Figure 8Saturating MET currents recorded from P6 Clrn2clarinet / + ( A ) and Clrn2clarinet / clarinet ( B ) apical - coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at − 121 mV and + 99 mV . The driver voltage ( DV ) signal of ± 40 V to the fluid jet is shown above the traces ( positive deflections of the DV are excitatory ) . The holding potential was − 81 mV . Extracellular Ca2 + concentration was 1 . 3 mM . Arrows and arrowheads indicate the closure of the MET currents ( i . e . resting MET current ) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials , respectively . Dashed lines indicate the holding current , which is the current at the holding membrane potential . Average peak - to - peak current - voltage curves recorded from Clrn2clarinet / + ( grey , P6 , n = 7 ) and Clrn2clarinet / clarinet ( red , P6 - 7 , n = 9 ) apical coil OHCs . Resting open probability ( Po ) of the MET current at the membrane potential of − 121 mV and + 99 mV from apical - ( D ) and basal - coil ( E ) OHCs . Number of OHCs investigated is shown above the columns . Saturating MET currents recorded from a P7 Clrn2clarinet / + ( F ) and a P8 Clrn2clarinet / clarinet ( G ) apical - coil IHC using the same experimental protocol described above . Average Po of the MET current measured in apical coil IHCs at the membrane potential of − 121 mV and + 99 mV from Clrn2clarinet / + ( P7 , n = 3 ) and Clrn2clarinet / clarinet ( P7 - 8 , n = 6 ) apical - coil IHCs .

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

Figure 8Saturating MET currents recorded from P6 Clrn2clarinet/+ (A) and Clrn2clarinet/clarinet (B) apical-coil OHCs by applying sinusoidal force stimuli of 50 Hz to the hair bundles at −121 mV and +99 mV. The driver voltage (DV) signal of ± 40 V to the fluid jet is shown above the traces (positive deflections of the DV are excitatory). The holding potential was −81 mV. Extracellular Ca2+ concentration was 1.3 mM. Arrows and arrowheads indicate the closure of the MET currents (i.e. resting MET current) elicited during inhibitory bundle displacements at hyperpolarized and depolarized membrane potentials, respectively. Dashed lines indicate the holding current, which is the current at the holding membrane potential.Average peak-to-peak current-voltage curves recorded from Clrn2clarinet/+ (grey, P6, n=7) and Clrn2clarinet/clarinet (red, P6-7, n=9) apical coil OHCs.Resting open probability (Po) of the MET current at the membrane potential of −121 mV and + 99 mV from apical- (D) and basal-coil (E) OHCs. Number of OHCs investigated is shown above the columns.Saturating MET currents recorded from a P7 Clrn2clarinet/+ (F) and a P8 Clrn2clarinet/clarinet (G) apical-coil IHC using the same experimental protocol described above.Average Po of the MET current measured in apical coil IHCs at the membrane potential of −121 mV and + 99 mV from Clrn2clarinet/+ (P7, n=3) and Clrn2clarinet/clarinet (P7-8, n=6) apical-coil IHCs.


Figure 9Potassium currents recorded from mature Clrn2clarinet / + ( A , P15 ) and Clrn2clarinet / clarinet ( B , P16 ) apical - coil OHCs . Currents were elicited by depolarizing voltage steps ( 10 mV nominal increments ) from - 144 mV to more depolarized values from the holding potential of - 84 mV . Note that the current characteristic of mature OHCs , The size of IK , n , measured in isolation as the deactivating tail currents ( difference between instantaneous and steady - state inward currents ) for voltage steps from the holding potential to − 124 mV , was 545 ± 115 pA , ( n = 5 ) in Clrn2clarinet / + and 595 ± 67 pA , ( n = 7 ) in Clrn2clarinet / clarinet OHCs . Average peak current - voltage relation for the total K + current recorded from Clrn2clarinet / + ( P15 - 16 , n = 7 ) and Clrn2clarinet / clarinet ( P15 - 16 , n = 5 ) OHCs . Potassium currents recorded from P22 mature Clrn2clarinet / + and Clrn2clarinet / clarinet apical - coil IHCs , respectively , using the same voltage protocol described above . Holding potential of - 64 mV . Peak current - voltage relation for the total K + current recorded from the IHCsVoltage responses from P22 Clrn2clarinet / + ( G ) and Clrn2clarinet / clarinet ( H , I ) IHCs . Note that Ca2 + - dependent action potentials could be induced in mature IHCs ( I ) .

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 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 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 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

Figure 9Potassium currents recorded from mature Clrn2clarinet/+ (A, P15) and Clrn2clarinet/clarinet (B, P16) apical-coil OHCs. Currents were elicited by depolarizing voltage steps (10 mV nominal increments) from -144 mV to more depolarized values from the holding potential of -84 mV. Note that the current characteristic of mature OHCs, The size of IK,n, measured in isolation as the deactivating tail currents (difference between instantaneous and steady-state inward currents) for voltage steps from the holding potential to −124 mV, was 545 ± 115 pA, (n=5) in Clrn2clarinet/+ and 595 ± 67 pA, (n=7) in Clrn2clarinet/clarinet OHCs.Average peak current-voltage relation for the total K+ current recorded from Clrn2clarinet/+ (P15-16, n=7) and Clrn2clarinet/clarinet (P15-16, n=5) OHCs.Potassium currents recorded from P22 mature Clrn2clarinet/+ and Clrn2clarinet/clarinet apical-coil IHCs, respectively, using the same voltage protocol described above. Holding potential of -64 mV.Peak current-voltage relation for the total K+ current recorded from the IHCsVoltage responses from P22 Clrn2clarinet/+ (G) and Clrn2clarinet/clarinet (H,I) IHCs. Note that Ca2+-dependent action potentials could be induced in mature IHCs (I).


Figure 1 ( A - D ) A . castellanii was infected ( MOI 5 ) with L . pneumophila JR32 producing ( A ) GFP under the control of PflaA ( Ptac - mCherry - PflaA - gfp ) and mCherry constitutively , ( B ) GFP under the control of PralF ( PralF - gfp ) and stained with DAPI , ( C ) GFP under the control of PsidC ( PsidC - gfp ) and stained with DAPI , or ( D ) Timer constitutively ( pNP107 ) . The infected amoebae were fixed at different time points post - infection , analyzed by confocal microscopy and 3D reconstructed ( scale bars : 5 µm ) . Motile and virulent bacteria ( GFP - positive ) emerge at the colony cluster periphery only after bacterial replication has ceased ( red fluorescent Timer ) . ( G ) Transmission electron micrographs of A . castellanii infected with L . pneumophila . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila wild - type strain JR32 and subjected to high - pressure freezing and electron microscopy . Many LCVs were still intact at 42 h post infection ( left panel ; arrow : intact LCV membrane , arrowhead plasma membrane ) , and occasionally , the bacteria were flagellated ( middle panel , arrow ) . Some LCVs were already ruptured , and the bacteria were released into the host cell cytosol ( right panel ; arrows : ruptured LCV membrane ) . Scale bars : 2 µm , 0 . 5 µm ( insets ) .

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 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

Figure 1(A-D) A. castellanii was infected (MOI 5) with L. pneumophila JR32 producing (A) GFP under the control of PflaA (Ptac-mCherry-PflaA-gfp) and mCherry constitutively, (B) GFP under the control of PralF (PralF-gfp) and stained with DAPI, (C) GFP under the control of PsidC (PsidC-gfp) and stained with DAPI, or (D) Timer constitutively (pNP107). The infected amoebae were fixed at different time points post-infection, analyzed by confocal microscopy and 3D reconstructed (scale bars: 5 µm). Motile and virulent bacteria (GFP-positive) emerge at the colony cluster periphery only after bacterial replication has ceased (red fluorescent Timer).(G) Transmission electron micrographs of A. castellanii infected with L. pneumophila. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila wild-type strain JR32 and subjected to high-pressure freezing and electron microscopy. Many LCVs were still intact at 42 h post infection (left panel; arrow: intact LCV membrane, arrowhead plasma membrane), and occasionally, the bacteria were flagellated (middle panel, arrow). Some LCVs were already ruptured, and the bacteria were released into the host cell cytosol (right panel; arrows: ruptured LCV membrane). Scale bars: 2 µm, 0.5 µm (insets).


Figure 2 ( A ) L . pneumophila subpopulation proteomics . A . castellanii was infected ( MOI 5 , 42 h ) with L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) . After cell lysis , released intracellular bacteria were sorted by flow cytometry in mCherry - positive / GFP - negative and mCherry - positive / GFP - positive L . pneumophila subpopulations , and their proteome was determined and compared . Protein abundance in each subset is depicted as a volcano plot . Proteins enriched in the GFP - producing subpopulation have a positive log2 ratio ( q - value ) . ( D , E ) Expression of select L . pneumophila genes in amoebae . A . castellanii was infected ( MOI 5 ; 6 h and 48 h ) with L . pneumophila JR32 harboring reporter constructs for ( D ) PfleQ - mCherry - PflaA - gfp or ( E ) PlegC8 - mCherry - PflaA - gfp , respectively . The infected amoebae were fixed and imaged by confocal microscopy ( scale bars : 5 µm ) .

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 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

Figure 2(A) L. pneumophila subpopulation proteomics. A. castellanii was infected (MOI 5, 42 h) with L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp). After cell lysis, released intracellular bacteria were sorted by flow cytometry in mCherry-positive/GFP-negative and mCherry-positive/GFP-positive L. pneumophila subpopulations, and their proteome was determined and compared. Protein abundance in each subset is depicted as a volcano plot. Proteins enriched in the GFP-producing subpopulation have a positive log2 ratio (q-value).(D, E) Expression of select L. pneumophila genes in amoebae. A. castellanii was infected (MOI 5; 6 h and 48 h) with L. pneumophila JR32 harboring reporter constructs for (D) PfleQ-mCherry-PflaA-gfp or (E) PlegC8-mCherry-PflaA-gfp, respectively. The infected amoebae were fixed and imaged by confocal microscopy (scale bars: 5 µm).


Figure 3 ( A - E ) Representative live cell microscopy images of D . discoideum infected with PflaA - GFP - producing L . pneumophila . D . discoideum Ax3 producing cytosolic mCherry ( pDM1042 ) was infected ( MOI 5 ) with ( A , B ) wild - type L . pneumophila JR32 ( Ptac - mCherry - PflaA - gfp ) , ( C , D ) JR32 ( Ptac - mCerulean - PflaA - gfp ) or ( E ) ∆ flaA ( Ptac - mCerulean - PflaA - gfp ) and embedded in agarose for 48 h . PflaA - GFP - positive L . pneumophila ( A ) escapes the LCV ( 0 sec ) , followed by other bacteria , which are contained in the cytosol and localize to the plasma membrane , where a PflaA - GFP - positive L . pneumophila first leaves the ruptured host cell ( 185 sec ) , or ( B ) triggers host cell death ( loss of cytosolic mCherry ) upon exit of the amoeba . PflaA - GFP - positive L . pneumophila ( C ) escape the LCV after 75 sec and spread within the cytosol for several minutes , and ( D ) are the first to exit the host cell , leading to the loss of cytosolic mCherry and host cell death . ( E ) L . pneumophila ∆ flaA lyses the LCV and host cell less vigorously . Scale bars : 5 µm .

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

Figure 3(A-E) Representative live cell microscopy images of D. discoideum infected with PflaA-GFP-producing L. pneumophila. D. discoideum Ax3 producing cytosolic mCherry (pDM1042) was infected (MOI 5) with (A, B) wild-type L. pneumophila JR32 (Ptac-mCherry-PflaA-gfp), (C, D) JR32 (Ptac-mCerulean-PflaA-gfp) or (E) ∆flaA (Ptac-mCerulean-PflaA-gfp) and embedded in agarose for 48 h. PflaA-GFP-positive L. pneumophila (A) escapes the LCV (0 sec), followed by other bacteria, which are contained in the cytosol and localize to the plasma membrane, where a PflaA-GFP-positive L. pneumophila first leaves the ruptured host cell (185 sec), or (B) triggers host cell death (loss of cytosolic mCherry) upon exit of the amoeba. PflaA-GFP-positive L. pneumophila (C) escape the LCV after 75 sec and spread within the cytosol for several minutes, and (D) are the first to exit the host cell, leading to the loss of cytosolic mCherry and host cell death. (E) L. pneumophila ∆flaA lyses the LCV and host cell less vigorously. Scale bars: 5 µm.


Figure 4 ( B ) L . pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae . A . castellanii amoebae were infected ( MOI 5 , 52 h ) with L . pneumophila JR32 , ∆ flaA or ∆ lqsA harboring Ptac - mCherry - PflaA - gfp and embedded in 0 . 5 % agarose / PYG medium . The fluorescence images are representative for the L . pneumophila strains indicated : JR32 ( n = 25 ) , ∆ flaA ( n = 13 ) or ∆ lqsA ( n = 19 ) . Scale bars : 20 µm .

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 4(B) L. pneumophila bacteria lacking flaA or lqsA are impaired for spreading from amoebae. A. castellanii amoebae were infected (MOI 5, 52 h) with L. pneumophila JR32, ∆flaA or ∆lqsA harboring Ptac-mCherry-PflaA-gfp and embedded in 0.5% agarose/PYG medium. The fluorescence images are representative for the L. pneumophila strains indicated: JR32 (n = 25), ∆flaA (n = 13) or ∆lqsA (n = 19). Scale bars: 20 µm.


Figure 5 ( A , B ) Role of the Lqs system for PflaA expression in intracellular L . pneumophila . A . castellanii was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , and ( A ) fixed and analyzed by confocal microscopy ( representative 3D reconstructions , scale bars : 10 µm ) , or ( B ) lysed , fixed and analyzed by flow cytometry . ( C - F ) Role of the Lqs system for LCV escape and cytosolic localization of L . pneumophila . ( C , D ) A . castellanii or ( E , F ) D . discoideum was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , or ∆ lqsS - ∆ lqsT harboring Ptac - mCherry - PflaA - gfp , fixed and treated with an anti ‐ ubiquitin antibody . Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy . Representative images of ( C ) A . castellanii ( scale bars : 10 µm ) or ( E ) D . discoideum ( scale bars : 5 μm ) infected with L . pneumophila JR32 escaping or not from the LCV are shown . The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells : ( D ) JR32 : n = 482 ; ΔlqsA : n = 834 ; ΔlqsR : n = 572 ; ΔlqsS : n = 729 ; ΔlqsT : n = 1469 ; ΔlqsS - ΔlqsT : n = 693 ; ( F ) : JR32 : n = 136 ; ΔlqsA : n = 201 ; ΔlqsR : n = 303 ; ΔlqsS : n = 183 ; ΔlqsT : n = 534 ; ΔlqsS - ΔlqsT : n = 180 . ( G , H ) D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - GFP and cytosolic - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila JR32 , ∆ lqsA , ∆ lqsR , ∆ lqsS , ∆ lqsT , ∆ lqsS - ∆ lqsT , or ∆ flaA harboring Ptac - mCerulean . Representative images of ( G ) D . discoideum infected with L . - pneumophila JR32 in intact cells with intact LCV ( 18 . 5 % ) , lysed cells and lysed LCVs ( 60 . 2 % ) , intact cells with lysed LCVs ( 15 . 5 % ) and lysed cells with intact LCVS ( 5 . 8 % ) are shown ( scale bars : 5 µm ) . ( H ) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane : JR32 : n = 426 ; ΔlqsA : n = 468 ; ΔlqsR : n = 344 ; ΔlqsS : n = 325 ; ΔlqsT : n = 287 ; ΔlqsS - ΔlqsT : n = 289 ; ΔflaA : n = 248 .

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

Figure 5(A, B) Role of the Lqs system for PflaA expression in intracellular L. pneumophila. A. castellanii was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, and (A) fixed and analyzed by confocal microscopy (representative 3D reconstructions, scale bars: 10 µm), or (B) lysed, fixed and analyzed by flow cytometry.(C-F) Role of the Lqs system for LCV escape and cytosolic localization of L. pneumophila. (C, D) A. castellanii or (E, F) D. discoideum was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, or ∆lqsS-∆lqsT harboring Ptac-mCherry-PflaA-gfp, fixed and treated with an anti‐ubiquitin antibody. Amoebae containing bacteria decorated with ubiquitin were quantified by confocal microscopy. Representative images of (C) A. castellanii (scale bars: 10 µm) or (E) D. discoideum (scale bars: 5 μm) infected with L. pneumophila JR32 escaping or not from the LCV are shown. The percentage of amoebae containing ubiquitinated bacteria was quantified by counting the total number of infected cells and the number of ubiquitinated cells: (D) JR32: n = 482; ΔlqsA: n = 834; ΔlqsR: n = 572; ΔlqsS: n = 729; ΔlqsT: n = 1469; ΔlqsS-ΔlqsT: n = 693; (F): JR32: n = 136; ΔlqsA: n = 201; ΔlqsR: n = 303; ΔlqsS: n = 183; ΔlqsT: n = 534; ΔlqsS-ΔlqsT: n = 180.(G, H) D. discoideum producing the LCV/PtdIns(4)P probe P4C-GFP and cytosolic-mCherry was infected (MOI 5, 48 h) with L. pneumophila JR32, ∆lqsA, ∆lqsR, ∆lqsS, ∆lqsT, ∆lqsS-∆lqsT, or ∆flaA harboring Ptac-mCerulean. Representative images of (G) D. discoideum infected with L.-pneumophila JR32 in intact cells with intact LCV (18.5%), lysed cells and lysed LCVs (60.2%), intact cells with lysed LCVs (15.5%) and lysed cells with intact LCVS (5.8%) are shown (scale bars: 5 µm). (H) The percentage of amoebae with intact LCVs was quantified by counting the total number of infected cells and the number of cells with intact LCV membrane: JR32: n = 426; ΔlqsA: n = 468; ΔlqsR: n = 344; ΔlqsS: n = 325; ΔlqsT: n = 287; ΔlqsS-ΔlqsT: n = 289; ΔflaA: n = 248.


Figure 6D . discoideum producing the LCV / PtdIns ( 4 ) P probe P4C - mCherry was infected ( MOI 5 , 48 h ) with L . pneumophila wild - type strains ( JR32 , Corby ) , or phospholipase triple mutants ( ∆ plcABC , ∆ plaACD ) harboring the Ptac - mCerulean - PflaA - gfp reporter construct , and fixed and treated with an anti ‐ ubiquitin antibody , LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact , P4C - mCherry - positive LCVs or ubiquitin - decorated , cytosolic bacteria . ( A ) Representative images of amoebae harboring L . pneumophila confined within LCVs or released from the LCV to the cytosol are shown ( scale bars : 5 μm ) .

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

Figure 6D. discoideum producing the LCV/PtdIns(4)P probe P4C-mCherry was infected (MOI 5, 48 h) with L. pneumophila wild-type strains (JR32, Corby), or phospholipase triple mutants (∆plcABC, ∆plaACD) harboring the Ptac-mCerulean-PflaA-gfp reporter construct, and fixed and treated with an anti‐ubiquitin antibody, LCV escape and cytosolic localization of the bacteria was assessed by confocal microscopy through quantification of amoeba containing intact, P4C-mCherry-positive LCVs or ubiquitin-decorated, cytosolic bacteria. (A) Representative images of amoebae harboring L. pneumophila confined within LCVs or released from the LCV to the cytosol are shown (scale bars: 5 μm).


figf1 ( a , b ) Histograms of TDP43 ( WT ) - EGFP ( a ) and TDP43A315T - EGFP ( b ) levels in individual neurons . Increasing amounts of DNA significantly shifted the distribution of log - transformed expression levels toward higher values ( P 0 . 01 for all comparisons , two - sided Kolmogorov - Smirnov test ) . TDP43 ( WT ) - EGFP , n = 1 , 287 neurons , with 290 - 342 cells per group ; TDP43A315T - EGFP , n = 1 , 290 , with 315 - 332 neurons per group . Values were pooled from 12 wells per condition , with experiments performed in duplicate . ( c ) Automated fluorescence microscopy . Primary rodent cortical neurons were transfected with mApple and TDP43 - EGFP , and survival was determined by repeated imaging at regular intervals . The last time at which the cell was noted to be alive ( red arrows ) was used as the time of death . Cells that survive the entire length of the experiment ( cyan arrow ) were censored . Scale bar , 25 μm . ( d , e ) Cumulative risk of death over time for neurons transfected with EGFP , TDP43 ( WT ) - EGFP ( d ) or TDP43A315T - neurons ( e ) . Supplementary Table 1 lists the number of neurons , hazard ratios , 95 % CI and P values for each condition , as determined by Cox proportional hazards analysis . Results were pooled from 12 wells per condition , with experiments performed in duplicate . f , g ) Linear regression of calculated hazard ratio ( ± s . e . m . ) as a function of total TDP43 ( WT ) - EGFP ( R2 = 0 . 9353 ) ( f ) or TDP43A315T - EGFP ( R2 = 0 . 9574 ) ( g ) level . The total TDP43 level was determined by quantitative immunocytochemistry using TDP43 - specific antibodies ( Supplementary Fig . 3 ) and normalized to the amount of TDP43 in nontransfected neurons . Dashed lines , reference ( HR = 1 ) .

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 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 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 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

figf1(a,b) Histograms of TDP43(WT)-EGFP (a) and TDP43A315T-EGFP (b) levels in individual neurons. Increasing amounts of DNA significantly shifted the distribution of log-transformed expression levels toward higher values (P 0.01 for all comparisons, two-sided Kolmogorov-Smirnov test). TDP43(WT)-EGFP, n = 1,287 neurons, with 290-342 cells per group; TDP43A315T-EGFP, n = 1,290, with 315-332 neurons per group. Values were pooled from 12 wells per condition, with experiments performed in duplicate.(c) Automated fluorescence microscopy. Primary rodent cortical neurons were transfected with mApple and TDP43-EGFP, and survival was determined by repeated imaging at regular intervals. The last time at which the cell was noted to be alive (red arrows) was used as the time of death. Cells that survive the entire length of the experiment (cyan arrow) were censored. Scale bar, 25 μm.(d,e) Cumulative risk of death over time for neurons transfected with EGFP, TDP43(WT)-EGFP (d) or TDP43A315T-neurons (e). Supplementary Table 1 lists the number of neurons, hazard ratios, 95% CI and P values for each condition, as determined by Cox proportional hazards analysis. Results were pooled from 12 wells per condition, with experiments performed in duplicate.f,g) Linear regression of calculated hazard ratio (± s.e.m.) as a function of total TDP43(WT)-EGFP (R2 = 0.9353) (f) or TDP43A315T-EGFP (R2 = 0.9574) (g) level. The total TDP43 level was determined by quantitative immunocytochemistry using TDP43-specific antibodies (Supplementary Fig. 3) and normalized to the amount of TDP43 in nontransfected neurons. Dashed lines, reference (HR = 1).


figf2 ( a ) Primary neurons were labeled with 35S - labeled methionine , and TDP43 was immunoprecipitated using anti - TDP43 antibodies ( IP - TDP43 ) or nonspecific IgG antibodies ( IP - IgG ) . ( b ) Log - normal plot of normalized TDP43 levels . TDP43 half - life was determined by fitting a first - order exponential curve to the data ( R2 = 0 . 8984 ) . Values were pooled from five independent experiments . ( c ) Potential confounders in half - life analyses . Top , total protein ideally decreases over time in a predictable manner ( top right panel ) . Middle , toxicity reduces the amount of measured protein , potentially shortening the calculated half - life ( middle right panel ) . Bottom , protein aggregation might also shorten half - life estimations if the protein cannot be immunoprecipitated ( black arrows , bottom right panel ) or lengthen it if the protein is more stable and successfully immunoprecipitated ( gray arrows , bottom right panel ) . ( d ) OPL . Primary cortical neurons were transfected with EGFP and TDP43 ( WT ) - Dendra2 , photoconverted then imaged by AFM . Dead cells ( arrows ) were excluded at the time of death . Pre , before photoconversion . ( e ) The half - life of TDP43 ( WT ) - Dendra2 was determined by fitting a first - order exponential curve to the data ( R2 = 0 . 9558 ) . ( f ) Including cells with aggregates prolonged TDP43 ( WT ) - Dendra2 half - life ( pink line , R2 = 0 . 9003 ; * P 0 . 0001 , F 15 . 34 , extra sum - of - squares F - test ) . Excluding cells that died over the 36 - h experiment also prolonged half - life ( cyan line , R2 = 0 . 7171 ; * * P 0 . 0001 , extra sum - of - squares F - test ) . Error bars represent s . e . m . Values were pooled from eight wells per condition , with experiments performed in quadruplicate .

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 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

figf2(a) Primary neurons were labeled with 35S-labeled methionine, and TDP43 was immunoprecipitated using anti-TDP43 antibodies (IP-TDP43) or nonspecific IgG antibodies (IP-IgG). (b) Log-normal plot of normalized TDP43 levels. TDP43 half-life was determined by fitting a first-order exponential curve to the data (R2 = 0.8984). Values were pooled from five independent experiments.(c) Potential confounders in half-life analyses. Top, total protein ideally decreases over time in a predictable manner (top right panel). Middle, toxicity reduces the amount of measured protein, potentially shortening the calculated half-life (middle right panel). Bottom, protein aggregation might also shorten half-life estimations if the protein cannot be immunoprecipitated (black arrows, bottom right panel) or lengthen it if the protein is more stable and successfully immunoprecipitated (gray arrows, bottom right panel).(d) OPL. Primary cortical neurons were transfected with EGFP and TDP43(WT)-Dendra2, photoconverted then imaged by AFM. Dead cells (arrows) were excluded at the time of death. Pre, before photoconversion. (e) The half-life of TDP43(WT)-Dendra2 was determined by fitting a first-order exponential curve to the data (R2 = 0.9558). (f) Including cells with aggregates prolonged TDP43(WT)-Dendra2 half-life (pink line, R2 = 0.9003; *P 0.0001, F 15.34, extra sum-of-squares F-test). Excluding cells that died over the 36-h experiment also prolonged half-life (cyan line, R2 = 0.7171; **P 0.0001, extra sum-of-squares F-test). Error bars represent s.e.m. Values were pooled from eight wells per condition, with experiments performed in quadruplicate.


figf3 ( a ) Scatter plot depicting half - life measurements for individual cells expressing TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ) and TDP43A315T - Dendra2 ( red , n = 251 ) . * P = 0 . 0029 , U = 11 , 797 , Mann - Whitney test . b ) Probability density plot for single - cell half - life measurements , demonstrating a reduced half - life of TDP43A315T - Dendra2 ( red , n = 251 ) , compared to TDP43 ( WT ) - Dendra2 ( cyan , n = 206 ; * * P = 0 . 0007 , two - sided Kolmogorov - Smirnov test ) . Values were pooled from eight wells per condition , with experiments performed in quadruplicate . ( c , d ) Probability density plots of single - cell half - life measurements for TDP43 ( WT ) - Dendra2 ( c ) and TDP43A315T - Dendra2 ( d ) . At 0 . 5 μM , all three of the compounds significantly reduced TDP43 ( WT ) - Dendra2 half - life compared to vehicle control ( DMSO , n = 110 ; FPZ , n = 96 , f * P = 1 × 10 − 4 ; MTM , n = 129 , m * P = 2 × 10 − 7 ; NCP , n = 111 , n * P = 2 × 10 − 5 , two - sided Kolmogorov - Smirnov test ) . The compounds also reduced TDP43A315T - Dendra2 half - life , in comparison to vehicle control ( DMSO , n = 120 ; FPZ , n = 155 , f * P = 2 × 10 − 9 ; MTM , n = 152 , m * P = 0 . 004 ; NCP , n = 111 , n * P = 0 . 005 , two - sided Kolmogorov - Smirnov test ) . Colored hash marks represent values from individual neurons in b - d . Values were obtained from eight wells per condition , with experiments performed in triplicate .

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 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

figf3(a) Scatter plot depicting half-life measurements for individual cells expressing TDP43(WT)-Dendra2 (cyan, n = 206) and TDP43A315T-Dendra2 (red, n = 251). *P = 0.0029, U = 11,797, Mann-Whitney test.b) Probability density plot for single-cell half-life measurements, demonstrating a reduced half-life of TDP43A315T-Dendra2 (red, n = 251), compared to TDP43(WT)-Dendra2 (cyan, n = 206; **P = 0.0007, two-sided Kolmogorov-Smirnov test). Values were pooled from eight wells per condition, with experiments performed in quadruplicate.(c,d) Probability density plots of single-cell half-life measurements for TDP43(WT)-Dendra2 (c) and TDP43A315T-Dendra2 (d). At 0.5 μM, all three of the compounds significantly reduced TDP43(WT)-Dendra2 half-life compared to vehicle control (DMSO, n = 110; FPZ, n = 96, f* P = 1 × 10−4; MTM, n = 129, m* P = 2 × 10−7; NCP, n = 111, n* P = 2 × 10−5, two-sided Kolmogorov-Smirnov test). The compounds also reduced TDP43A315T-Dendra2 half-life, in comparison to vehicle control (DMSO, n = 120; FPZ, n = 155, f* P = 2 × 10−9; MTM, n = 152, m* P = 0.004; NCP, n = 111, n* P = 0.005, two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons in b-d. Values were obtained from eight wells per condition, with experiments performed in triplicate.


figf4 ( a ) Each of the compounds induced LC3 - II in primary neurons , as determined by western blotting . At 5 μM but not 0 . 5 μM , the compounds significantly increased LC3 - II / GAPDH ( b ) and LC3 - II / LC3 - I ( c ) ratios ( nonsignificant ( NS ) , P > 0 . 05 ; 0 . 05 * P 0 . 01 , * * P 0 . 01 ; F = 13 . 74 ( in b ) and 9 . 74 ( in c ) , one - way ANOVA with Dunnett ' s post - test ) . Error bars represent s . e . m . Values were pooled from five independent experiments . ( d ) Optical pulse labeling of LC3 - Dendra2 in rodent primary cortical neurons . Scale bars , 25 μm . ( e ) The change in red fluorescence intensity over time was used to calculate LC3 - Dendra2 half - life , as in Figure 2 ( DMSO , R2 = 0 . 7333 ; Beclin , R2 = 0 . 6968 , FPZ , R2 = 0 . 8752 ; MTM , R2 = 0 . 8447 ; NCP , R2 = 0 . 8357 ; * P 0 . 0001 for global trend , F = 12 . 6 , extra sum - of - squares F - test ) . Error bars represent s . e . m . ( . ( f ) The median single - cell half - life of LC3 - Dendra2 was significantly reduced by beclin expression or treatment with 0 . 5 μM NCP , MTM or FPZ ( * P 0 . 01 , * * P 0 . 001 , Kruskal Wallis statistic = 52 . 56 , Kruskal - Wallis with Dunn ' s test ) . ( g ) Probability density plot of LC3 - Dendra2 half - life measurements from individual neurons treated with vehicle control ( DMSO , n = 140 ) , NCP ( n = 128 ) , MTM ( n = 91 ) , FPZ ( n = 77 ) or those expressing beclin ( n = 101 ) . n * , P = 6 × 10 − 4 ; m * , P = 1 × 10 − 10 ; f * , P = 5 × 10 − 9 ; b * , P = 2 × 10 − 3 ; two - sided Kolmogorov - Smirnov test ) . Colored hash marks represent values from individual neurons . Values were pooled from eight wells per condition , with experiments performed in triplicate .

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

figf4(a) Each of the compounds induced LC3-II in primary neurons, as determined by western blotting. At 5 μM but not 0.5 μM, the compounds significantly increased LC3-II/GAPDH (b) and LC3-II/LC3-I (c) ratios (nonsignificant (NS), P > 0.05; 0.05 *P 0.01, **P 0.01; F = 13.74 (in b) and 9.74 (in c), one-way ANOVA with Dunnett's post-test). Error bars represent s.e.m. Values were pooled from five independent experiments.(d) Optical pulse labeling of LC3-Dendra2 in rodent primary cortical neurons. Scale bars, 25 μm.(e) The change in red fluorescence intensity over time was used to calculate LC3-Dendra2 half-life, as in Figure 2 (DMSO, R2 = 0.7333; Beclin, R2 = 0.6968, FPZ, R2 = 0.8752; MTM, R2 = 0.8447; NCP, R2 = 0.8357; *P 0.0001 for global trend, F = 12.6, extra sum-of-squares F-test). Error bars represent s.e.m. (. (f) The median single-cell half-life of LC3-Dendra2 was significantly reduced by beclin expression or treatment with 0.5 μM NCP, MTM or FPZ (*P 0.01, **P 0.001, Kruskal Wallis statistic = 52.56, Kruskal-Wallis with Dunn's test).(g) Probability density plot of LC3-Dendra2 half-life measurements from individual neurons treated with vehicle control (DMSO, n = 140), NCP (n = 128), MTM (n = 91), FPZ (n = 77) or those expressing beclin (n = 101). n*, P = 6 × 10−4; m*, P = 1 × 10−10; f*, P = 5 × 10−9; b*, P = 2 × 10−3; two-sided Kolmogorov-Smirnov test). Colored hash marks represent values from individual neurons. Values were pooled from eight wells per condition, with experiments performed in triplicate.


figf5 ( a ) FPZ ( n = 490 ) and MTM ( n = 580 ) but not NCP ( n = 523 ) lowered total TDP43 ( WT ) - EGFP levels in comparison to vehicle ( DMSO , n = 1 , 007 ) . ( b ) Histogram of normalized , log - transformed TDP43 ( WT ) - EGFP levels . ( c ) Each compound also reduced TDP43A315T - EGFP levels ( DMSO , n = 983 ; FPZ , n = 550 ; MTM , n = 542 ; NCP , n = 493 ) . ( d ) Histogram of single - cell TDP43 ( A315T ) - EGFP levels . Asterisks in b and d denote median values for cells exposed to vehicle ( gray ) , FPZ ( green ) , MTM ( blue ) or NCP ( purple ) . For FPZ , MTM and NCP versus DMSO in a and d , P 1 × 10 − 3 by two - sided Kolmogorov - Smirnov test . e ) No compound reduced TDP43 ( WT ) - EGFP inclusions 48 h after transfection ( DMSO , n = 360 ; FPZ , n = 194 ; MTM , n = 234 ; NCP , n = 203 ) . ( f ) FPZ and MTM but not NCP reduced TDP43A315T - EGFP inclusions at 48 h ( DMSO , n = 327 ; FPZ , n = 226 ; MTM , n = 262 ; NCP , n = 253 ) . ( g ) No compound affected TDP43 ( WT ) - EGFP localization 24 h after transfection ( DMSO , n = 256 ; FPZ , n = 212 ; MTM , n = 250 ; NCP , n = 241 ) . ( h , i ) Each compound ( h ) prevented TDP43A315T - EGFP cytoplasmic mislocalization ( DMSO , n = 233 ; FPZ , n = 268 ; MTM , n = 263 ; NCP , n = 270 ) and reduced cytoplasmic TDP43 ( A315T ) - EGFP levels ( DMSO , n = 125 ; FPZ , n = 102 ; MTM , n = 121 ; NCP , n = 144 . * P 0 . 05 ; NS , P > 0 . 05 ) ( i ) . For a , c and e - i , * P 0 . 05 ; nonsignificant ( NS ) , P > 0 . 05 , one - way ANOVA with Dunnett ' s test . Error bars represent s . e . m . Values in a - d were pooled from 8 - 12 wells per condition from six experiments , values in e - h were pooled from eight wells per condition from three experiments , and values in i were pooled from five wells per condition from two experiments .

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

figf5(a) FPZ (n = 490) and MTM (n = 580) but not NCP (n = 523) lowered total TDP43(WT)-EGFP levels in comparison to vehicle (DMSO, n = 1,007).(b) Histogram of normalized, log-transformed TDP43(WT)-EGFP levels.(c) Each compound also reduced TDP43A315T-EGFP levels (DMSO, n = 983; FPZ, n = 550; MTM, n = 542; NCP, n = 493).(d) Histogram of single-cell TDP43(A315T)-EGFP levels. Asterisks in b and d denote median values for cells exposed to vehicle (gray), FPZ (green), MTM (blue) or NCP (purple). For FPZ, MTM and NCP versus DMSO in a and d, P 1 × 10−3 by two-sided Kolmogorov-Smirnov test.e) No compound reduced TDP43(WT)-EGFP inclusions 48 h after transfection (DMSO, n = 360; FPZ, n = 194; MTM, n = 234; NCP, n = 203).(f) FPZ and MTM but not NCP reduced TDP43A315T-EGFP inclusions at 48 h (DMSO, n = 327; FPZ, n = 226; MTM, n = 262; NCP, n = 253). (g) No compound affected TDP43(WT)-EGFP localization 24 h after transfection (DMSO, n = 256; FPZ, n = 212; MTM, n = 250; NCP, n = 241). (h,i) Each compound (h) prevented TDP43A315T-EGFP cytoplasmic mislocalization (DMSO, n = 233; FPZ, n = 268; MTM, n = 263; NCP, n = 270) and reduced cytoplasmic TDP43(A315T)-EGFP levels (DMSO, n = 125; FPZ, n = 102; MTM, n = 121; NCP, n = 144. *P 0.05; NS, P > 0.05) (i). For a, c and e-i, *P 0.05; nonsignificant (NS), P > 0.05, one-way ANOVA with Dunnett's test. Error bars represent s.e.m. Values in a-d were pooled from 8-12 wells per condition from six experiments, values in e-h were pooled from eight wells per condition from three experiments, and values in i were pooled from five wells per condition from two experiments.


figf6 ( a , b ) Primary neurons transfected with TDP43 ( WT ) - EGFP ( a ) or TDP43A315T - EGFP ( b ) were treated with autophagy inducers and survival determined by AFM . Data were obtained from eight wells per condition , with experiments performed in triplicate . Supplementary Table 2 lists the number of neurons per condition , hazard ratios , 95 % CI and P values , as determined by Cox hazards analysis . ( c ) Autophagic induction improved survival in TDP43M337V HB9 - positive human iPSC - derived MNs . ( d ) FPZ and MTM also reduced the risk of death in MAP2 - positive human iPSC - derived MNs Values in c and d were pooled from 18 wells per condition , with experiments performed in triplicate . ( e ) Astrocytes differentiated from human iPSCs exhibit mutant TDP43 - related toxicity that is mitigated by FPZ and MTM . Values were pooled from 18 wells per condition , performed in triplicate . Supplementary Table 3 lists the number of human iPSC - derived neurons and astrocytes per condition , hazard ratios , 95 % CI and P values , as determined by Cox hazards analysis .

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

figf6(a,b) Primary neurons transfected with TDP43(WT)-EGFP (a) or TDP43A315T-EGFP (b) were treated with autophagy inducers and survival determined by AFM. Data were obtained from eight wells per condition, with experiments performed in triplicate. Supplementary Table 2 lists the number of neurons per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis. (c) Autophagic induction improved survival in TDP43M337V HB9-positive human iPSC-derived MNs. (d) FPZ and MTM also reduced the risk of death in MAP2-positive human iPSC-derived MNs Values in c and d were pooled from 18 wells per condition, with experiments performed in triplicate. (e) Astrocytes differentiated from human iPSCs exhibit mutant TDP43-related toxicity that is mitigated by FPZ and MTM. Values were pooled from 18 wells per condition, performed in triplicate. Supplementary Table 3 lists the number of human iPSC-derived neurons and astrocytes per condition, hazard ratios, 95% CI and P values, as determined by Cox hazards analysis.


Figure 1B . qRT - PCR expression analysis of lncMN2 isoforms ( left panel ) and embedded miRNAs ( right panel ) in mESC and EBs differentiated for 6 days ( EB6 ) . Data , that are expressed as mean ( error bars represent SD ) , were normalized over Atp50 mRNA ( left ) and snoRNA U25 ( right ) levels and reported as RNA fold over the control ( mESC ) , set as 1 . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) . C . qRT - PCR expression analysis of lncMN2 - 202 / 203 / 204 isoforms ( left panel ) , miR - 325 - 3p ( middle panel ) and miR - 384 - 5p ( right panel ) in WT , KO1 and KO2 EB6 . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) . D . Cytofluorimetric analysis of cells from dissociated EB6 . The left diagram reports the distribution of cells ( percentage of the peak ) according to GFP levels , from WT ( gray ) , KO1 ( orange ) and KO2 ( blue ) EB . In the histogram aside , the percentage of GFP + cells in WT , KO1 and KO2 is reported . Data are expressed as mean and error bars represent SD . N = 3 biological replicates . Data information : histogram Histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) . E . Left : representative immunofluorescence of the MN marker Isl1 ( red signals ) in cells maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . . Right : box plot shows the distribution of Isl1 + nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants ( clone 1 and clone 2 ) . Data are expressed as mean and error bars represent SD . The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value . N = 3 biological replicates and 10 fields were analyzed for each replicate . Scale bar = 100 mm . Data information : * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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

Figure 1B. qRT-PCR expression analysis of lncMN2 isoforms (left panel) and embedded miRNAs (right panel) in mESC and EBs differentiated for 6 days (EB6). Data, that are expressed as mean (error bars represent SD), were normalized over Atp50 mRNA (left) and snoRNA U25 (right) levels and reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).C. qRT-PCR expression analysis of lncMN2-202/203/204 isoforms (left panel), miR-325-3p (middle panel) and miR-384-5p (right panel) in WT, KO1 and KO2 EB6. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).D. Cytofluorimetric analysis of cells from dissociated EB6. The left diagram reports the distribution of cells (percentage of the peak) according to GFP levels, from WT (gray), KO1 (orange) and KO2 (blue) EB. In the histogram aside, the percentage of GFP+ cells in WT, KO1 and KO2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data information: histogram Histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).E. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm.. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in lncMN2 KO mutants (clone 1 and clone 2). Data are expressed as mean and error bars represent SD. The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Scale bar=100 mm. Data information: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


Figure 3B . qRT - PCR expression analysis of lncMN2 - 203 isoform , lncMN2 - 202 / 204 , miR - 325 - 3p and miR - 384 - 5p embedded miRNAs in WT , ∆ SP1 and ∆ SP2 EB6 . Data , that are expressed as mean ( error bars represent SD ) , are normalized over Atp5o mRNA levels ( for lncRNA isoform quantification ) or over snoRNA U25 levels ( for miRNA quantification ) and are reported as RNA fold over the control ( mESC ) , set as 1 . N = 3 biological replicates . Data are expressed as mean and error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) . C . Cytofluorimetric analysis of cells from dissociated EB6 . The right diagram reports the cell distribution ( percentage of the peak ) according to GFP levels , from WT ( gray line ) , ∆ SP1 ( purple line ) and ∆ SP2 ( blue line ) EB6 . In the histogram aside , the percentage of GFP + cells in WT , ∆ SP1 and ∆ SP2 is reported . Data are expressed as mean and error bars represent SD . N = 3 biological replicates . Data are expressed as mean and error bars represent SD . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired Student ' s t - test ) . D . Left : representative immunofluorescence of the MN marker Isl1 ( red signals ) in cells maintained in vitro for 14 days after EB6 dissociation ( DIV14 ) . DAPI counterstaining in blue . Scale bar = 100 mm . Right : box plot shows the distribution of Isl1 + nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆ SP mutants ( clone 1 and clone 2 ) . The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value . N = 3 biological replicates and 10 fields were analyzed for each replicate . Data are expressed as mean and error bars represent SD . Scale bar = 100 mm . Data information : histogram dots represent single fields replicates . * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , n . s . > 0 . 05 ( two - tailed , unpaired 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 3B. qRT-PCR expression analysis of lncMN2-203 isoform, lncMN2-202/204, miR-325-3p and miR-384-5p embedded miRNAs in WT, ∆SP1 and ∆SP2 EB6. Data, that are expressed as mean (error bars represent SD), are normalized over Atp5o mRNA levels (for lncRNA isoform quantification) or over snoRNA U25 levels (for miRNA quantification) and are reported as RNA fold over the control (mESC), set as 1. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).C. Cytofluorimetric analysis of cells from dissociated EB6. The right diagram reports the cell distribution (percentage of the peak) according to GFP levels, from WT (gray line), ∆SP1 (purple line) and ∆SP2 (blue line) EB6. In the histogram aside, the percentage of GFP+ cells in WT, ∆SP1 and ∆SP2 is reported. Data are expressed as mean and error bars represent SD. N=3 biological replicates. Data are expressed as mean and error bars represent SD. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).D. Left: representative immunofluorescence of the MN marker Isl1 (red signals) in cells maintained in vitro for 14 days after EB6 dissociation (DIV14). DAPI counterstaining in blue. Scale bar=100 mm. Right: box plot shows the distribution of Isl1+ nuclei with respect to the total cell number marked with DAPI staining in WT and in ∆SP mutants (clone 1 and clone 2). The central band represents median and the whiskers represent the distribution that goes from the minimum value to the lower quartile and than from the upper quartile to the maximum value. N=3 biological replicates and 10 fields were analyzed for each replicate. Data are expressed as mean and error bars represent SD. Scale bar=100 mm. Data information: histogram dots represent single fields replicates. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.> 0.05 (two-tailed, unpaired Student's t-test).


Figure 1B . Volcano plot showing DE 1hr after NB1 ( top ) and IMR32 ( bottom ) cell treatment with ALKAL2 . Dashed lines show DE thresholds . Up - / downregulated genes indicated in blue . Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red . D . Temporal dynamics of ALKAL2 - induced transcription of ARC , EGR1 - 3 , FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib , as indicated . E . Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells . Cells were treated for 0 , 1 and 6h in the presence and absence of lorlatinib as indicated . G . Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells . Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated . RNA - Seq analysis was performed using 3 biological repeats . Immunoblots are representative of at least 3 independent experiments .

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 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 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 1B. Volcano plot showing DE 1hr after NB1 (top) and IMR32 (bottom) cell treatment with ALKAL2. Dashed lines show DE thresholds. Up-/downregulated genes indicated in blue. Six genes that are DE in both cell lines and sensitive to the ALK inhibitor lorlatinib are indicated and labelled in red.D. Temporal dynamics of ALKAL2-induced transcription of ARC, EGR1-3, FOS and FOSB in NB1 and IMR32 cells in the presence and absence of the lorlatinib, as indicated.E. Immunoblot validation of ALKAL2 induction of EGR1 and FOS at the protein level in NB1 cells. Cells were treated for 0, 1 and 6h in the presence and absence of lorlatinib as indicated.G. Immunoblot analysis of ALKAL2 induction of pSRF in NB1 cells. Cells were treated for 0 and 1h in the presence and absence of lorlatinib as indicated. RNA-Seq analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.


Figure 2B . Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 + / - lorlatinib stimulation as indicated . Dashed lines indicate differential expression thresholds . Differentially expressed proteins indicated in blue . Most pronounced responding proteins indicated in black and labelled . C , D . Immunoblot analysis of VGF protein in NB cells . ( C ) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib . ( D ) CLB - BAR and CLB - GE cells after 24h inhibition with lorlatinib . Dashed lines indicate differential expression thresholds . Differentially expressed proteins indicated in blue . Most pronounced responding proteins indicated in black and labelled . E . Volcano plots showing differential phosphorylation . Labelling colors as in ( B ) . G . Graphical representation of FOXO3 dynamics , indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation , in the presence or absence of lorlatinib . H - I . Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated . The slower migrating FOXO3a band in SDS - PAGE in ( H ) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib . Proteomic analysis was performed using 3 biological repeats . Immunoblots are representative of at least 3 independent experiments .

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 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 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

Figure 2B. Volcano plots showing differential protein expression 1hr and 24hr after ALKAL2 +/- lorlatinib stimulation as indicated. Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled.C, D. Immunoblot analysis of VGF protein in NB cells. (C) NB1 cells after 24h stimulation with ALKAL2 in the presence or absence of lorlatinib. (D) CLB-BAR and CLB-GE cells after 24h inhibition with lorlatinib.Dashed lines indicate differential expression thresholds. Differentially expressed proteins indicated in blue. Most pronounced responding proteins indicated in black and labelled. E. Volcano plots showing differential phosphorylation. Labelling colors as in (B).G. Graphical representation of FOXO3 dynamics, indicating S253 phosphorylation and total FOXO3 protein levels in response to ALKAL2 stimulation, in the presence or absence of lorlatinib.H-I. Immunoblot validation of FOXO3a and STAT3 in response to ALKAL2 stimulation in the presence or absence of lorlatinib as indicated. The slower migrating FOXO3a band in SDS-PAGE in (H) likely reflects FOXO3a phosphorylation that is not seen in the presence of lorlatinib. Proteomic analysis was performed using 3 biological repeats. Immunoblots are representative of at least 3 independent experiments.


Figure 3A . Hematoxylin and eosine staining of longitudinal sections of P9 pups at the central part of the left celiac ganglion . B . Quantification of the area of celiac ganglia cross - section . Largest sections from the central part of left celiac ganglions of different individuals were chosen for the analysis . * * p < 0 . 01 , one - way ANOVA followed by Tukey multiple comparison test . Data shown represents mean ± SD . C . Hyperplasia quantification in central sections of P9 left celiac ganglions shown as a percent of hyperplastic regions areas per ganglion cross - section . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Tukey multiple comparison test . Data shown represents mean ± SD . D . Kaplan - Meier survival curve of mice resulting from intercrosses of Th - MYCN hemizygotes and Alk - F1178S heterozygote mice ( p < 0 . 0001 ; log - rank test ) . Wild type littermates were excluded . E . Gross appearance , hematoxylin and eosin as well as Ki67 stained sections of representative Th - MYCN and Alk - F1178S ; Th - MYCN tumours . Scale bars indicate 250μm . F . Ki67 immunohistochemically staining of P9 celiac ganglia in mice of the indicated genotype ( quantified in G ) . G . Quantification of hyperplastic areas ( shown as percent of hyperplastic regions per ganglion cross - section ) and Ki67 expression ( shown as positive for Ki67 staining areas per total area of the section through the ganglion central part at P9 ) . ( * p < 0 . 05 , * * * p < 0 . 001 , * * * * p < 0 . 0001 , one - way ANOVA followed by Tukey multiple comparison test . Data shown represents mean ± SD ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3A. Hematoxylin and eosine staining of longitudinal sections of P9 pups at the central part of the left celiac ganglion. B. Quantification of the area of celiac ganglia cross-section. Largest sections from the central part of left celiac ganglions of different individuals were chosen for the analysis. **p<0.01, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD. C. Hyperplasia quantification in central sections of P9 left celiac ganglions shown as a percent of hyperplastic regions areas per ganglion cross-section. **p<0.01, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD. D. Kaplan-Meier survival curve of mice resulting from intercrosses of Th-MYCN hemizygotes and Alk-F1178S heterozygote mice (p<0.0001; log-rank test). Wild type littermates were excluded.E. Gross appearance, hematoxylin and eosin as well as Ki67 stained sections of representative Th-MYCN and Alk-F1178S;Th-MYCN tumours. Scale bars indicate 250μm.F. Ki67 immunohistochemically staining of P9 celiac ganglia in mice of the indicated genotype (quantified in G). G. Quantification of hyperplastic areas (shown as percent of hyperplastic regions per ganglion cross-section) and Ki67 expression (shown as positive for Ki67 staining areas per total area of the section through the ganglion central part at P9). (*p<0.05, ***p<0.001, ****p<0.0001, one-way ANOVA followed by Tukey multiple comparison test. Data shown represents mean ± SD).


Figure 4A . The oncogenic activity of MYCN is potentiated by overexpression of ALKAL2 . Kaplan - Meier survival curves for Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN and Th - MYCN mice . Also shown are Rosa26 _ Alkal2N , Alk - F1178S and control ( Ctrl ) mice . Comparison of survival of Th - MYCN alone and Rosa26 _ Alkal2 ; Th - MYCN curves showed a significant difference ( p = 0 . 003 ; log - rank test ) . Tumours harvested from Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN and Th - MYCN mice express NB markers . Tumours from all three genotypes were large , in most cases filling the abdominal cavity ( B ) . Dissection post - mortem revealed that the majority of Rosa26 _ Alkal2 ; Th - MYCN ( 18 / 20 ) and Alk - F1178S ; Th - MYCN ( 7 / 10 ) tumours did not involve the adrenal glands ( C ) . Histological examination of Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN and Th - MYCN tumours revealed positive staining for NCAM1 , synaptophysin ( SYP ) and Chromogranin A ( CGA ) . Scale bars indicate 100μm . ( D ) Histological examination of Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN and Th - MYCN tumours was confirmed for NCAM1 and SYP along with MYCN , ALK and ALKAL2 by immunoblotting ( E ) . Immunoblots are representative of 3 independent technical analyses . F . Accumulated Kaplan - Meier survival curves are shown for all monitored Rosa26 _ Alkal2 ; Th - MYCN , Alk - F1178S ; Th - MYCN and Th - MYCN mice over time , estimating tumour penetrance ( p < 0 . 001 ; log - rank test ) .

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Figure 4A. The oncogenic activity of MYCN is potentiated by overexpression of ALKAL2. Kaplan-Meier survival curves for Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice. Also shown are Rosa26_Alkal2N, Alk-F1178S and control (Ctrl) mice. Comparison of survival of Th-MYCN alone and Rosa26_Alkal2;Th-MYCN curves showed a significant difference (p=0.003; log-rank test).Tumours harvested from Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice express NB markers. Tumours from all three genotypes were large, in most cases filling the abdominal cavity (B).Dissection post-mortem revealed that the majority of Rosa26_Alkal2;Th-MYCN (18/20) and Alk-F1178S;Th-MYCN (7/10) tumours did not involve the adrenal glands (C).Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours revealed positive staining for NCAM1, synaptophysin (SYP) and Chromogranin A (CGA). Scale bars indicate 100μm. (D)Histological examination of Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN tumours was confirmed for NCAM1 and SYP along with MYCN, ALK and ALKAL2 by immunoblotting (E). Immunoblots are representative of 3 independent technical analyses.F. Accumulated Kaplan-Meier survival curves are shown for all monitored Rosa26_Alkal2;Th-MYCN, Alk-F1178S;Th-MYCN and Th-MYCN mice over time, estimating tumour penetrance (p<0.001; log-rank test).


Figure 5RNA - Seq based differential gene expression analysis of tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( Alkal2 ) [ n = 6 ] , Alk - F1178S ; Th - MYCN ( AlkF1174S ) [ n = 6 ] and Th - MYCN ( MYCN ) mice [ n = 4 ] . See Table S4 for detailed results . A . Read coverage of the codon - optimised Alkal2 transgene , confirming Alkal2 expression in Alkal2 tumours . C - D . Volcano plot showing differential expression ( DE ) between Alkal2 and MYCN tumours . Differentially expressed genes are shown in blue ( DE in Alkal2 tumours only ) or black ( DE in both Alkal2 and AlkF1174S tumours , as shown in ( D ) ) . Top ranked genes labelled . Dashed lines represent DE cut - offs . E . DE heatmap based on unsupervised hierarchical clustering of 400 DE genes ( rows ) and 16 samples ( columns , as indicated on top ) . Sample color legend as in ( A ) . Color key shown on top left . F . Boxplot showing Vgf expression in the 3 tumour types as indicated . Box plots indicate median values and lower / upper quartiles with whiskers extending to 1 . 5 times the interquartile range . P values calculated using Wald test as reported by DESeq2 . G . Histological examination of Th - MYCN , Rosa26 _ Alkal2 ; Th - null and Alk - F1178S ; Th - MYCN tumours revealing positive staining for VGF . Scale bars indicate 100μm . H . Kaplan - Meier relapse free ( RF ) and event free ( EF ) survival probability curves from two different NB cohorts , the Versteeg 88 cohort ( upper panel ) and the Kocak 649 cohort ( lower panel ) , as derived from the R2 platform . Patients with higher VGF expression are highlighted in blue , whereas patients with lower expression are highlighted in red . The log - rank test P values are indicated .

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Figure 5RNA-Seq based differential gene expression analysis of tumours arising in Rosa26_Alkal2;Th-MYCN (Alkal2) [n=6], Alk-F1178S;Th-MYCN (AlkF1174S) [n=6] and Th-MYCN (MYCN) mice [n=4]. See Table S4 for detailed results. A. Read coverage of the codon-optimised Alkal2 transgene, confirming Alkal2 expression in Alkal2 tumours. C-D. Volcano plot showing differential expression (DE) between Alkal2 and MYCN tumours. Differentially expressed genes are shown in blue (DE in Alkal2 tumours only) or black (DE in both Alkal2 and AlkF1174S tumours, as shown in (D)). Top ranked genes labelled. Dashed lines represent DE cut-offs.E. DE heatmap based on unsupervised hierarchical clustering of 400 DE genes (rows) and 16 samples (columns, as indicated on top). Sample color legend as in (A). Color key shown on top left.F. Boxplot showing Vgf expression in the 3 tumour types as indicated. Box plots indicate median values and lower/upper quartiles with whiskers extending to 1.5 times the interquartile range. P values calculated using Wald test as reported by DESeq2.G. Histological examination of Th-MYCN, Rosa26_Alkal2;Th-null and Alk-F1178S;Th-MYCN tumours revealing positive staining for VGF. Scale bars indicate 100μm.H. Kaplan-Meier relapse free (RF) and event free (EF) survival probability curves from two different NB cohorts, the Versteeg 88 cohort (upper panel) and the Kocak 649 cohort (lower panel), as derived from the R2 platform. Patients with higher VGF expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P values are indicated.


Figure 6Murine NB cell lines were generated from tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) and Alk - F1178S ; Th - MYCN ( # 3456 ) mice . A . Alkal2 expression in cells derived from Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) and Alk - F1178S ; Th - MYCN ( # 3456 ) NB . Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines . Whole cell lysates ( 30μg ) were analysed in each lane . B . The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) and Alk - F1178S ; Th - MYCN ( # 3456 ) cell lines . Data is presented as mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - tailed paired student t - test . C - D . Brigatinib suppressed tumour spheroid formation and spheroid viability . Cells ( # 3456 or # 3540 ) were treated with brigatinib ( 0 , 150nM ) for 4d in ultra - low attachment plates . The spheroid number was analyzed by IncuCyte ® Live Cell Analysis Systems . Data are presented as means ± SEM from three independent experiments . * p < 0 . 05 , two - tailed unpaired student t - test . Scale bar ( C ) is 200μm . E . Tumour spheroids formed from either Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) or Alk - F1178S ; Th - MYCN ( # 3456 ) were formed at indicated cell number in ultra - low attachment plates for 3d and followed by brigatinib ( 0 , 150nM ) for 10d . Inhibitor was re - fed every other day . Cell viability was determined by CellTiter - Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments . * p < 0 . 05 , * * * * p < 0 . 0001 , two - tailed unpaired student t - test . F . Cell viability . Mouse tumour derived cell lines # 3540 ( Rosa26 _ Alkal2 ; Th - MYCN ) and # 3456 ( Alk - F1178S ; Th - MYCN ) were treated with brigatinib ( 125nM and 500nM ) and viability was evaluated by using a resazurin - based assay . Data is presented as mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - tailed paired student t - test . G . Brigatinib treatment ( 0 or 150 nM ) for 6h resulted in inhibition of ALK phosphorylation , and of activation of downstream signaling ( ERK1 / 2 ) , as well as MYCN expression . Cell lysates ( Rosa26 _ Alkal2 ; Th - MYCN ( # 3540 ) and Alk - F1178S ; Th - MYCN ( # 3456 ) ) were immunoblotted with the indicated antibodies .

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Figure 6Murine NB cell lines were generated from tumours arising in Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) mice. A. Alkal2 expression in cells derived from Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) NB. Immunoblotting analysis for ALKAL2 and tubulin in the indicated mouse NB cell lines. Whole cell lysates (30μg) were analysed in each lane. B. The effect of increasing concentrations of brigatintib on cell confluence was analyzed by IncuCyte Live Cell Analysis of both Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456) cell lines. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.C-D. Brigatinib suppressed tumour spheroid formation and spheroid viability. Cells (#3456 or #3540) were treated with brigatinib (0, 150nM) for 4d in ultra-low attachment plates. The spheroid number was analyzed by IncuCyte® Live Cell Analysis Systems. Data are presented as means ± SEM from three independent experiments. *p<0.05, two-tailed unpaired student t-test. Scale bar (C) is 200μm.E. Tumour spheroids formed from either Rosa26_Alkal2;Th-MYCN (#3540) or Alk-F1178S;Th-MYCN (#3456) were formed at indicated cell number in ultra-low attachment plates for 3d and followed by brigatinib (0, 150nM) for 10d. Inhibitor was re-fed every other day. Cell viability was determined by CellTiter-Glo 3D cell viability kit and data is presented as mean ± SEM from five independent experiments. *p<0.05, ****p<0.0001, two-tailed unpaired student t-test.F. Cell viability. Mouse tumour derived cell lines #3540 (Rosa26_Alkal2;Th-MYCN) and #3456 (Alk-F1178S;Th-MYCN) were treated with brigatinib (125nM and 500nM) and viability was evaluated by using a resazurin-based assay. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.G. Brigatinib treatment (0 or 150 nM) for 6h resulted in inhibition of ALK phosphorylation, and of activation of downstream signaling (ERK1/2), as well as MYCN expression. Cell lysates (Rosa26_Alkal2;Th-MYCN (#3540) and Alk-F1178S;Th-MYCN (#3456)) were immunoblotted with the indicated antibodies.


Figure 7A . Cells derived from tumours arising in Rosa26 _ Alkal2 ; Th - MYCN ( # 4953 ) and Alk - F1178S ; Th - MYCN ( # 4938 ) mice are sensitive to both lorlatinib and brigatinib . The effect of increasing concentrations of each ALK TKI ( as indicated ) on cell confluence was analyzed by IncuCyte Live Cell Analysis . Data is presented as mean ± SEM from three independent experiments . * p < 0 . 05 , * * p < 0 . 005 ; two - tailed paired student t - test . B . Tumour volume changes over time for Rosa26 _ Alkal2 ; Th - MYCN mice treated with lorlatinib ( 10 mg / kg ; twice daily ) or vehicle control . Tumour volume was measured by ultrasound on Day 0 and 7 , and by direct measurement at Day 14 . Day 0 ( lorlatinib n = 7 , Ctrl n = 6 ) , Day 7 ( lorlatinib n = 2 , Ctrl n = 5 ) and Day 14 ( lorlatinib n = 7 , Ctrl n = 6 ) . Data shown represents mean ± SD . * * p < 0 . 005 ; two - tailed unpaired student t - test . C . Rosa26 _ Alkal2 ; Th - MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls . Data shown represents mean ± SD . D . Representative ultrasound images of tumours observed in Rosa26 _ Alkal2 ; Th - MYCN mice with annotated measurements at Day 0 and Day 7 . Tumours arise in the retroperitoneal space ventral to the aorta , Ao . E . Representative MRI imaging of Rosa26 _ Alkal2 ; Th - MYCN tumours in response to lorlatinib at 4d and 14d . F . Rosa26 _ Alkal2 ; Th - MYCN tumours from lorlatinib or vehicle controls were analysed for pHis . A representative field of view for each tumour at 40x ( 175740μm2 ) were manually counted . Data shown represents mean ± 95 % CI . p = 0 . 0286 ; Mann - Whitney test .

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Figure 7A. Cells derived from tumours arising in Rosa26_Alkal2;Th-MYCN (#4953) and Alk-F1178S;Th-MYCN (#4938) mice are sensitive to both lorlatinib and brigatinib. The effect of increasing concentrations of each ALK TKI (as indicated) on cell confluence was analyzed by IncuCyte Live Cell Analysis. Data is presented as mean ± SEM from three independent experiments. *p<0.05, **p<0.005; two-tailed paired student t-test.B. Tumour volume changes over time for Rosa26_Alkal2;Th-MYCN mice treated with lorlatinib (10 mg/kg; twice daily) or vehicle control. Tumour volume was measured by ultrasound on Day 0 and 7, and by direct measurement at Day 14. Day 0 (lorlatinib n=7, Ctrl n=6), Day 7 (lorlatinib n=2, Ctrl n=5) and Day 14 (lorlatinib n=7, Ctrl n=6). Data shown represents mean ± SD. **p<0.005; two-tailed unpaired student t-test.C. Rosa26_Alkal2;Th-MYCN animals treated with lorlatinib did not display any significant loss of body weight compared with vehicle controls. Data shown represents mean ± SD.D. Representative ultrasound images of tumours observed in Rosa26_Alkal2;Th-MYCN mice with annotated measurements at Day 0 and Day 7. Tumours arise in the retroperitoneal space ventral to the aorta, Ao.E. Representative MRI imaging of Rosa26_Alkal2;Th-MYCN tumours in response to lorlatinib at 4d and 14d.F. Rosa26_Alkal2;Th-MYCN tumours from lorlatinib or vehicle controls were analysed for pHis. A representative field of view for each tumour at 40x (175740μm2) were manually counted. Data shown represents mean ± 95% CI. p=0.0286; Mann-Whitney test.


Figure 1Hoxb8 - FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1 - inhibitors PF - 477736 ( PF ) or CHIR - 124 ( CHIR ) . Representative histograms of CHK1i - treated ( grey ) or DMSO - treated ( black ) cells , analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown . WT Hoxb8 - FL cells were treated with CHIR - 124 in the presence or absence of the pan - caspase inhibitor QVD . Cell death was assessed after 24h using flow cytometry as in ( A ) . Western blot of CHIR - treated Hoxb8 - FL WT and Bax - / - Bak - / - cells using the indicated antibodies . WT and Bax - / - Bak - / - Hoxb8 - FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1 - inhibition by real - time RT - qPCR . Bars represent means ± S . D . of independent experiments ( n = 4 - 6 ) , performed in duplicates . 50 . 000 Hoxb8 - FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1 - inhibition . Cells were counted 48h post seeding .

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Figure 1Hoxb8-FL cells generated from bone marrow of mice of the indicated genotypes were treated for 24h with graded doses of the CHK1-inhibitors PF-477736 (PF) or CHIR-124 (CHIR). Representative histograms of CHK1i-treated (grey) or DMSO-treated (black) cells, analyzed for cell cycle distribution and cell death using Nicoletti staining and flow cytometry are shown.WT Hoxb8-FL cells were treated with CHIR-124 in the presence or absence of the pan-caspase inhibitor QVD. Cell death was assessed after 24h using flow cytometry as in (A).Western blot of CHIR-treated Hoxb8-FL WT and Bax-/-Bak-/- cells using the indicated antibodies.WT and Bax-/-Bak-/- Hoxb8-FL cells were treated for up to 8h with 500 nM CHIR and p21 mRNA levels were compared to Hprt in response to CHK1-inhibition by real-time RT-qPCR. Bars represent means ± S.D. of independent experiments (n=4-6), performed in duplicates.50.000 Hoxb8-FL cells of the indicated genotypes were seeded to assess cell growth in response to CHK1-inhibition. Cells were counted 48h post seeding.


Figure 2LSK ( Lin - Sca1 + cKit + ) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13 . 5 or the bone marrow of adult mice and treated for 48h with PF - 477736 [ 1 . 5 μM ] . Survival was assessed using AnnexinV - staining and flow cytometry . Bars represent means ± S . E . M . from n = 6 wild type and n = 3 Vav - BCL2 fetal liver LSK cells ( left ) and n = 3 bone marrow LSK cells ( right ) . WT and Vav - BCL2 derived E14 . 5 total fetal liver cells ( B ) were treated for 72h ( B ) with different doses of CHK1i . Survival was assessed using Nicoletti staining and flow cytometry . Bars represent means ± S . E . M . ( n = 3 biological replicates per genotype ) . WT and Vav - BCL2 derived E14 . 5 total bone marrow cells ( C ) were treated for up to 48h with different doses of CHK1i . Survival was assessed using Nicoletti staining and flow cytometry . Bars represent means ± S . E . M . ( n = 3 biological replicates per genotype ) .

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Figure 2LSK (Lin- Sca1+ cKit+) cells of the indicated genotypes were isolated by cell sorting from the fetal liver at embryonic day E13.5 or the bone marrow of adult mice and treated for 48h with PF-477736 [1.5 μM]. Survival was assessed using AnnexinV-staining and flow cytometry. Bars represent means ± S.E.M. from n=6 wild type and n=3 Vav-BCL2 fetal liver LSK cells (left) and n=3 bone marrow LSK cells (right).WT and Vav-BCL2 derived E14.5 total fetal liver cells (B) were treated for 72h (B) with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).WT and Vav-BCL2 derived E14.5 total bone marrow cells (C) were treated for up to 48h with different doses of CHK1i. Survival was assessed using Nicoletti staining and flow cytometry. Bars represent means ± S.E.M. (n=3 biological replicates per genotype).


Figure 3MACS - purified CD34 + human cord - blood derived HSPC were treated with PF - 477736 or CHIR - 124 ± the caspase - inhibitor QVD ( 50 μM ) . Cell death was assessed using AnnexinV / 7 - AAD staining and flow cytometry . CD34 + cells were transduced with a BCL2 - encoding lentivirus or a control virus , along with IRES - GFP with a MOI of 10 for two consecutive days , followed by CHK1i application for 48 h . Survival was assessed using Nicoletti staining and flow cytometry . Percentage of living GFP + transduced human CD34 + HSPCs , cultured in the absence or presence of CHK1i for 48 hours . Colony formation potential of human CD34 + HSPCs transduced with empty control or BCL2 encoding virus . Colonies were counted 10 days post seeding of 1000 CD34 + cells per plate / sample . " Myeloid colonies " include granulocyte / monocyte , monocyte , granulocyte and GEMM ( granulocyte , erythroid , monocyte / macrophage and megakaryocyte ) colonies .

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Figure 3MACS-purified CD34+ human cord-blood derived HSPC were treated with PF-477736 or CHIR-124 ± the caspase-inhibitor QVD (50 μM). Cell death was assessed using AnnexinV/7-AAD staining and flow cytometry.CD34+ cells were transduced with a BCL2-encoding lentivirus or a control virus, along with IRES-GFP with a MOI of 10 for two consecutive days, followed by CHK1i application for 48 h. Survival was assessed using Nicoletti staining and flow cytometry.Percentage of living GFP+ transduced human CD34+ HSPCs, cultured in the absence or presence of CHK1i for 48 hours.Colony formation potential of human CD34+ HSPCs transduced with empty control or BCL2 encoding virus. Colonies were counted 10 days post seeding of 1000 CD34+ cells per plate/sample. "Myeloid colonies" include granulocyte/monocyte, monocyte, granulocyte and GEMM (granulocyte, erythroid, monocyte/macrophage and megakaryocyte) colonies.


Figure 4Representative pictures of E13 . 5 and E18 . 5 embryos of the indicated genotypes . Scale bar E13 . 5 = 1 mm , E18 . 5 = 5 mm . Quantification of fetal liver cell number at E13 . 5 . Bars represent means ± S . E . M . ( N = 4 for Chk1fl / + , N = 3 for Chk1fl / + Vav - Cre and N = 4 for Chk1fl / - Vav - Cre ) . Representative pictures of isolated fetal livers at E13 . 5 . Scale bar = 1 mm . Representative H & E - staining of fetal liver sections from E13 . 5 embryos . Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl / - Vav - Cre fetal livers . Scale bar = 500 µM . Single cell suspensions of fetal livers from Chk1fl / + , Chk1fl / + Vav - Cre and Chk1fl / - Vav - Cre embryos were stained with antibodies recognizing ckit , CD71 , Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry . Large ' EryA ' erythroblasts ( CD71high Ter119high FSChigh ) , smaller , more mature ' EryB ' erythroblasts ( CD71high Ter119high FSClow ) ; most mature erythroblast subset is EryC ( CD71low Ter119high FSClow ) . Bars represent means ± S . E . M . ctrl N = 5 ( pooled N = 4 Chk1fl / + and N = 1 Chk1fl / + Vav - Cre ) , N = 3 for Chk1fl / - Vav - Cre .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4Representative pictures of E13.5 and E18.5 embryos of the indicated genotypes. Scale bar E13.5 = 1 mm, E18.5 = 5 mm. Quantification of fetal liver cell number at E13.5. Bars represent means ± S.E.M. (N=4 for Chk1fl/+, N=3 for Chk1fl/+ Vav-Cre and N=4 for Chk1fl/- Vav-Cre).Representative pictures of isolated fetal livers at E13.5. Scale bar = 1 mm.Representative H&E-staining of fetal liver sections from E13.5 embryos. Black arrows indicate lack of erythroid cells in the blood vessels of Chk1fl/- Vav-Cre fetal livers. Scale bar = 500 µM.Single cell suspensions of fetal livers from Chk1fl/+, Chk1fl/+ Vav-Cre and Chk1fl/- Vav-Cre embryos were stained with antibodies recognizing ckit, CD71, Ter119 or CD45 to assess primitive and definitive erythropoiesis by flow cytometry. Large 'EryA' erythroblasts (CD71high Ter119high FSChigh), smaller, more mature 'EryB' erythroblasts (CD71high Ter119high FSClow); most mature erythroblast subset is EryC (CD71low Ter119high FSClow). Bars represent means ± S.E.M. ctrl N=5 (pooled N=4 Chk1fl/+ and N=1 Chk1fl/+ Vav-Cre), N=3 for Chk1fl/- Vav-Cre.


Figure 5Representative dot - plots of the LSK cell phenotype of Chk1fl / + and Chk1fl / - Vav - Cre embryos observed in E13 . 5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells , as well as LKlow and LSKlow cells , found in the fetal liver on E13 . 5 . Bars represent means ± S . E . M . N = 4 for Chk1fl / + and N = 4 for Chk1fl / - Vav - Cre . Colony formation potential of FACS - sorted LSK cells ( left panel , N = 4 for Chk1fl / + and N = 5 for Chk1fl / - Vav - Cre ) and total fetal liver cells ( right panel , N = 3 for Chk1fl / + and N = 6 for Chk1fl / - Vav - Cre ) in methyl - cellulose assays . Bars represent means ± S . E . M . Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 ( N = 4 for Chk1fl / + and N = 4 for Chk1fl / - Vav - Cre ) , to discriminate LT - and ST - HSC from MPP within the LSK - subset . Bars represent means ± S . E . M .

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

Figure 5Representative dot-plots of the LSK cell phenotype of Chk1fl/+ and Chk1fl/- Vav-Cre embryos observed in E13.5 fetal livers and relative distribution / absolute numbers of LKhi and LSKhi cells, as well as LKlow and LSKlow cells, found in the fetal liver on E13.5. Bars represent means ± S.E.M. N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre.Colony formation potential of FACS-sorted LSK cells (left panel, N=4 for Chk1fl/+ and N=5 for Chk1fl/- Vav-Cre) and total fetal liver cells (right panel, N=3 for Chk1fl/+ and N=6 for Chk1fl/- Vav-Cre) in methyl-cellulose assays. Bars represent means ± S.E.M.Flow cytometric analysis of LSKhi or LSKlow cells stained with antibodies specific for CD34 and Flt3 (N=4 for Chk1fl/+ and N=4 for Chk1fl/- Vav-Cre), to discriminate LT- and ST-HSC from MPP within the LSK-subset. Bars represent means ± S.E.M.