Figure 2 ( A ) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy - related genes in HCT116 cells . Lysates were extracted for immunoprecipitation with CHK2 - specific antibody or control IgG , followed by probing with antibodies specific for Ulk1 , Atg5 , Beclin1 , Atg7 or LC3 . ( B ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after HBSS starvation in the cytoplasmic fractions . ( C ) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions ( t = 0 ) and after H2O2 ( 500μM ) stimulation in the cytoplasmic fractions . ( D ) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation ( E ) The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 ( 500μM ) stimulation ( F ) . The cytoplasmic lysates were subjected to immunoprecipitation with anti - Beclin 1 antibody followed by immunoblotting with anti - CHK2 and anti - Beclin 1 antibody . The expression of p - CHK2 Thr68 , CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions . Tubulin was used as a loading control . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were untreated ( G ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with HBSS starvation ( H ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . HCT116 cells were transiently transfected with the expression plasmids as indicated . After 36 hr post - transfection , cells were treated with H2O2 ( 500μM ) stimulation ( I ) for 1hr and then collected for immunoprecipitation and western blotting analysis . Immunoprecipitation was performed using anti - Beclin 1 antibody followed by immunoblotting with anti - Flag or anti - Beclin 1 antibody . ( J ) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 ( 500 μM ) cultured for 3 hr . ( K ) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 ( 500μM ) stimulation . Western blot detection of p - CHK2 Thr68 , CHK2 , p62 and LC3 .

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

Figure 2(A) Immunoprecipitation assays testing the physical interaction between CHK2 and proteins encoded by autophagy-related genes in HCT116 cells. Lysates were extracted for immunoprecipitation with CHK2-specific antibody or control IgG, followed by probing with antibodies specific for Ulk1, Atg5, Beclin1, Atg7 or LC3.(B) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after HBSS starvation in the cytoplasmic fractions.(C) Interaction between endogenous CHK2 and Beclin 1 in HCT116 cells under fed conditions (t=0) and after H2O2 (500μM) stimulation in the cytoplasmic fractions.(D) HCT116 cells were pretreated with NAC in RPMI 1640 complete medium for 4 h and then cultured for 1 hr in HBSS starvation. The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr and then cultured for 1 hr in HBSS starvation (E) The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were pretreated with CHK2 inhibitorⅡ in RPMI 1640 complete medium for 4 hr or H2O2 (500μM) stimulation (F). The cytoplasmic lysates were subjected to immunoprecipitation with anti-Beclin 1 antibody followed by immunoblotting with anti-CHK2 and anti-Beclin 1 antibody. The expression of p-CHK2 Thr68, CHK2 and Beclin 1 were monitored by immunoblotting in the cytoplasmic fractions. Tubulin was used as a loading control.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were untreated (G) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with HBSS starvation (H) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.HCT116 cells were transiently transfected with the expression plasmids as indicated. After 36 hr post-transfection, cells were treated with H2O2 (500μM) stimulation (I) for 1hr and then collected for immunoprecipitation and western blotting analysis. Immunoprecipitation was performed using anti-Beclin 1 antibody followed by immunoblotting with anti-Flag or anti-Beclin 1 antibody.(J) Western blot detection of p62 and LC3 in H1299 cells transfected with indicated plasmid in normal medium or after H2O2 (500 μM) cultured for 3 hr.(K) H1299 cells were pretreated with CHK2 inhibitorⅡ in complete medium for 4 h and then cultured for 3 hr in H2O2 (500μM) stimulation. Western blot detection of p-CHK2 Thr68, CHK2, p62 and LC3.


Figure 3 ( A ) The indicated recombinant fragments of GST - Beclin1 were incubated with recombinant CHK2 in the presence of 32P - labeled ATP for in vitro kinase assays . MBP was used as a positive control . Ponceau staining indicates the expression of GST - Beclin 1 fragments for in vitro kinase assays . The asterisks in the blot indicate the domain of phosphorylated Beclin 1 . ( C ) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST - Beclin 1 - WT , S9093A ( AA ) , S90A and S93A protein . Reactions were analyzed by SDS - PAGE followed by autoradiography . The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants . Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation ( D ) Western blot detection of p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 ( 500μM ) stimulation ( E ) . Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation ( F ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 ( 500μM ) stimulation ( G ) . ( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation .

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

Figure 3(A) The indicated recombinant fragments of GST-Beclin1 were incubated with recombinant CHK2 in the presence of 32P-labeled ATP for in vitro kinase assays. MBP was used as a positive control. Ponceau staining indicates the expression of GST-Beclin 1 fragments for in vitro kinase assays. The asterisks in the blot indicate the domain of phosphorylated Beclin 1.(C) In vitro kinase assays to test the ability of recombinant CHK2 to phosphorylate recombinant GST-Beclin 1-WT, S9093A (AA), S90A and S93A protein. Reactions were analyzed by SDS-PAGE followed by autoradiography. The asterisks in the blot indicate the phosphorylation of Beclin 1 mutants.Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or after 1 hr HBSS starvation (D)Western blot detection of p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in MEFs indicated genotype in normal medium or H2O2 (500μM) stimulation (E).Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or after HBSS starvation (F)Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with indicated shRNA in normal medium or H2O2 (500μM) stimulation (G).(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells pretreated with NAC for 4 h and then cultured for 1 hr in HBSS starvation.


Figure 4 ( A ) The effect of Beclin 1 Ser90 / 93 phosphorylation on the interaction between Beclin 1 and Bcl - 2 . GST - Beclin1 WT , AA mutant and DD mutant ( bait ) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag - Bcl - 2 ( preys ) . ( B ) Immunoprecipitation of Bcl - 2 with Flag - Beclin 1 WT , AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 ( 500μM ) treatment for 1h . ( C - D ) Autophagy levels were determined in Beclin 1 - depleted H1299 cells with reconstituted expression of Beclin 1 WT , AA mutant or DD mutant in normal medium or after H2O2 ( 500μM ) treatment for 3h in the presence ( C ) or absence ( D ) of CHK2 . ( E ) Representative electron microscopic image of an autophagosome ( arrow , left panel , AP ) and an autolysosome ( arrow , right panel , AL ) in H1299 Beclin 1 WT cells treated with H2O2 ( 500μM ) for 6 hr . Scale bars , 500 nm . Electron microscopic quantification of autophagosomes ( AP ) and autolysosomes ( AL ) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 ( 500μM ) treatment . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 001 compared to Beclin 1 WT ; # P < 0 . 001 compared to Beclin 1 WT treated with H2O2 ; & P < 0 . 001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2 . ( F ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 , Beclin 1 , p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 ( 500μM ) treatment for 3h .

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

Figure 4(A) The effect of Beclin 1 Ser90/93 phosphorylation on the interaction between Beclin 1 and Bcl-2. GST-Beclin1 WT, AA mutant and DD mutant (bait) were incubated with or without recombinant CHK2 in the presence of ATP and then incubated with flag-Bcl-2 (preys).(B) Immunoprecipitation of Bcl-2 with Flag-Beclin 1 WT, AA mutant and DD mutant in HCT116 cells in normal medium or after H2O2 (500μM) treatment for 1h.(C-D) Autophagy levels were determined in Beclin 1-depleted H1299 cells with reconstituted expression of Beclin 1 WT, AA mutant or DD mutant in normal medium or after H2O2 (500μM) treatment for 3h in the presence (C) or absence (D) of CHK2.(E) Representative electron microscopic image of an autophagosome (arrow, left panel, AP) and an autolysosome (arrow, right panel, AL) in H1299 Beclin 1 WT cells treated with H2O2 (500μM) for 6 hr. Scale bars, 500 nm. Electron microscopic quantification of autophagosomes (AP) and autolysosomes (AL) in H1299 cells with the indicated plasmids cultured for 6 hr in normal or H2O2 (500μM) treatment. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.001 compared to Beclin 1 WT; #P < 0.001 compared to Beclin 1 WT treated with H2O2; &P < 0.001 compared to Beclin 1 WT treated with H2O2 in the absence of CHK2.(F) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93, Beclin 1, p62 and LC3 in H1299 cells transfected with the indicated shRNA in normal medium or after H2O2 (500μM) treatment for 3h.


Figure 5 ( A ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation . ( B ) Western blot detection of p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia . ( C ) Western blot detection of ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation . ( D ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia . ( E ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to hypoxia with or without pretreatment with NAC . ( F ) P - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , AMPKα , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells , and the cells were subjected to glucose starvation with or without pretreatment with NAC . ( G ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 , and AMPKα in H1299 cells in normal medium or after H2O2 ( 25μM ) stimulation . ( H ) Western blot detection of p - ATM Ser1981 , ATM , p - CHK2 Thr68 , CHK2 , p - AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG ( 5mM ) and oligomycin ( 2 . 5μM ) treatment .

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

Figure 5(A) Western blot detection of p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.(B) Western blot detection of p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.(C) Western blot detection of ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after glucose starvation.(D) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in H1299 cells transfected with the indicated shRNA in normal medium or after hypoxia.(E) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to hypoxia with or without pretreatment with NAC.(F) P-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, AMPKα, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 were analyzed in H1299 cells, and the cells were subjected to glucose starvation with or without pretreatment with NAC.(G) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172, and AMPKα in H1299 cells in normal medium or after H2O2 (25μM) stimulation.(H) Western blot detection of p-ATM Ser1981, ATM, p-CHK2 Thr68, CHK2, p-AMPKα Thr172 and AMPKα in H1299 cells in normal medium or after 2DG (5mM) and oligomycin (2.5μM) treatment.


Figure 6Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation ( Glu - ) Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia ( B ) . ( C ) Western blot detection of CHK2 , HSP60 , TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation ( Glu - ) or 36 hr hypoxia . ( D - E ) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation ( Glu - ) medium or hypoxia . The yellow asterisks mark mitochondria . The red arrows indicate autophagic structures . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * * * P < 0 . 001 compared to shNC treated with H2O2 or hypoxia ( Mann - Whitney test ) ; Scale bar , 1 μm . ( F ) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm . Scale bar , 10 μm . The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation ( G ) All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) . The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia ( H ) . All results are from three independent experiments . All quantitative data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 ( Mann - Whitney test ) .

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

Figure 6Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 12hr glucose starvation (Glu-)Intracellular ROS levels detected in H1299 cells transfected with the indicated shRNA in normal medium or after 36hr hypoxia (B).(C) Western blot detection of CHK2, HSP60, TOM20 and TIM23 in H1299 cells transfected with the indicated shRNA in normal medium or after12 hr glucose starvation (Glu-) or 36 hr hypoxia.(D-E) Transmission electron microscopy of indicated H1299 cells cultured in glucose starvation (Glu-) medium or hypoxia. The yellow asterisks mark mitochondria. The red arrows indicate autophagic structures. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; ***P < 0.001 compared to shNC treated with H2O2 or hypoxia (Mann-Whitney test); Scale bar, 1 μm.(F) Representative confocal images of the shift in the fluorescence emission of Keima from 458 to 561 nm. Scale bar, 10 μm.The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or after 18 hr glucose starvation (G) All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).The mitochondrial membrane potential analysis of 293 cells transfected with the indicated shRNA in normal medium or 48 hr hypoxia (H). All results are from three independent experiments. All quantitative data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 (Mann-Whitney test).


Figure 7H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation ( A ) Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments ; * p < 0 . 05 compared to NC glucose starvation ( A ) ( Student ' s t - test ) . H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation ( B ) . Representative FACS analysis of apoptosis . Data are presented as mean ± s . e . m . from 3 independent experiments * * p < 0 . 01 compared to NC hypoxia ( B ) ( Student ' s t - test ) . H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation ( C ) Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) . H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation ( D ) . Representative FACS analysis of apoptosis . Results from three independent experiments are presented as a histogram . Data are presented as mean ± s . e . m . from 3 independent experiments ; * P < 0 . 05 compared to Beclin 1 S9093A ( AA ) treated with H2O2 or hypoxia ( Mann - Whitney test ) . ( E ) CHK2 + / + and CHK2 - / - mice were subjected to MCAO for 1 hr and reperfusion for 12 h . Contralateral ( C ) and ipsilateral ( I ) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC . ( F ) The infarct volume was determined by measuring infarct size relative to normal in the slice . ( n = 5 mice ) . Data are presented as mean ± s . e . m . ; * * P < 0 . 01 ( Student ' s t - test ) . ( G - J ) Immunoblots for p62 , p - Beclin 1 Ser90 , p - Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion - treated CHK2 + / + and CHK2 - / - mice . Quantification of p62 , p - Beclin 1 Ser90 and p - Beclin 1 Ser93 protein levels ( n = 3 mice ) . Data are presented as mean ± s . e . m . ; * P < 0 . 05 , * * P < 0 . 01 ( Student ' s t - test ) .

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

Figure 7H1299 cells transfected with the indicated shRNA in normal medium or after 12h glucose starvation (A) Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments; *p < 0.05 compared to NC glucose starvation (A) (Student's t-test).H1299 cells transfected with the indicated shRNA in normal medium or 48h hypoxia stimulation (B). Representative FACS analysis of apoptosis. Data are presented as mean ± s.e.m. from 3 independent experiments **p < 0.01 compared to NC hypoxia (B) (Student's t-test).H1299 cells transfected with the indicated plasmids in normal medium or after 12h glucose starvation (C) Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).H1299 cells transfected with the indicated plasmids in normal medium or 48h hypoxia stimulation (D). Representative FACS analysis of apoptosis. Results from three independent experiments are presented as a histogram. Data are presented as mean ± s.e.m. from 3 independent experiments; *P < 0.05 compared to Beclin 1 S9093A (AA) treated with H2O2 or hypoxia (Mann-Whitney test).(E) CHK2+/+ and CHK2-/- mice were subjected to MCAO for 1 hr and reperfusion for 12 h. Contralateral (C) and ipsilateral (I) tissues of the mouse brain were coronally sectioned and stained with 2 % TTC.(F) The infarct volume was determined by measuring infarct size relative to normal in the slice. (n = 5 mice). Data are presented as mean ± s.e.m.; **P < 0.01 (Student's t-test).(G-J) Immunoblots for p62, p-Beclin 1 Ser90, p-Beclin 1 Ser93 and Beclin 1 in the cortical extracts from ischemia and reperfusion-treated CHK2+/+ and CHK2-/- mice. Quantification of p62, p-Beclin 1 Ser90 and p-Beclin 1 Ser93 protein levels (n = 3 mice). Data are presented as mean ± s.e.m.; *P < 0.05, **P < 0.01 (Student's t-test).


Figure 1 ( A ) NPM1 expression in CD34 + Lin - cells from myelodysplastic syndrome ( MDS ) patients compared to healthy volunteers ( n = 29 ) . ( B ) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia ( AML ) with multi - lineage dysplasia ( MLD ) . Insets show representative staining in blasts [ B ] and in differentiated myeloid cells [ e . g . , neutrophil , M ] with a high magnification . ( C ) Percentages of patient samples with NPM1 - low and NPM1 - normal in healthy , MDS and MLD in AML groups .

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Figure 1(A) NPM1 expression in CD34+Lin- cells from myelodysplastic syndrome (MDS) patients compared to healthy volunteers (n = 29).(B) Representative staining of NPM1 protein levels in bone marrow samples of MDS or acute myeloid leukemia (AML) with multi-lineage dysplasia (MLD). Insets show representative staining in blasts [B] and in differentiated myeloid cells [e.g., neutrophil, M] with a high magnification.(C) Percentages of patient samples with NPM1-low and NPM1-normal in healthy, MDS and MLD in AML groups.


Figure 2 ( C ) Representative distribution of Tubulin and CDC42 in Ctrl , 4 - months old Npm1 cKO and 2 - years old WT HSCs determined by IF . Scale bars , 5 μm . ( F ) HSCs of Ctrl , 4 - months old Npm1 cKO and 2 - years old WT mice were sorted and stained for TOM20 . Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) in single cells ( n = 58 cells in Ctrl , n = 70 cells in cKO and n = 23 cells in Old , from 3 mice per group ) , identified by volume rendering of TOM20 . Scale bars , 2 μm . ( J ) Representative images ( top ) and quantification of mitochondrial volume and number ( bottom ) , identified by volume rendering of TOM20 , in MDS - L cells transfected with siNPM1 or control ( siCTRL ) ( n = 58 cells per group , from 3 transfected MDS - L cultures ) . Scale bars , 2 μm .

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Figure 2(C) Representative distribution of Tubulin and CDC42 in Ctrl, 4-months old Npm1 cKO and 2-years old WT HSCs determined by IF. Scale bars, 5 μm.(F ) HSCs of Ctrl, 4-months old Npm1 cKO and 2-years old WT mice were sorted and stained for TOM20. Representative images (top) and quantification of mitochondrial volume and number (bottom) in single cells (n = 58 cells in Ctrl, n = 70 cells in cKO and n = 23 cells in Old, from 3 mice per group), identified by volume rendering of TOM20. Scale bars, 2 μm.(J) Representative images (top) and quantification of mitochondrial volume and number (bottom), identified by volume rendering of TOM20, in MDS-L cells transfected with siNPM1 or control (siCTRL) (n = 58 cells per group, from 3 transfected MDS-L cultures). Scale bars, 2 μm.


Figure 3 ( F ) Representative peripheral blood ( PB ) smears . Insets show the differentiated granulocytes ( a , b ) in control mice , and anisocytosis ( c ) , dysplastic myeloid cells ( d - g ) in Npm1 cKO mice ( Npm1F / FVav1Cre + ) . Npm1 cKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ; schistocyte , blue - outlined arrowhead ; tear drop , Howell - Jolly bodies , black arrowhead ; black - outlined arrowhead ) , dysplastic monocytes ( black arrow ) , and dysplastic neutrophils ( d - f ) . Scale bars , 10 μm .

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Figure 3(F) Representative peripheral blood (PB) smears. Insets show the differentiated granulocytes (a, b) in control mice, and anisocytosis (c), dysplastic myeloid cells (d-g) in Npm1 cKO mice (Npm1F/FVav1Cre+). Npm1 cKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead; schistocyte, blue-outlined arrowhead; tear drop, Howell-Jolly bodies, black arrowhead; black-outlined arrowhead), dysplastic monocytes (black arrow), and dysplastic neutrophils (d-f). Scale bars, 10 μm.


Figure 4 ( C ) Npm1 and p53 protein levels in KSL cells of Ctrl ( Npm1F / FVav1Cre - ) , and cKO ( Npm1F / FVav1Cre + ) mice . ( F ) Representative PB smears of DKO mice . Insets show anisocytosis ( a ) , poikilocytosis ( b ) and dysplastic myeloid cell ( c ) . DKO mice show dysplastic erythroid cells ( polychromatophilic , blue arrowhead ) , Howell - Jolly bodies , ( black arrowhead in insert a ) , dysplastic monocytes ( black arrow , and i - v ) , dysplastic neutrophils ( vi ) , a pseudo - Pelger - Huet anomaly ( ii ) , and dysplastic platelets ( giant platelet , iv and vii ) . Scale bars , 10μm . ( I ) Representative H & E staining of sections of the spleen of 2 - month - old DKO mice with myeloid disorders . Arrowheads ( right ) indicate the infiltrating myeloid blasts . Scale bars , 250 μm ( left ) and 10μm ( right ) , respectively .

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

Figure 4(C) Npm1 and p53 protein levels in KSL cells of Ctrl (Npm1F/FVav1Cre-), and cKO (Npm1F/FVav1Cre+) mice.(F) Representative PB smears of DKO mice. Insets show anisocytosis (a), poikilocytosis (b) and dysplastic myeloid cell (c). DKO mice show dysplastic erythroid cells (polychromatophilic, blue arrowhead), Howell-Jolly bodies, (black arrowhead in insert a), dysplastic monocytes (black arrow, and i-v), dysplastic neutrophils (vi), a pseudo-Pelger-Huet anomaly (ii), and dysplastic platelets (giant platelet, iv and vii). Scale bars, 10μm.(I) Representative H&E staining of sections of the spleen of 2-month-old DKO mice with myeloid disorders. Arrowheads (right) indicate the infiltrating myeloid blasts. Scale bars, 250 μm (left) and 10μm (right), respectively.


Figure 2B . Growth comparisons of selected kinases with the kinase ‑ dead mutant match . 24h growth curves ( OD595 ) from liquid culture of yeast strains ( W303 ) expressing human kinase in the presence or absence of 3 . 2 % ( v / v ) DMSO . Upper panel : BUB1 : BUB1 wild type , BUB1 - KD : BUB1 ( K821M ) ; upper middle panel : NEK6 : NEK6 wild type , NEK6 - KD : NEK6 ( K74M & K75M ) ; lower middle panel : PKCα - ac : PKCα ( A25E ) , PKCα - KD : PKCα ( K368R ) ; lower panel : Control with empty plasmid . Error bars represent SD from three biological replicates . C . Human protein kinases phosphorylate large sets of yeast proteins . Upper panel : In total , 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho - motif antibodies on yeast proteins in comparison to other human kinases , kinase ‑ dead mutant versions or vector control samples . Commercially available pS / T - antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases . Lower panel : Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded . When developed with the indicated phospho - motif recognizing antibodies , immunoreactive bands indicated phosphorylation of yeast proteins .

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Figure 2B. Growth comparisons of selected kinases with the kinase‑dead mutant match. 24h growth curves (OD595) from liquid culture of yeast strains (W303) expressing human kinase in the presence or absence of 3.2% (v/v) DMSO. Upper panel: BUB1: BUB1 wild type, BUB1-KD: BUB1(K821M); upper middle panel: NEK6: NEK6 wild type, NEK6-KD: NEK6(K74M&K75M); lower middle panel: PKCα-ac: PKCα(A25E), PKCα-KD: PKCα(K368R); lower panel: Control with empty plasmid. Error bars represent SD from three biological replicates.C. Human protein kinases phosphorylate large sets of yeast proteins. Upper panel: In total, 27 human protein kinases showed activity towards yeast proteins visible on western blots through enhanced reactivity with phospho-motif antibodies on yeast proteins in comparison to other human kinases, kinase‑dead mutant versions or vector control samples. Commercially available pS/T-antibodies with relatively low sequence specificities were chosen to enable detection of phosphorylation of the yeast proteome by human kinases. Lower panel: Examples of western blots with equal amount of whole yeast cell lysate from strains expressing human kinases loaded. When developed with the indicated phospho-motif recognizing antibodies, immunoreactive bands indicated phosphorylation of yeast proteins.


Figure 5C . Y2H interaction experiment with human AAR2 ( FL ) as bait and PRP8 ( aa1755 - 2335 ) as prey . Growth on selective media is abolished when AAR2 carries a S284E phospho - mimicry amino acid substitution . Mutation of second candidate phospho - site , AAR2 ( T356 ) , does not influence the AAR2 - PRP8 protein interaction . D . Interaction of AAR2 and PRP8 in human cells . HA ‑ tagged PRP8 ( aa1755 - 2335 ) was co - expressed in HEK293 cells with either wildtype AAR2 , AAR2 ( S284A ) or AAR2 ( S284E ) mutant versions . Immunoprecipitation of PRP8 led to a co - precipitation of wild typeAAR2 and phospho - null AAR2 ( S284A ) . In contrast , the phospho - mimicry AAR2 ( S284E ) version did not bind PRP8 . E . Results of the phospho - Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8 . Growth on selective media ( SD5 ) , of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co - expression of either SGK2 or CK2α1 ( CSNK2A1 ) . Growth reduction is visible in a 1 : 10 dilution series on selective agar ( SD5 ) in comparison to the control media ( SD3 ) . Ctrl : non - related kinases . F . Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases . Experiment as in D . Co - expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co - immunoprecipitated with RPR8 .

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

Figure 5C. Y2H interaction experiment with human AAR2 (FL) as bait and PRP8 (aa1755-2335) as prey. Growth on selective media is abolished when AAR2 carries a S284E phospho-mimicry amino acid substitution. Mutation of second candidate phospho-site, AAR2(T356), does not influence the AAR2-PRP8 protein interaction.D. Interaction of AAR2 and PRP8 in human cells. HA‑tagged PRP8 (aa1755-2335) was co-expressed in HEK293 cells with either wildtype AAR2, AAR2(S284A) or AAR2(S284E) mutant versions. Immunoprecipitation of PRP8 led to a co-precipitation of wild typeAAR2 and phospho-null AAR2(S284A). In contrast, the phospho-mimicry AAR2(S284E) version did not bind PRP8.E. Results of the phospho-Y2H screen to identify kinases modulating the interaction of AAR2 and PRP8. Growth on selective media (SD5), of strains carrying AAR2 bait and PRP8 prey plasmids is reduced through co-expression of either SGK2 or CK2α1(CSNK2A1). Growth reduction is visible in a 1:10 dilution series on selective agar (SD5) in comparison to the control media (SD3). Ctrl: non-related kinases.F. Interaction of AAR2 and PRP8 in human cells in the presences of SGK and CK2α1 kinases. Experiment as in D. Co-expression of wild type SGK2 and CK2α1 reduces the amount of AAR2 co-immunoprecipitated with RPR8.


Figure 6B . Results of the phospho - Y2H screen to identify kinases modulating the interaction of the ERα with 14 ‑ 3 ‑ 3β . The interaction of ERα ( prey ) with 14 ‑ 3 ‑ 3β ( bait ) is promoted by the co - expression of wildtype kinases of PAK5 , CDK17 , BUB1 and NEK6 , visible by the yeast growth on selective media in comparison to the control ( three biological replicas are shown ) . The coexpression of the kinase ‑ dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14 ‑ 3 ‑ 3β . In contrast the kinase ‑ dead mutant versions of BUB1 ( K821M ) and NEK6 ( K74M & K75M ) did not promote the Y2H interaction . C . Phosphorylation of ERα in mammalian cells . ERα was co - expressed with the indicated wildtype kinases or kinase ‑ dead versions in HEK293 cells and immunoprecipitated . Phosphorylation status of ERα was analyzed with an in - gel phosphostain ( Pro - Q ® Diamond phosphoprotein gel stain , Invitrogen ) , relative to the whole protein stain ( SYPRO ® Ruby protein gel stain , Invitrogen ) . D . Interaction of ERα and 14 ‑ 3 ‑ 3β in a GST pull - down assay . GST ‑ tagged 14 ‑ 3 ‑ 3β was expressed in E . coli and immobilized on beads . PA ‑ tagged ERα was co - expressed with either wildtype PAK5 or NEK6 , the kinase ‑ dead ( KD ) version or without a kinase in HEK293 cells . Immobilized 14 ‑ 3 ‑ 3β was used to pull PA ‑ tagged ERα from the HEK lysates . Co - precipitation of ERα was successful in the presence of wild kinases , but not in their absence or with kinase ‑ dead mutant versions . E . Effect of NEK6 co - expression on ERα gene activation . Relative luciferase activity of ERα in the presence and absence of 1 nM E2 ( 17β - estradiol ) was measured . A dose ‑ dependent reduction ( ng plasmid transfected ) in E2 - Erα ‑ dependent luciferase expression was observed in HEK293T , MCF - 7 and US - OS cancer cell lines . This effect was not observed with the NEK6 ( K74M & K75M ) kinase ‑ dead version . Graphs represent ≥ 5 biological replicates , each derived from averaging 6 technical replicates . Error bars indicate SD across the biological replicates .

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

Figure 6B. Results of the phospho-Y2H screen to identify kinases modulating the interaction of the ERα with 14‑3‑3β. The interaction of ERα (prey) with 14‑3‑3β (bait) is promoted by the co-expression of wildtype kinases of PAK5, CDK17, BUB1 and NEK6, visible by the yeast growth on selective media in comparison to the control (three biological replicas are shown). The coexpression of the kinase‑dead mutants of PAK5 and CDK17 also promoted the interaction of ERα and 14‑3‑3β. In contrast the kinase‑dead mutant versions of BUB1(K821M) and NEK6(K74M&K75M) did not promote the Y2H interaction.C. Phosphorylation of ERα in mammalian cells. ERα was co-expressed with the indicated wildtype kinases or kinase‑dead versions in HEK293 cells and immunoprecipitated. Phosphorylation status of ERα was analyzed with an in-gel phosphostain (Pro-Q® Diamond phosphoprotein gel stain, Invitrogen), relative to the whole protein stain (SYPRO®Ruby protein gel stain, Invitrogen).D. Interaction of ERα and 14‑3‑3β in a GST pull-down assay. GST‑tagged 14‑3‑3β was expressed in E. coli and immobilized on beads. PA‑tagged ERα was co-expressed with either wildtype PAK5 or NEK6, the kinase‑dead (KD) version or without a kinase in HEK293 cells. Immobilized 14‑3‑3β was used to pull PA‑tagged ERα from the HEK lysates. Co-precipitation of ERα was successful in the presence of wild kinases, but not in their absence or with kinase‑dead mutant versions.E. Effect of NEK6 co-expression on ERα gene activation. Relative luciferase activity of ERα in the presence and absence of 1 nM E2 (17β-estradiol) was measured. A dose‑dependent reduction (ng plasmid transfected) in E2-Erα‑dependent luciferase expression was observed in HEK293T, MCF-7 and US-OS cancer cell lines. This effect was not observed with the NEK6(K74M&K75M) kinase‑dead version. Graphs represent ≥ 5 biological replicates, each derived from averaging 6 technical replicates. Error bars indicate SD across the biological replicates.


Figure 1 ( A ) Unlabeled or Cy5 - labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase ( dsRNA - RL ) or the GFP ( dsRNA - GFP ) coding sequences visualised on gel stained with ethidium bromide ( EtBr - stained , top ) or by in - gel fluorescence ( bottom ) . ( B ) Gating strategy used in the flow cytometry - based assay . First , cells were selected ( side scatter ( SSC ) vs . forward scatter ( FSC ) ) and gated for singlets ( SSC - Width ( W ) vs . FSC ) to hence exclude doublets or cell aggregates . The single cell gate was then further analysed for DAPI , gating on the live cells ( DAPI negative ) . DsRNA - transfected cells ( Cy5 - positive ) were then gated and the level of GFP expression analysed within that population . Flow cytometry counting beads ( gated on SSC vs . FSC plot ) were added to quantify cell number in each sample . ( C ) mESCs stably expressing GFP ( mESCs - GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and the level of GFP in Cy5 + cells was analysed 3 days later and is represented as histograms ( left ) . Right panel , bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control ( D ) mESCs stably expressing d2GFP ( mESCs - d2GFP ) were mock transfected or transfected with the indicated Cy5 - labeled dsRNA . The level of d2GFP was analysed at the indicated timepoints post - transfection and displayed as in ( C ) . ( E ) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5 - labeled dsRNA and d2GFP level was measured as in ( D ) .

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

Figure 1(A) Unlabeled or Cy5-labeled dsRNA corresponding to the first 200 nucleotides of either the Renilla luciferase (dsRNA-RL) or the GFP (dsRNA-GFP) coding sequences visualised on gel stained with ethidium bromide (EtBr-stained, top) or by in-gel fluorescence (bottom).(B) Gating strategy used in the flow cytometry-based assay. First, cells were selected (side scatter (SSC) vs. forward scatter (FSC)) and gated for singlets (SSC-Width (W) vs. FSC) to hence exclude doublets or cell aggregates. The single cell gate was then further analysed for DAPI, gating on the live cells (DAPI negative). DsRNA-transfected cells (Cy5-positive) were then gated and the level of GFP expression analysed within that population. Flow cytometry counting beads (gated on SSC vs. FSC plot) were added to quantify cell number in each sample.(C) mESCs stably expressing GFP (mESCs-GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA and the level of GFP in Cy5+cells was analysed 3 days later and is represented as histograms (left). Right panel, bar graphs displaying the percentage of GFP mean fluorescence intensity relative to mock control (D) mESCs stably expressing d2GFP (mESCs-d2GFP) were mock transfected or transfected with the indicated Cy5-labeled dsRNA. The level of d2GFP was analysed at the indicated timepoints post-transfection and displayed as in (C).(E) L929 cells stably expressing d2GFP were mock transfected or transfected with the indicated Cy5-labeled dsRNA and d2GFP level was measured as in (D).


Figure 2 ( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs ( top panels ) or siRNAs ( bottom panels ) . GFP level was measured in Cy5 + cells by flow cytometry 72 hours ( hrs ) post - transfection . ( B ) Mavs + / - and Mavs - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs . The level of d2GFP in Cy5 + cells was measured at various timepoints post - transfection as indicated . Histograms plots show d2GFP level 48 hrs post - transfection . Bar graphs show 3 timepoints as indicated . ( C ) WT , Ifnar1 + / - or Ifnar1 - / - MEFs stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry at 48 hrs post - transfection .

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

Figure 2(A) Mavs+/- and Mavs-/- MEFs stably expressing GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs (top panels) or siRNAs (bottom panels). GFP level was measured in Cy5+ cells by flow cytometry 72 hours (hrs) post-transfection.(B) Mavs+/- and Mavs-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs. The level of d2GFP in Cy5+ cells was measured at various timepoints post-transfection as indicated. Histograms plots show d2GFP level 48 hrs post-transfection. Bar graphs show 3 timepoints as indicated.(C) WT, Ifnar1+/- or Ifnar1-/- MEFs stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry at 48 hrs post-transfection.


Figure 3 ( A ) Mavs + / - and Mavs - / - MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR ( α - IFNAR Ab ) , an isotype control antibody ( IgG1 ) or no antibody ( no Ab ) . Cells were then were transfected or not ( mock ) with Cy5 - labeled dsRNA as indicated and GFP level monitored in Cy5 + cells 72 hrs post - transfection . ( B ) Mavs - / - MEFs stably expressing d2GFP were treated with recombinant IFN ( 200 U / ml ) for 24 hrs prior to transfection or not ( mock ) with Cy5 - labeled dsRNA , as indicated . IFN treatment was maintained for 2 days post - transfection and then GFP level monitored in Cy5 + cells .

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

Figure 3(A) Mavs+/- and Mavs-/-MEFs stably expressing GFP were incubated with either a neutralising antibody against IFNAR (α-IFNAR Ab), an isotype control antibody (IgG1) or no antibody (no Ab). Cells were then were transfected or not (mock) with Cy5-labeled dsRNA as indicated and GFP level monitored in Cy5+cells 72 hrs post-transfection.(B) Mavs-/-MEFs stably expressing d2GFP were treated with recombinant IFN (200 U/ml) for 24 hrs prior to transfection or not (mock) with Cy5-labeled dsRNA, as indicated. IFN treatment was maintained for 2 days post-transfection and then GFP level monitored in Cy5+cells.


Figure 4 ( A ) Northern blot analysis of Ifnar1 - / - MEFs at 24 h . p . t with the indicated dsRNA or transfection reagent alone ( mock ) using a probe specific for dsRNA - GFP ( top ) or dsRNA - RL ( bottom ) . Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG - human Dicer were loaded in parallel . Two lanes ( delimited by dashed lines ) were left empty between the Ifnar1 - / - MEFs transfected samples and the in vitro dicing assay reactions . A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17 , 21 and 25 - nt in length was run in parallel . Endogenous U6 was used as a loading control . Signals corresponding to the dsRNA - derived siRNAs are indicated with an arrow . The membrane was first probed for dsRNA - GFP ( top ) , then probed for the miRNA marker ( top left ) and subsequently stripped and reprobed for dsRNA - RL ( middle ) and finally stripped and reprobed for both U6 ( bottom ) and for the miRNA marker again ( middle left ) . ( B ) Western blot of Dicer in d2GFP - expressing Mavs - / - MEFs either not transduced ( NT ) or transduced to express a non - targeting shRNA control ( CTL shRNA ) or two different Dicer - specific targeting shRNAs ( DICER shRNA 1 and 2 ) . P97 served as loading control . ( C ) Cells described in ( B ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later . Please note that the biphasic pattern of d2GFP level observed in dsRNA - GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP ( D ) Western blots of Ago2 in parental Mavs - / - MEFs stably expressing d2GFP and three Mavs - / - Ago2 - / - clones generated by CRISPR / CAS9 - mediated genome engineering . P97 served as loading control . ( E ) Mavs - / - ( parental line ) and Mavs - / - Ago2 - / - MEFs ( clone 5 . 1 ) stably expressing d2GFP were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

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

Figure 4(A) Northern blot analysis of Ifnar1-/- MEFs at 24 h.p.t with the indicated dsRNA or transfection reagent alone (mock) using a probe specific for dsRNA-GFP (top) or dsRNA-RL (bottom). Reactions from a dicing assay using the indicated dsRNA incubated in vitro with immunoprecipitated FLAG-human Dicer were loaded in parallel. Two lanes (delimited by dashed lines) were left empty between the Ifnar1-/- MEFs transfected samples and the in vitro dicing assay reactions. A miRNA marker containing 3 synthetic ssRNA oligonucleotides of 17, 21 and 25-nt in length was run in parallel. Endogenous U6 was used as a loading control. Signals corresponding to the dsRNA-derived siRNAs are indicated with an arrow. The membrane was first probed for dsRNA-GFP (top), then probed for the miRNA marker (top left) and subsequently stripped and reprobed for dsRNA-RL (middle) and finally stripped and reprobed for both U6 (bottom) and for the miRNA marker again (middle left).(B) Western blot of Dicer in d2GFP-expressing Mavs-/- MEFs either not transduced (NT) or transduced to express a non-targeting shRNA control (CTL shRNA) or two different Dicer-specific targeting shRNAs (DICER shRNA 1 and 2). P97 served as loading control.(C) Cells described in (B) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later. Please note that the biphasic pattern of d2GFP level observed in dsRNA-GFP transfected cells is caused by a lower transfection efficiency of dsRNA in these cells giving rise to significant amounts of lowly transfected cells that do not display a decrease level of d2GFP(D) Western blots of Ago2 in parental Mavs-/- MEFs stably expressing d2GFP and three Mavs-/- Ago2-/- clones generated by CRISPR/CAS9-mediated genome engineering. P97 served as loading control.(E) Mavs-/- (parental line) and Mavs-/- Ago2-/- MEFs (clone 5.1) stably expressing d2GFP were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


Figure 5 ( A ) Western blots for Ago2 ( top ) and HA tag ( bottom ) of parental Mavs - / - MEFs and two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ; see Fig 4A ) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA - tagged wild - type ( HA - mAgo2 WT ) or catalytic mutant ( HA - mAgo2 D597A ) versions of mouse Ago2 . β - actin served as loading control . The membrane was first analysed for Ago2 and β - actin , stripped and then analysed for HA tag . ( B ) Parental non - transduced ( NT ) Mavs - / - MEFs and complemented Mavs - / - Ago2 - / - MEFs as described in ( A ) were transfected or not ( mock ) with the indicated Cy5 - labeled dsRNAs and d2GFP level in Cy5 + cells was monitored by flow cytometry 48 hrs later .

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

Figure 5(A) Western blots for Ago2 (top) and HA tag (bottom) of parental Mavs-/- MEFs and two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3; see Fig 4A) stably expressing d2GFP and transduced with an empty vector or a vector encoding HA-tagged wild-type (HA-mAgo2 WT) or catalytic mutant (HA-mAgo2 D597A) versions of mouse Ago2. β-actin served as loading control. The membrane was first analysed for Ago2 and β-actin, stripped and then analysed for HA tag.(B) Parental non-transduced (NT) Mavs-/- MEFs and complemented Mavs-/- Ago2-/- MEFs as described in (A) were transfected or not (mock) with the indicated Cy5-labeled dsRNAs and d2GFP level in Cy5+cells was monitored by flow cytometry 48 hrs later.


Figure 6 ( A ) Ifnar1 - / - MEFs were transfected with the indicated Cy5 - labeled dsRNA and the next day were infected with SFV - Rluc at the indicated multiplicities of infection ( MOI ) . Renilla luciferase activity was measured 24 hours later . Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold - reduction in viral activity achieved with dsRNA - RL vs . dsRNA - GFP treatment . ( B ) As in ( a ) but SFV - Rluc virus titre was measured by plaque assay and expressed as plaque forming units ( PFU ) per ml of supernatant at 24 h post - infection ( MOI = 0 . 1 ) . ( C ) As in ( a ) but with Ifnar1 - / - or Mavs - / - MEFs and a single dose of SFV - Rluc ( MOI = 0 . 1 ) . ( D ) As in ( a ) but with two Mavs - / - Ago2 - / - clones ( clone 5 . 2 and 5 . 3 ) individually complemented to express wild - type ( HA - mAgo2 WT ) or a catalytic mutant ( HA - mAgo2 D597A ) version of mAgo2 or transduced with a control vector ( as described in Fig 5 ) . SFV - Rluc was used at an MOI = 0 . 1 .

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

Figure 6(A) Ifnar1-/- MEFs were transfected with the indicated Cy5-labeled dsRNA and the next day were infected with SFV-Rluc at the indicated multiplicities of infection (MOI). Renilla luciferase activity was measured 24 hours later. Each bar represents mean + SD of biological duplicates and numbers above each pair of bars depict the fold-reduction in viral activity achieved with dsRNA-RL vs. dsRNA-GFP treatment.(B) As in (a) but SFV-Rluc virus titre was measured by plaque assay and expressed as plaque forming units (PFU) per ml of supernatant at 24 h post-infection (MOI=0.1).(C) As in (a) but with Ifnar1-/- or Mavs-/- MEFs and a single dose of SFV-Rluc (MOI=0.1).(D) As in (a) but with two Mavs-/- Ago2-/- clones (clone 5.2 and 5.3) individually complemented to express wild-type (HA-mAgo2 WT) or a catalytic mutant (HA-mAgo2 D597A) version of mAgo2 or transduced with a control vector (as described in Fig 5). SFV-Rluc was used at an MOI=0.1.


Figure 1 ( C ) Relative mRNA expression from real - time PCR analysis of the GnRH transcript in GFP - positive cells , in comparison with GFP - negative cells . The GnRH transcript appears to be selectively expressed in GFP - positive cells . Mann - Whitney U test , n = 4 mice from 2 litters . ( D ) Relative mRNA expression from real - time PCR analysis of Neuropilin - 1 ( Nrp1 ) , Nrp2 , Plexin A1 ( PlxnA1 ) , and PlxnA4 transcripts in FACS - isolated GFP - positive GnRH neurons from control ( Nrp1loxP / loxP ; grey ) and mutant ( Gnrh : : cre ; Nrp1loxP / loxP ; red ) P0 mice . Unpaired t - test , n = 5 to 6 mice per group from 3 litters . ( E ) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1 - immunoreactive vomeronasal / terminal nerve fibers ( empty arrowheads ) at the nose - brain junction in sagittal slices from E14 . 5 control and mutant embryos . GnRH neurons ( green ) themselves express Nrp1 ( red , white arrows ) in control Nrp1loxP / loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh : : cre ; Nrp1loxP / loxP littermates ( black arrows ) . Scale bar : 50μm .

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

Figure 1(C) Relative mRNA expression from real-time PCR analysis of the GnRH transcript in GFP-positive cells, in comparison with GFP-negative cells. The GnRH transcript appears to be selectively expressed in GFP-positive cells. Mann-Whitney U test, n=4 mice from 2 litters.(D) Relative mRNA expression from real-time PCR analysis of Neuropilin-1 (Nrp1), Nrp2, Plexin A1 (PlxnA1), and PlxnA4 transcripts in FACS-isolated GFP-positive GnRH neurons from control (Nrp1loxP/loxP; grey) and mutant (Gnrh::cre; Nrp1loxP/loxP; red) P0 mice. Unpaired t-test, n=5 to 6 mice per group from 3 litters.(E) Representative immunofluorescence images showing GnRH neurons migrating along Nrp1-immunoreactive vomeronasal/terminal nerve fibers (empty arrowheads) at the nose-brain junction in sagittal slices from E14.5 control and mutant embryos. GnRH neurons (green) themselves express Nrp1 (red, white arrows) in control Nrp1loxP/loxP mice whereas Nrp1 is undetectable in GnRH neurons from mutant Gnrh::cre; Nrp1loxP/loxP littermates (black arrows). Scale bar: 50μm.


Figure 2 ( A ) Quantification of total number of GnRH neurons during embryogenesis , at E12 . 5 , E14 . 5 and E18 . 5 . More GnRH neurons were found in E14 . 5 and E18 . 5 mutant embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 to 6 mouse embryos from at least 3 litters . ( B ) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14 . 5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose , the olfactory bulb ( OB ) and the ventral forebrain ( VFB ) in control and mutant embryos . Scale bar : 50μm . VNO , vomeronasal organ ; OE , olfactory epithelium ; OB , olfactory bulbs ; VFB , ventral forebrain . ( C ) Quantitative analysis showing distribution of GnRH neurons in the nose , OB and VFB of mutant and control embryos . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 5 and 6 mouse embryos for mutants and controls , respectively , from at least 3 litters . ( D ) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs , in a control ( left panel ) and a mutant ( right panel ) postnatal brain . Knock - out animals show an accumulation of GnRH neurons in the accessory olfactory bulb ( circle formed by the GnRH labelling , right panel ) . AOB , accessory olfactory bulb . Scale bar : 300μm . ( E ) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis ( OVLT ) in the median preoptic area , from a control ( left ) and a mutant ( right ) female postnatal brain . Scale bar : 500μm . Oc , optic chiasma . ( F ) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates . OB , olfactory bulb ; DBB , diagonal band of Broca ; 3V , periventricular region of the preoptic area ; Tot , total . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 adult mice from 3 litters .

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

Figure 2(A) Quantification of total number of GnRH neurons during embryogenesis, at E12.5, E14.5 and E18.5. More GnRH neurons were found in E14.5 and E18.5 mutant embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 to 6 mouse embryos from at least 3 litters.(B) Schematic representation of the areas containing GnRH migrating cells during embryogenesis at E14.5 and representative immunofluorescence showing migrating GnRH neurons at the level of the nose, the olfactory bulb (OB) and the ventral forebrain (VFB) in control and mutant embryos. Scale bar: 50μm. VNO, vomeronasal organ; OE, olfactory epithelium; OB, olfactory bulbs; VFB, ventral forebrain.(C) Quantitative analysis showing distribution of GnRH neurons in the nose, OB and VFB of mutant and control embryos. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=5 and 6 mouse embryos for mutants and controls, respectively, from at least 3 litters.(D) Representative image of cleared brains and immunolabeling for GnRH at the level of the olfactory bulbs, in a control (left panel) and a mutant (right panel) postnatal brain. Knock-out animals show an accumulation of GnRH neurons in the accessory olfactory bulb (circle formed by the GnRH labelling, right panel). AOB, accessory olfactory bulb. Scale bar: 300μm.(E) Representative image of cleared brains and immunolabeling for GnRH at the level of the organum vasculosum of the lamina terminalis (OVLT) in the median preoptic area, from a control (left) and a mutant (right) female postnatal brain. Scale bar: 500μm. Oc, optic chiasma.(F) Quantitative analysis of the distribution of GnRH neurons in cleared brains of mutant and control littermates. OB, olfactory bulb; DBB, diagonal band of Broca; 3V, periventricular region of the preoptic area; Tot, total. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 adult mice from 3 litters.


Figure 3 ( A ) Schematic representation of the transwell assay used to assess the effect of Sema3A , which was placed in the lower chamber , on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst ( grey ) in the lower part of the transwell membrane after overnight incubation in SFM ( left panel ) , SFM with 100ng / ml Sema3A ( middle panel ) and SFM with 100ng / ml Sema3A + 1μg Nrp1Ab ( right panel ) . Scale bar : 100μm . ( B ) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber ( SFM ) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber . Kruskal - Wallis test , n = 5 to 6 wells in two independent experiments . ( C ) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11 . 5 mouse embryos after 8 days in vitro . NE , nasal epithelium ; NMC , nasal midline cartilage . Scale bar 500μm ( 150μm in inset ) . ( D ) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos , quantified after 8 days in culture . Mann - Whitney U test , n = 3 to 5 embryos from at least 3 litters . ( E ) Distance travelled by GnRH neurons from the olfactory epithelia ; Cells were quantified within a radius of 1 to 6 mm . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 3 to 5 explants . ( F ) Representative immunofluorescence for GnRH - ( red ) and BrdU - ( green ) positive cells in control and mutant E14 . 5 embryos arising from progenitors born between E9 . 5 and E10 . 5 . BrdU - positive GnRH neurons ( white arrowheads ) were more abundant in mutant than in control embryos . Scale bar : 20μm . ( G ) Number of BrdU - positive GnRH neurons in the nose , olfactory bulb ( OB ) and ventral forebrain ( VFB ) at E14 . 5 and total number of cells in control and mutant embryos . Double - labeled cells were more numerous in the mutant VFB . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 4 embryos from 2 litters . ( I ) Number of GnRH neurons in E14 . 5 embryos injected with neutralizing antibodies to Neuropilin - 1 ( Nrp1Ab ) compared to an isotype goat antibody , or the antiapoptotic compound ZVAD compared to its vehicle , together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age . Note that the data are represented as fold change , as raw values are not comparable with transgenic animals that did not undergo surgery . Unpaired t - test , n = 5 to 7 embryos from at least 2 litters .

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

Figure 3(A) Schematic representation of the transwell assay used to assess the effect of Sema3A, which was placed in the lower chamber, on GN11 cell migration and representative microphotograph of cell nuclei stained with Hoechst (grey) in the lower part of the transwell membrane after overnight incubation in SFM (left panel), SFM with 100ng/ml Sema3A (middle panel) and SFM with 100ng/ml Sema3A + 1μg Nrp1Ab (right panel). Scale bar: 100μm.(B) Quantitative analyses of the mean number of GN11 cells that migrated from the upper chamber (SFM) through the perforated membrane in the presence or absence of the indicated doses of Sema3A and Nrp1Ab in the lower chamber. Kruskal-Wallis test, n= 5 to 6 wells in two independent experiments.(C) Schematic illustration and representative photomicrograph of an organotypic culture of a nasal explant from E11.5 mouse embryos after 8 days in vitro. NE, nasal epithelium; NMC, nasal midline cartilage. Scale bar 500μm (150μm in inset).(D) Total number of GnRH neurons that migrated out of nasal explants isolated from control and mutant embryos, quantified after 8 days in culture. Mann-Whitney U test, n=3 to 5 embryos from at least 3 litters.(E) Distance travelled by GnRH neurons from the olfactory epithelia; Cells were quantified within a radius of 1 to 6 mm. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=3 to 5 explants.(F) Representative immunofluorescence for GnRH- (red) and BrdU- (green) positive cells in control and mutant E14.5 embryos arising from progenitors born between E9.5 and E10.5. BrdU-positive GnRH neurons (white arrowheads) were more abundant in mutant than in control embryos. Scale bar: 20μm.(G) Number of BrdU-positive GnRH neurons in the nose, olfactory bulb (OB) and ventral forebrain (VFB) at E14.5 and total number of cells in control and mutant embryos. Double-labeled cells were more numerous in the mutant VFB. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=4 embryos from 2 litters.(I) Number of GnRH neurons in E14.5 embryos injected with neutralizing antibodies to Neuropilin-1 (Nrp1Ab) compared to an isotype goat antibody, or the antiapoptotic compound ZVAD compared to its vehicle, together with fold change in GnRH neuron number as well as mutant and control embryos at the same embryonic age. Note that the data are represented as fold change, as raw values are not comparable with transgenic animals that did not undergo surgery. Unpaired t-test, n=5 to 7 embryos from at least 2 litters.


Figure 4 ( A ) Olfactory sex preference test in P20 ( left panel ) and adult ( right panel ) control and mutant female mice . Half of the mutant females at P20 showed a preference for males , similar to adults of both genotypes , whereas the other half showed an even more pronounced preference for females than their control littermates at P20 . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters . ( B ) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates . One - way ANOVA , Fisher ' s LSD multiple - comparison test for P20 mice and unpaired t - test for adult mice ; n = 4 to 6 from at least 3 litters . ( C ) Representative traces of spontaneous firing in GnRH neurons from control ( left , black ) and mutant ( right , red ) animals at week 2 ( upper panels ) and week 4 ( lower panels ) ; lower traces show an enlargement of the upper trace . ( D ) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2 , 3 and 4 . Individual males and females used for this analysis are represented by blue and grey dots , respectively . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 8 to 14 neurons from at least 3 animals from different litters . Note that individual neurons from male ( n = 3 to 8 ) and female mice ( n = 5 to 8 ) used for this analysis are represented by blue and grey dots , respectively . ( E ) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps . Two - way repeated - measures ANOVA , n = 10 to 11 neurons from at least 3 mice from different litters .

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

Figure 4(A) Olfactory sex preference test in P20 (left panel) and adult (right panel) control and mutant female mice. Half of the mutant females at P20 showed a preference for males, similar to adults of both genotypes, whereas the other half showed an even more pronounced preference for females than their control littermates at P20. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.(B) Time spent sniffing after the introduction of both male and female odors into the cage of control and mutant littermates. One-way ANOVA, Fisher's LSD multiple-comparison test for P20 mice and unpaired t-test for adult mice; n=4 to 6 from at least 3 litters.(C) Representative traces of spontaneous firing in GnRH neurons from control (left, black) and mutant (right, red) animals at week 2 (upper panels) and week 4 (lower panels); lower traces show an enlargement of the upper trace.(D) Average spontaneous firing frequency in GnRH neurons from control and mutant mice at weeks 2, 3 and 4. Individual males and females used for this analysis are represented by blue and grey dots, respectively. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=8 to 14 neurons from at least 3 animals from different litters. Note that individual neurons from male (n=3 to 8) and female mice (n=5 to 8) used for this analysis are represented by blue and grey dots, respectively.(E) Quantification of evoked firing in GnRH neurons from control and mutant mice in response to current steps. Two-way repeated-measures ANOVA, n=10 to 11 neurons from at least 3 mice from different litters.


Figure 5 ( A ) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age . Mutant females show a significantly higher body weight from P21 . The grey lines mark body weight at puberty onset . Two - way ANOVA , Fisher ' s LSD multiple - comparison test , n = 7 to 17 mice from at least 4 litters . ( B ) Fat mass or volume of white adipose tissue ( WAT ) . Adult mutant females showed higher WAT volumes . Unpaired t - test , n = 8 mice . ( C ) Volume of lean tissue . Mann - Whitney U test , n = 8 mice . ( D ) Cumulative percentage of the age at vaginal opening , n = 13 mice . ( E ) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n = 13 mice . ( F ) Bar graph showing body weight at P45 , which is the mean age at puberty in mutants . Unpaired t - test , n = 9 to 14 mice . ( G ) Plasma LH levels at P45 in control and mutant mice . Mann - Whitney U test , n = 5 to 6 mice . ( H ) Mean uterine weight normalized to body weight ( mg / g BW ) . Unpaired t - test , n = 6 to 10 mice .

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

Figure 5(A) Evolution of body weight in control and mutant female littermates from 6 to 100 days of age. Mutant females show a significantly higher body weight from P21. The grey lines mark body weight at puberty onset. Two-way ANOVA, Fisher's LSD multiple-comparison test, n=7 to 17 mice from at least 4 litters.(B) Fat mass or volume of white adipose tissue (WAT). Adult mutant females showed higher WAT volumes. Unpaired t-test, n=8 mice.(C) Volume of lean tissue. Mann-Whitney U test, n=8 mice.(D) Cumulative percentage of the age at vaginal opening, n=13 mice.(E) Cumulative percentage of animals undergoing first ovulation as assessed by the occurrence of first estrus at different ages n=13 mice.(F) Bar graph showing body weight at P45, which is the mean age at puberty in mutants. Unpaired t-test, n=9 to 14 mice.(G) Plasma LH levels at P45 in control and mutant mice. Mann-Whitney U test, n=5 to 6 mice.(H) Mean uterine weight normalized to body weight (mg/g BW). Unpaired t-test, n=6 to 10 mice.


Figure 1PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( B ) Immunohistochemistry for dystrophin expression in body - wide muscles from mdx mice treated with PMO in saline ( PMO - S ) , PMO - G ( IV ) or PMO - G ( D ) ( scale bar = 100μm ) . PMO - G ( IV ) refers to intravenous injection of PMO in combination with intravenous injection of 5 % glycine every other day for 3 weeks . PMO - G ( D ) represents intravenous injection of PMO supplemented with 1 % glycine orally . PMO was administered intravenously into adult mdx mice at 25 or 50 mg / kg / week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration . ( C ) Western blot for dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( IV ) , PMO - G ( D ) or PMO - S . 0 . 5µg , 2 . 5 µg , 5µg , 10µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded . α - actinin was used as the loading control . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , D - diaphragm and A - abdominal muscle . ( D ) Quantitative analysis of dystrophin expression in body - wide muscles from mdx mice treated with PMO - G ( i . v . , n = 4 ) , PMO - G ( D25 and D50 , n = 3 ) and PMO - S ( n = 3 ) ( * P < 0 . 05 , * * P < 0 . 001 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

Figure 1PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO in saline (PMO-S), PMO-G(IV) or PMO-G (D)(scale bar=100μm).PMO-G (IV) refers to intravenous injection of PMO in combination with intravenous injection of 5% glycine every other day for 3 weeks. PMO-G (D) represents intravenous injection of PMO supplemented with 1% glycine orally.PMO was administered intravenously into adult mdx mice at 25 or 50 mg/kg/week for 3 weeks with dietary supplementation of glycine for 3 consecutive days per week prior to PMO administration. (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (IV), PMO-G (D) or PMO-S. 0.5µg, 2.5 µg, 5µg, 10µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated mdx mice were loaded. α-actinin was used as the loading control. TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, D-diaphragm and A-abdominal muscle. (D) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated with PMO-G (i.v., n=4), PMO-G (D25 and D50, n=3) and PMO-S (n=3) (*P<0.05,**P<0.001; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.


Figure 2PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( A ) Muscle function was assessed to determine the physical improvement with grip strength test ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified . PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( B ) Measurement of bodyweight changes of DKO mice treated with PMO - G or PMO - S ( n = 3 ; * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified . PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( C ) Measurement of TA muscle mass of DKO mice treated with PMO - G or PMO - S ( n = 3 , * P < 0 . 05 ; one - way ANOVA post - hoc Student - Newman - Keuls test ) . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified . PMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( D ) Immunohistochemistry for dystrophin expression in treated DKO mice ( scale bar = 100 μm ) . TA - tibialis anteriorPMO was administered intravenously into 3 - week old DKO mice at 50 mg / kg / week for 3 weeks with dietary supplementation of 1 % glycine for 3 consecutive days per week prior to PMO introduction ( PMO - G ) . ( E , F ) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice ( n = 3 , * P < 0 . 05 ; One way - ANOVA post hoc Student - Newman - Keuls test ) . TA - tibialis anterior , Q - quadriceps , G - gastrocnemius , T - triceps , A - abdominal muscle and D - diaphragm . 2 . 5 µg , 5µg and 15µg total protein from C57BL / 6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded . α - actinin was used as the loading control . Data information : Error bars indicate s . e . m . Data shown are representative of biological replicates as specified .

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

Figure 2PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (A) Muscle function was assessed to determine the physical improvement with grip strength test (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (B) Measurement of bodyweight changes of DKO mice treated with PMO-G or PMO-S (n=3; *P<0.05; One way-ANOVA post hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (C) Measurement of TA muscle mass of DKO mice treated with PMO-G or PMO-S (n = 3, *P < 0.05; one-way ANOVA post-hoc Student-Newman-Keuls test). Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.PMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (D) Immunohistochemistry for dystrophin expression in treated DKO mice (scale bar=100 μm). TA-tibialis anteriorPMO was administered intravenously into 3-week old DKO mice at 50 mg/kg/week for 3 weeks with dietary supplementation of 1% glycine for 3 consecutive days per week prior to PMO introduction (PMO-G). (E, F) Western blot and quantitative analysis for the dystrophin protein in treated DKO mice (n=3, *P<0.05; One way-ANOVA post hoc Student-Newman -Keuls test). TA-tibialis anterior, Q-quadriceps, G-gastrocnemius, T-triceps, A-abdominal muscle and D-diaphragm. 2.5 µg, 5µg and 15µg total protein from C57BL/6 and 50 µg of muscle samples from untreated and treated DKO mice were loaded. α-actinin was used as the loading control. Data information: Error bars indicate s.e.m. Data shown are representative of biological replicates as specified.