - 2 - OS ( Flp - In T - Rex ) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min . Extracts were subjected to immunoprecipitation with antibodies against CDKL5 ( G ) or PAR ( H ) ( or non - specific IgG as control ) . Precipitates , and input lysates , were analyzed by western blotting using the indicated antibodies . One of two independent experiments is

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(G-H) .U-2-OS (Flp-In T-Rex) cells stably expressing CDKL5 were either mock treated or treated with 500 µM H2O2 for 30 min. Extracts were subjected to immunoprecipitation with antibodies against CDKL5 (G) or PAR (H) (or non-specific IgG as control). Precipitates, and input lysates, were analyzed by western blotting using the indicated antibodies. One of two independent experiments is shown


A . ( Top ) BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro - irradiation ( 405 nm ) . ( Bottom ) Quantitation of spot intensities . Data represent the mean ± SEM of two independent experiments ; > 50 micro - irradiated cells per point . Scale bar is 10

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A. (Top) BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5 were treated with indicated transcription inhibitors before subjecting to spot micro-irradiation (405 nm). (Bottom) Quantitation of spot intensities. Data represent the mean ± SEM of two independent experiments; > 50 micro-irradiated cells per point. Scale bar is 10 μm


BrdU - sensitized U - 2 - OS ( Flp - In T - Rex ) cells stably expressing GFP - NLS - CDKL5 , mCherry - XRCC1 or mCherry - FAN1 were pre - incubated with indicated transcription inhibitors prior to line micro - irradiation ( 355 nm ) and time lapse imaging . One of three independent experiments is shown . Scale bar is 10

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BrdU-sensitized U-2-OS (Flp-In T-Rex) cells stably expressing GFP-NLS-CDKL5, mCherry-XRCC1 or mCherry-FAN1 were pre-incubated with indicated transcription inhibitors prior to line micro-irradiation (355 nm) and time lapse imaging. One of three independent experiments is shown. Scale bar is 10 μm


E . Same as B - D . except that BrdU - sensitized cells stably expressing GFP - NLS - CDKL5 were also pre - incubated with olaparib as control . Cells were subjected to line micro - irradiation , fixed and then subjected to indirect immunofluorescence using antibodies against GFP , PAR and γH2AX . Scale bar is 10

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E. Same as B-D. except that BrdU-sensitized cells stably expressing GFP-NLS-CDKL5 were also pre-incubated with olaparib as control. Cells were subjected to line micro-irradiation, fixed and then subjected to indirect immunofluorescence using antibodies against GFP, PAR and γH2AX. Scale bar is 10 μm


F . Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging . Scale bar is 10

F. Stable cell lines were permeabilized and incubated with RNase A or PBS before irradiation and imaging. Scale bar is 10 μm


B . Boxplots showing VSN - normalised intensity of phospho - peptides corresponding to EP400 pSer729 , ELOA pSer311and TTDN1 pSer40 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . In all cases the data was derived from 3 biological replicates .

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B. Boxplots showing VSN-normalised intensity of phospho-peptides corresponding to EP400 pSer729, ELOA pSer311and TTDN1 pSer40 from the experiment in A. The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. In all cases the data was derived from 3 biological replicates.


C . Boxplots of the VSN - adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG - EP400 , FLAG - ELOA and FLAG - TTDN1 from the experiment in A . The central band of the boxplot indicates the median value , while the hinges represent the first and third quartile ( bottom and top of boxplot , respectively ) . The whiskers extend to the largest / smallest ( upper or lower whisker , respectively ) datapoint not further than 1 . 5 times the interquartile range from their respective hinge . Datapoints further removed than the whiskers are plotted individually . The experiment was conducted using three biological replicates within each respective group , each TMT channel represents a single biological replicate .

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C. Boxplots of the VSN-adjusted TMT reporter ion intensities for all peptides for each TMT label in the case of FLAG-EP400, FLAG-ELOA and FLAG-TTDN1 from the experiment in A.The central band of the boxplot indicates the median value, while the hinges represent the first and third quartile (bottom and top of boxplot, respectively). The whiskers extend to the largest/smallest (upper or lower whisker, respectively) datapoint not further than 1.5 times the interquartile range from their respective hinge. Datapoints further removed than the whiskers are plotted individually. The experiment was conducted using three biological replicates within each respective group, each TMT channel represents a single biological replicate.


A . HEK293 cells were co - transfected with CDKL5 ( wild type " WT " or kinase - dead " KD " K42R mutant ) fused to an NLS , and FLAG - ELOA ( wild type " WT " or a S311A mutant " SA " ) . Anti - FLAG precipitates or cell extracts were probed with the antibodies indicated . One of three independent experiments is shown . B . Same as A . showing a range of pathogenic ( red ) and benign ( blue ) CDKL5 variants .

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A. HEK293 cells were co-transfected with CDKL5 (wild type "WT" or kinase-dead "KD" K42R mutant) fused to an NLS, and FLAG-ELOA (wild type "WT" or a S311A mutant "SA"). Anti-FLAG precipitates or cell extracts were probed with the antibodies indicated. One of three independent experiments is shown. B. Same as A. showing a range of pathogenic (red) and benign (blue) CDKL5 variants.


( C - E ) . Wild type ( WT ) , CDKL5 - disrupted ( CDKL5Δ / Δ ) or siRNA - transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks . Quantification of ELOA - pSer311 signal at the laser tracks is shown . Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated ( n ) . For simplicity , only intensities greater than zero are shown . Statistical significance was assessed by one - way - ANOVA - test or unpaired t test with Welch ' s correction . Asterisks * * * * indicate P - values of < 0 . 0001 . Scale bar is 10

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(C-E). Wild type (WT), CDKL5-disrupted (CDKL5Δ/Δ) or siRNA-transfected cells were subjected to indirect immunofluorescence analysis with the indicated antibodies at laser tracks. Quantification of ELOA-pSer311 signal at the laser tracks is shown. Data represent mean ± SD of total pELOA Ser311 intensities in different biological replicates as indicated (n). For simplicity, only intensities greater than zero are shown. Statistical significance was assessed by one-way-ANOVA-test or unpaired t test with Welch's correction. Asterisks **** indicate P-values of <0.0001. Scale bar is 10 μm


D . ( Left ) Quantification of transcription in U - 2 - OS 263 IFII reporter cells from experiment in B . > 150 cells were analysed per condition per experiment . The mean ± SD from four independent experiments is shown . ( Right ) Quantitative RT - PCR analysis of YFP - MS2 mRNA in U - 2 - OS 263 reporter cells . Data represent mean ± SD in different biological replicates as indicated ( n ) . Statistical significance for all the data was assessed by two - way - ANOVA - test , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant .

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D. (Left) Quantification of transcription in U-2-OS 263 IFII reporter cells from experiment in B. >150 cells were analysed per condition per experiment. The mean ± SD from four independent experiments is shown. (Right) Quantitative RT-PCR analysis of YFP-MS2 mRNA in U-2-OS 263 reporter cells. Data represent mean ± SD in different biological replicates as indicated (n). Statistical significance for all the data was assessed by two-way-ANOVA-test, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant.


B , C . Quantitative PCR with reverse transcription ( qRT - PCR ) analysis of SLCO5a1 ( B ) and RYR2 expression levels ( C ) ( left panels ) U - 2 - OS HA - ER - I - PpoI cells depleted of CDKL5 transiently transfected with FLAG - tagged CDKL5 wild type ( WT ) or a K42R - mutated kinase dead ( KD ) mutant , or empty vector , at the times indicated after inducing I - PpoI . The mean ± SD from two qPCR replicates of two independent experiments is shown . Statistical significance for all the data was assessed by two - way - ANOVA - test * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; ns - not significant . I - PpoI - mediated cutting efficiency in the relevant gene is shown in the right - hand

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. Quantitative PCR with reverse transcription (qRT-PCR) analysis of SLCO5a1 (B) and RYR2 expression levels (C) (left panels) U-2-OS HA-ER-I-PpoI cells depleted of CDKL5 transiently transfected with FLAG-tagged CDKL5 wild type (WT) or a K42R-mutated kinase dead (KD) mutant, or empty vector, at the times indicated after inducing I-PpoI. The mean ± SD from two qPCR replicates of two independent experiments is shown. Statistical significance for all the data was assessed by two-way-ANOVA-test *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001; ns - not significant. I-PpoI-mediated cutting efficiency in the relevant gene is shown in the right-hand panel


A ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles . Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points . Statistical analysis comparing Tbk1 - / - MEFs and cells expressing TBK1 alleles . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs stably expressing the indicated TBK1 alleles. Intracellular bacteria were enumerated by their ability to form colonies on agar plates following cell lysis at the indicated time points. Statistical analysis comparing Tbk1-/- MEFs and cells expressing TBK1 alleles. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


B ) Replication of S . Typhimurium in Atg5 - / - MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 ( 10 nM ) or DMSO . Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation ( p . i . ) following cell lysis .

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

B) Replication of S. Typhimurium in Atg5-/- MEFs cells complemented with ATG5 or mock and treated with the TBK1 kinase inhibitor MRT68843 (10 nM) or DMSO. Fold replication was determined by counting bacterial colonies on agar plates at 2 and 8 h post inoculation (p.i.) following cell lysis.


C ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium . Percentage of S . Typhimurium coated with ubiquitin ( detected by FK2 antibody ) at 1 or 4 h p . i . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

C) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium. Percentage of S. Typhimurium coated with ubiquitin (detected by FK2 antibody) at 1 or 4 h p.i. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : LC3B ( A ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:LC3B (A). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with the indicated GFP : WIPI alleles ( B ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with the indicated GFP:WIPI alleles (B). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S . Typhimurium ( C - H ) .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Confocal microscopy of MEFs expressing the indicated GFP fusion proteins or immunolabeled for WIPI2 and infected with mCherry expressing S. Typhimurium (C-H).


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : DFCP1 ( FYVE * denotes a PI ( 3 ) P - binding mutant of DFCP1 ) ( D - E ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

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

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:DFCP1 (FYVE* denotes a PI(3)P-binding mutant of DFCP1) (D-E). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - H ) Analysis of Tbk1 - / - MEFs stably expressing the indicated TBK1 alleles and infected with S . Typhimurium at 1 h p . i . Percentage of S . Typhimurium coated with GFP : ML1N * 2 , a probe for PI ( 3 , 5 ) P2 ( G ) . Where indicated , Wortmannin ( Wort ) was added at 100 nM .

O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O

A-H) Analysis of Tbk1-/- MEFs stably expressing the indicated TBK1 alleles and infected with S. Typhimurium at 1 h p.i. Percentage of S. Typhimurium coated with GFP:ML1N*2, a probe for PI(3,5)P2 (G). Where indicated, Wortmannin (Wort) was added at 100 nM.


A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI1 ( A ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . Western blot for GFP : WIPI1 upon the indicated siRNA treatments .

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

A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI1 (A). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. Western blot for GFP:WIPI1 upon the indicated siRNA treatments.


A - B ) Kinetics of S . Typhimurium replication in MEFs depleted of WIPI2 ( B ) . Intracellular bacteria were enumerated by their ability to form colonies on agar plates . quantitative RT - PCR for WIPI2 upon the indicated siRNA treatments .

A-B) Kinetics of S. Typhimurium replication in MEFs depleted of WIPI2 (B). Intracellular bacteria were enumerated by their ability to form colonies on agar plates. quantitative RT-PCR for WIPI2 upon the indicated siRNA treatments.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of four independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test .

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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of four independent experiments. >200 bacteria counted per coverslip. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test.


A - D ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - adaptor fusion proteins . S . Typhimurium replication kinetics ( A , D ) . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical significance to Tbk1 - / - MEFs expressing TBK1ΔC is indicated . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates .

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

A-D) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-adaptor fusion proteins. S. Typhimurium replication kinetics (A,D). Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical significance to Tbk1-/- MEFs expressing TBK1ΔC is indicated. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.


E ) Lumier binding assay . Binding of the indicated luciferase - tagged NDP52 variants to beads coated with GST , GST : galectin - 8 , or GST : ubiquitin . Coomassie stain of purified GST proteins . Western blot for luciferase - tagged NDP52 alleles in total cell lysates .

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

E) Lumier binding assay. Binding of the indicated luciferase-tagged NDP52 variants to beads coated with GST, GST:galectin-8, or GST:ubiquitin. Coomassie stain of purified GST proteins. Western blot for luciferase-tagged NDP52 alleles in total cell lysates.


F ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - NDP52 fusion proteins . Infected cells were lysed at the indicated times post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical comparison with Tbk1 - / - MEFs expressing TBK1ΔC is indicated . * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

F) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-NDP52 fusion proteins. Infected cells were lysed at the indicated times post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical comparison with Tbk1-/- MEFs expressing TBK1ΔC is indicated. ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . S . Typhimurium replication kinetics ( A ) . Infected cells were lysed at the indicated times post inoculation ( p . i . ) . and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 05 , * * * p < 0 . 001 , one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

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

A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. S. Typhimurium replication kinetics (A). Infected cells were lysed at the indicated times post inoculation (p.i.). and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.05, ***p<0.001, one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


A - B ) Analysis of Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC - galectin fusion proteins . ( B ) Percentage of GFP : WIPI1 positive S . Typhimurium at 1 h p . i . in the indicated complemented MEFs . Mean and s . e . m . of three independent experiments . > 200 bacteria counted per coverslip . * p < 0 . 05 , one - way ANOVA with Dunnett ' s multiple comparisons test .

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

A-B) Analysis of Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC-galectin fusion proteins. (B) Percentage of GFP:WIPI1 positive S. Typhimurium at 1 h p.i. in the indicated complemented MEFs. Mean and s.e.m. of three independent experiments. >200 bacteria counted per coverslip. *p<0.05, one-way ANOVA with Dunnett's multiple comparisons test.


A ) Binding of NDP52 to ubiquitin tetramers of different linkage types . Beads coated with the indicated GST - tagged proteins were incubated with pooled M1 - , K63 - , and K48 - linked tetra - ubiquitin chains . Anti - ubiquitin Western blot of input and proteins bound to beads .

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

A) Binding of NDP52 to ubiquitin tetramers of different linkage types. Beads coated with the indicated GST-tagged proteins were incubated with pooled M1-, K63-, and K48-linked tetra-ubiquitin chains. Anti-ubiquitin Western blot of input and proteins bound to beads.


B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclear cell lysates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclear cell lysates.


B - D ) Kinetics of S . Typhimurium replication in Tbk1 - / - MEFs complemented with the indicated Flag - tagged TBK1ΔC fusion proteins . Infected cells were lysed at the indicated time points post inoculation ( p . i . ) and bacteria were enumerated by their ability to form colonies on agar plates . Mean and s . d . of triplicate MEF cultures and duplicate colony counts . Data are representative of at least two repeats . Statistical differences to Tbk1 - / - MEFs expressing TBK1ΔC are shown . * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 one - way ANOVA with Dunnett ' s multiple comparisons test . Western blot for Flag - tagged TBK1 variants in post nuclearcell lysates .

B-D) Kinetics of S. Typhimurium replication in Tbk1-/- MEFs complemented with the indicated Flag-tagged TBK1ΔC fusion proteins. Infected cells were lysed at the indicated time points post inoculation (p.i.) and bacteria were enumerated by their ability to form colonies on agar plates. Mean and s.d. of triplicate MEF cultures and duplicate colony counts. Data are representative of at least two repeats. Statistical differences to Tbk1-/- MEFs expressing TBK1ΔC are shown. *p<0.5, **p<0.01, ***p<0.001 one-way ANOVA with Dunnett's multiple comparisons test. Western blot for Flag-tagged TBK1 variants in post nuclearcell lysates.


( B , C ) Representative immunoblots ( B ) and quantification of Tau degradation ( C ) from 14 DIV primary neurons transduced with EGFP or shTSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shTSG101 - expressing neurons exhibit markedly decreased Tau degradation compared to EGFP - expressing controls ( n = 18 - 19 per condition , unpaired student ' s t - test , * p = 0 . 0108 )

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

(B, C) Representative immunoblots (B) and quantification of Tau degradation (C) from 14 DIV primary neurons transduced with EGFP or shTSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shTSG101-expressing neurons exhibit markedly decreased Tau degradation compared to EGFP-expressing controls (n=18-19 per condition, unpaired student's t-test, *p=0.0108)


( D , E ) Representative immunoblots ( D ) and quantification of Tau degradation ( E ) from 14 DIV neurons transduced with mCh or mCh - TSG101 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . Overexpression of TSG101 increases Tau degradation ( n = 17 / condition , unpaired student ' s t - test , * p = 0 . 0115 )

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

(D, E) Representative immunoblots (D) and quantification of Tau degradation (E) from 14 DIV neurons transduced with mCh or mCh-TSG101, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. Overexpression of TSG101 increases Tau degradation (n=17/condition, unpaired student's t-test, *p=0.0115)


( F , G ) Representative immunoblots ( F ) and quantification of Tau degradation ( G ) from 14 DIV neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau and tubulin . shRab35 - expressing neurons exhibit markedly decreased Tau degradation compared to mCh - expressing controls ( n = 23 - 26 per condition , unpaired student ' s t - test , * * * p = 0 . 0008 )

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

(F, G) Representative immunoblots (F) and quantification of Tau degradation (G) from 14 DIV neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau and tubulin. shRab35-expressing neurons exhibit markedly decreased Tau degradation compared to mCh-expressing controls (n=23-26 per condition, unpaired student's t-test, ***p=0.0008)


( H , I ) Representative immunoblots ( H ) and quantification of Tau degradation ( I ) from 14 DIV neurons transduced with mCh or mCh - Rab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for Tau or tubulin . Rab35 overexpression increases Tau degradation ( n = 18 per condition , unpaired student ' s t - test , * p = 0 . 0126 )

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

(H, I) Representative immunoblots (H) and quantification of Tau degradation (I) from 14 DIV neurons transduced with mCh or mCh-Rab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for Tau or tubulin. Rab35 overexpression increases Tau degradation (n=18 per condition, unpaired student's t-test, *p=0.0126)


( J ) Flow cytometry distribution of N2a cells cotransfected with either HA vector ( black ) or HA - Rab35 ( green ) and medium fluoresence timer - tagged wild - type Tau ( FT - Tau ) ; blue fluorescence ( y axis ) indicates " younger " Tau protein while red fluorescence ( x axis ) indicates " older "

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

(J) Flow cytometry distribution of N2a cells cotransfected with either HA vector (black) or HA-Rab35 (green) and medium fluoresence timer-tagged wild-type Tau (FT-Tau); blue fluorescence (y axis) indicates "younger" Tau protein while red fluorescence (x axis) indicates "older" Tau


( K ) The ratio of cells expressing red to blue ( older : younger ) FT - Tau is reduced in cells overexpressing Rab35 , indicating faster Tau turnover ( n = 9 / condition , unpaired student ' s t - test , * * * * p < 0 . 0001 )

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

(K) The ratio of cells expressing red to blue (older:younger) FT-Tau is reduced in cells overexpressing Rab35, indicating faster Tau turnover (n=9/condition, unpaired student's t-test , ****p<0.0001)


( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) from 14 DIV primary neurons transduced with mCh or shRab35 , treated for 24 h with either DMSO ( CON ) or cycloheximide ( CHX ) , and probed for pSer396 / 404 - Tau ( PHF1 ) , pSer262 - Tau , or pSer202 - Tau ( CP13 ) and tubulin . shRab35 - expressing neurons exhibit markedly decreased pSer262 - and p396 / 404 - Tau degradation compared to mCh - expressing controls , while pSer202 - Tau degradation is unaffected ( n = 4 per condition for pSer396 / 404 - Tau and pSer202 - Tau , n = 3 for pSer262 - Tau , unpaired student ' s t - test , * * p = 0 . 0027 , * p = 0 . 0120 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) from 14 DIV primary neurons transduced with mCh or shRab35, treated for 24 h with either DMSO (CON) or cycloheximide (CHX), and probed for pSer396/404-Tau (PHF1), pSer262-Tau, or pSer202-Tau (CP13) and tubulin. shRab35-expressing neurons exhibit markedly decreased pSer262- and p396/404-Tau degradation compared to mCh-expressing controls, while pSer202-Tau degradation is unaffected (n=4 per condition for pSer396/404-Tau and pSer202-Tau, n=3 for pSer262-Tau, unpaired student's t-test, **p=0.0027, *p=0.0120)


( C ) Images of PLA signal ( green ) for Hrs / Tau interaction in N2a cells co - transfected with FLAG - Hrs and either mCh or mCh - Rab35 ( red ) , and probed with antibodies against FLAG and total ( DA9 ) or phospho - Tau species ( pSer396 / 404 - Tau , pSer262 - Tau , or pSer202 - Tau ) ; scale bar : 5 μm . ( D ) Rab35 overexpression significantly increases PLA puncta for total , pSer396 / 404 - Tau , pSer262 - Tau but not pSer202 - Tau ( n = 121 - 132 cells for total Tau , 178 - 195 cells for pSer396 / 404 - Tau , 71 - 98 cells for pSer262 - Tau , 25 - 29 cells for pSer202 - Tau ; Mann - Whitney test , * * * * p < 0 . 0001 )

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

(C) Images of PLA signal (green) for Hrs/Tau interaction in N2a cells co-transfected with FLAG-Hrs and either mCh or mCh-Rab35 (red), and probed with antibodies against FLAG and total (DA9) or phospho-Tau species (pSer396/404-Tau, pSer262-Tau, or pSer202-Tau); scale bar: 5 μm. (D) Rab35 overexpression significantly increases PLA puncta for total, pSer396/404-Tau, pSer262-Tau but not pSer202-Tau (n=121-132 cells for total Tau, 178-195 cells for pSer396/404-Tau, 71-98 cells for pSer262-Tau, 25-29 cells for pSer202-Tau; Mann-Whitney test, ****p<0.0001)


( A , B ) Representative immunoblots ( A ) and quantification of Hrs , TSG101 , CHMP2b , and Rab35 protein levels ( B ) from 14 DIV neurons treated with either DMSO ( CON ) or glucocorticoids ( GC ) . GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs , TSG101 , or CHMP2b ( n = 9 - 10 / condition , unpaired student ' s t - test , * p = 0 . 0221 )

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

(A, B) Representative immunoblots (A) and quantification of Hrs, TSG101, CHMP2b, and Rab35 protein levels (B) from 14 DIV neurons treated with either DMSO (CON) or glucocorticoids (GC). GC treatment selectively decreases Rab35 protein levels without affecting levels of Hrs, TSG101, or CHMP2b (n=9-10/condition, unpaired student's t-test, *p=0.0221)


( C ) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells ( for neurons , n = 9 / condition , unpaired student ' s t - test , * * p = 0 . 0036 ; for N2a cells , n = 6 / condition , unpaired student ' s t - test , * p = 0 . 0138 )

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

(C) Rab35 mRNA levels are decreased by GC in 14 DIV hippocampal neurons and N2a cells (for neurons, n=9/condition, unpaired student's t-test, **p=0.0036; for N2a cells, n=6/condition, unpaired student's t-test, *p=0.0138)


( E , F ) Representative immunoblots ( E ) and quantification of levels of different Rab proteins ( F ) in hippocampus of GC - treated and CON animals . Protein levels of Rab35 , but not other Rabs , are decreased in GC - treated animals compared to CON ones ( n = 5 animals / condition , unpaired student ' s t - test , * p = 0 . 0257 )

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

(E, F) Representative immunoblots (E) and quantification of levels of different Rab proteins (F) in hippocampus of GC-treated and CON animals. Protein levels of Rab35, but not other Rabs, are decreased in GC-treated animals compared to CON ones (n=5 animals/condition, unpaired student's t-test, *p=0.0257)


( G , H ) Immunofluorescence staining of Rab35 ( green ) and DAPI ( blue ) ( G ) showing that Rab35 fluorescence intensity is reduced in hippocampal area ( CA1 ) of GC - treated aniamls ( H ) ( n = 15 slices / condition ; unpaired student ' s t - test , * * * p = 0 . 0003 ) . Data information : All numeric data represent mean ± SEM

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

(G, H) Immunofluorescence staining of Rab35 (green) and DAPI (blue) (G) showing that Rab35 fluorescence intensity is reduced in hippocampal area (CA1) of GC-treated aniamls (H) (n=15 slices/condition; unpaired student's t-test, ***p=0.0003). Data information: All numeric data represent mean ± SEM


( A , B ) Representative immunoblots ( A ) and quantification of Tau degradation ( B ) in 14 DIV neurons expressing mCh - Rab35 or mCh , treated for 24 h with cycloheximide ( CHX ) or DMSO ( CON ) under GC conditions . GC significantly decreases Tau degradation , whereas Rab35 overexpression blocks this effect ( n = 12 - 14 / condition ; 1 - way ANOVA , Dunnet post - hoc analysis , * pmCh vs mCh GC = 0 , 0319 , * pmCh vs Rab35 GC = 0 , 047 )

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

(A, B) Representative immunoblots (A) and quantification of Tau degradation (B) in 14 DIV neurons expressing mCh-Rab35 or mCh, treated for 24 h with cycloheximide (CHX) or DMSO (CON) under GC conditions. GC significantly decreases Tau degradation, whereas Rab35 overexpression blocks this effect (n=12-14/condition; 1-way ANOVA, Dunnet post-hoc analysis, *pmCh vs mCh GC=0,0319, *pmCh vs Rab35 GC=0,047)


( D , E ) Representative immunoblots ( D of Tau levels ( E ) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP , but not Rab35 ( n = 5 - 6 animals / group , 2 replicates , 2 - way ANOVA , GC x Rab35 interaction F1 , 39 = 10 , 51 p = 0 . 002 ; Sidak posthoc analysis * p = 0 . 0168 )

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

(D, E) Representative immunoblots (D of Tau levels (E) in hippocampal synaptosomes reveal that GC increases total Tau levels in animals expressing EGFP, but not Rab35 (n=5-6 animals/group, 2 replicates, 2-way ANOVA, GC x Rab35 interaction F1, 39=10,51 p=0.002; Sidak posthoc analysis *p=0.0168)


( F ) Golgi - based 3D neuronal reconstruction of hippocampal pyramidal neurons ( CA1 area )

(F) Golgi-based 3D neuronal reconstruction of hippocampal pyramidal neurons (CA1 area)


( G ) GC treatment reduces the length of apical dendrites in animals expressing EGFP , but not EGFP - Rab35 ( n = 6 - 7 animals / group ; 6 - 8 neurons / animal , 2 - way ANOVA , GC x Rab35 interaction F1 , 155 = 3 . 969 p = 0 . 0481 , overall GC effect F1 , 155 = 8 . 998 p = 0 . 0031 , Sidak posthoc analysis * * p = 0 . 0021 )

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

(G) GC treatment reduces the length of apical dendrites in animals expressing EGFP, but not EGFP-Rab35 (n=6-7 animals/group; 6-8 neurons/animal, 2-way ANOVA , GC x Rab35 interaction F1, 155=3.969 p=0.0481, overall GC effect F1, 155=8.998 p=0.0031, Sidak posthoc analysis **p=0.0021)


( H ) GC treatment reduces mature spine density in animals expressing EGFP , but not EGFP - Rab35 ( 2 - way ANOVA , GC x Rab35 interaction F1 , 499 = 12 . 33 p = 0 . 0005 , overall GC effect F1 , 499 = 4 . 373 p = 0 . 0370 , Sidak posthoc analysis * * * p = 0 . 0003 ; n = 6 - 7 animals / group ; 6 - 8 neurons per animal )

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

(H) GC treatment reduces mature spine density in animals expressing EGFP, but not EGFP-Rab35 (2-way ANOVA, GC x Rab35 interaction F1, 499=12.33 p=0.0005, overall GC effect F1, 499=4.373 p=0.0370, Sidak posthoc analysis *** p=0.0003; n=6-7 animals/group; 6-8 neurons per animal)


( I , J ) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP - expressing animals ; however , this effect is not seen in EGFP - Rab35 - expressing animals ( 3 - way ANOVA , GC x Rab35 interaction F1 , 4212 = 14 , 926 p < 0 . 0001 , Simple effect analysis , Sidak test for multiple comparisons * p120 = 0 . 012 , * * p140 = 0 . 001 , * * p220 = 0 . 002 , * * * p240 < 0 . 001 , * * * p260 < 0 . 001 , * * p280 = 0 . 001 , * * p300 = 0 . 001 , * p320 = 0 . 021 , * p340 = 0 . 047 , n = 6 - 7 animals / group , 6 - 8 neurons per animal )

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

(I, J) Sholl analysis of apical dendrites in rat hippocampus shows reduced dendritic intersections after GC treatment in EGFP-expressing animals; however, this effect is not seen in EGFP-Rab35-expressing animals (3-way ANOVA, GC x Rab35 interaction F1, 4212= 14,926 p<0.0001, Simple effect analysis, Sidak test for multiple comparisons *p120=0.012, **p140=0.001, **p220=0.002, ***p240<0.001, ***p260<0.001, **p280=0.001, **p300=0.001, *p320=0.021, *p340=0.047, n=6-7 animals/group, 6-8 neurons per animal)


Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right . Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope - tagged proteins in the anti - Mta1 , Mta2 or Mta3 panels ; and a grey triangle shows the location of IgG bands in the anti - Mbd3 panel . 10 % of the input was loaded into " Input " lanes . Molecular weights in kDa are shown at left .

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

Tagged MTA proteins in heterozygously targeted cell lines were immunoprecipitated and subsequent western blots probed with antibodies indicated at right. Solid black triangles indicate the locations of untagged proteins and open triangles show the position of epitope-tagged proteins in the anti-Mta1, Mta2 or Mta3 panels; and a grey triangle shows the location of IgG bands in the anti-Mbd3 panel. 10% of the input was loaded into "Input" lanes. Molecular weights in kDa are shown at left.


Proteins co - purifying with Mta1 , Mta2 or Mta3 in IP - mass spectrometry experiments in wild type cells ( top ) or Mbd3 - null cells ( bottom ) . Proteins showing significant enrichment with the bait protein are located outside the dotted lines . For all panels the protein being immunoprecipitated is indicated in red , the other MTA proteins in purple , and other NuRD components in blue . Each IP / Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation .

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

Proteins co-purifying with Mta1, Mta2 or Mta3 in IP-mass spectrometry experiments in wild type cells (top) or Mbd3-null cells (bottom). Proteins showing significant enrichment with the bait protein are located outside the dotted lines. For all panels the protein being immunoprecipitated is indicated in red, the other MTA proteins in purple, and other NuRD components in blue. Each IP/Mass spec experiment set comprised three independent immunoprecipitations from one nuclear extract preparation.


Relative enrichment of indicated proteins in MTA pulldowns from wild type ( WT ) or Mbd3 - null ( Mbd3KO ) ES cells , normalised to 2x MTA proteins . NuRD components comprising the remodelling subunit are labelled in blue , those comprising the deacetylase subunit in red . As MTA proteins are the bait in these experiments , they may be isolated more efficiently than their interaction partners . Error bars represent standard deviation from three ( Mta1 and Mta2 ) or six ( Mta3 ) replicate pulldowns .

Relative enrichment of indicated proteins in MTA pulldowns from wild type (WT) or Mbd3-null (Mbd3KO) ES cells, normalised to 2x MTA proteins. NuRD components comprising the remodelling subunit are labelled in blue, those comprising the deacetylase subunit in red. As MTA proteins are the bait in these experiments, they may be isolated more efficiently than their interaction partners. Error bars represent standard deviation from three (Mta1 and Mta2) or six (Mta3) replicate pulldowns.


ChIP - seq peaks identified for Mta1 and Mta2 ( left ) , Mta1 and Mta3 ( middle ) or Mta2 and Mta3 ( right ) are plotted by enrichment for each protein . Peaks called for both proteins are indicated in the overlap .

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

ChIP-seq peaks identified for Mta1 and Mta2 (left), Mta1 and Mta3 (middle) or Mta2 and Mta3 (right) are plotted by enrichment for each protein. Peaks called for both proteins are indicated in the overlap.


ChIP - seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta - bound sites . Mta1 / 2 / 3 refers to peaks identified in all three ChIP - seq datasets , whereas " Mta1 Only " , " Mta2 Only " or " Mta3 Only " refer to peaks only called for that protein .

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

ChIP-seq enrichment for indicated proteins or histone modifications is plotted across different subsets of Mta-bound sites. Mta1/2/3 refers to peaks identified in all three ChIP-seq datasets, whereas "Mta1 Only", "Mta2 Only" or "Mta3 Only" refer to peaks only called for that protein.


Average enrichment of density plots are plotted for different subsets of Mta ChIP - seq peaks .

Average enrichment of density plots are plotted for different subsets of Mta ChIP-seq peaks.


Western blots of wild type ( Control ) , double or triple - null ES cell nuclear extract was probed with antibodies indicated at right . LaminB1 acts as a loading control . Approximate sizes are indicated in kDa .

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

Western blots of wild type (Control), double or triple-null ES cell nuclear extract was probed with antibodies indicated at right. LaminB1 acts as a loading control. Approximate sizes are indicated in kDa.


Phase - contrast images of wild type or Mta123 ∆ ES cells in 2i + LIF conditions . Scale bars represent

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

Phase-contrast images of wild type or Mta123∆ ES cells in 2i+LIF conditions. Scale bars represent 100µ


Western blots of anti - Chd4 immunoprecipitation of nuclear extract from indicated cell lines ( top ) probed with antibodies indicated at right . For the Gatad2b and Mbd3 blots , arrowheads indicate target proteins , while an asterisk marks a non - specific IgG bands . 10 % of the input was loaded into " Input " lanes . Approximate sizes are indicated in kDa .

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

Western blots of anti-Chd4 immunoprecipitation of nuclear extract from indicated cell lines (top) probed with antibodies indicated at right. For the Gatad2b and Mbd3 blots, arrowheads indicate target proteins, while an asterisk marks a non-specific IgG bands. 10% of the input was loaded into "Input" lanes. Approximate sizes are indicated in kDa.


Western blot of a time course of Mta3 deletion in Mta1 ∆ Mta2 ∆ Mta3Flox / Flox : Cre - ER ( Mta12 ∆ 3F / F ) or Control ( Mta12 ∆ 3F / F without Cre - ER ) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control ( αRNAPII ) . The time course is indicated at the top as Days + tamoxifen .

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

Western blot of a time course of Mta3 deletion in Mta1∆Mta2∆Mta3Flox/Flox: Cre-ER (Mta12∆3F/F) or Control (Mta12∆3F/F without Cre-ER) ES cells probed for indicated NuRD component proteins or RNA Polymerase II as a loading control (αRNAPII). The time course is indicated at the top as Days + tamoxifen.


Western blot showing rescue of Mta123 ∆ ES cells by ectopic expression of Mta1 , Mta2 or Mta3 from a transgene ( TG ) . Total RNA Polymerase II acts as a loading control ( αRNAPII ) . For all western blots molecular weight is indicated at left in kDa .

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

Western blot showing rescue of Mta123∆ ES cells by ectopic expression of Mta1, Mta2 or Mta3 from a transgene (TG). Total RNA Polymerase II acts as a loading control (αRNAPII). For all western blots molecular weight is indicated at left in kDa.


Comparison of gene expression in Mta123 ∆ ES cells and wild type ( WT ) ES cells . Each circle represents a gene : red indicates spike - in controls , blue indicates genes that are not differentially expressed to a significant degree , and green indicates differentially expressed genes ( 2404 increased , 1293 decreased ) defined with an adjusted p - value < 0 . 05 and a log2 fold - change > 1 . N = 3 for each genotype .

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

Comparison of gene expression in Mta123∆ ES cells and wild type (WT) ES cells. Each circle represents a gene: red indicates spike-in controls, blue indicates genes that are not differentially expressed to a significant degree, and green indicates differentially expressed genes (2404 increased, 1293 decreased) defined with an adjusted p-value < 0.05 and a log2 fold-change > 1. N=3 for each genotype.


Fold change in gene expression is plotted for different subsets of genes . All genes are plotted in black , while subsets of genes located nearest to ChIP - seq peaks for the indicated proteins + Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated . The number of genes in each of the Mta categories are : all genes ( 32271 ) , all differentially expressed ( n = 3701 ) , Mta1 + 2 + 3 ( n = 1738 ) , Mta1 + 2 ( n = 1460 ) , Mta1 ( n = 1020 ) and Mta2 ( n = 924 ) .

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

Fold change in gene expression is plotted for different subsets of genes. All genes are plotted in black, while subsets of genes located nearest to ChIP-seq peaks for the indicated proteins +Chd4 and which show significant changes in expression compared to wild type cells are plotted in dashed coloured lines as indicated. The number of genes in each of the Mta categories are: all genes (32271), all differentially expressed (n=3701), Mta1+2+3 (n=1738), Mta1+2 (n=1460), Mta1 (n=1020) and Mta2 (n=924).


Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self - renewing conditions ( 2i ) or after 48 hours in the absence of two inhibitors and LIF ( Diff48 ) . Each point represents a biological replicate .

Principal component analysis of RNAseq data from ES cell lines of indicated genotypes in either self-renewing conditions (2i) or after 48 hours in the absence of two inhibitors and LIF (Diff48). Each point represents a biological replicate.