D . Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12 % SDS - PAGE gel . E = LukE , D = LukD , Emut1 = LukEmut1 , Dmut = LukDmut . Bars indicate Mean ± SEM , with n = 4 donors .

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

D. Viability of primary human PMNs following incubation with WT and mutant toxin pairings using the viability dye CellTiter. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie Blue staining on a 12% SDS-PAGE gel. E = LukE, D = LukD, Emut1 = LukEmut1, Dmut = LukDmut. Bars indicate Mean ± SEM, with n = 4 donors.


E . Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED ( 78 . 12 nM per subunit ) and a dose response of WT or dominant - negative ( DN ) toxin subunits using the viability dye LukE . E = LukE , D = LukD , Emut1 = LukEmut1 , Emut2 = LukEmut2 , Emut1Dmut = LukEmut1LukDmut , Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM , with n = 6 donors .

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

E. Viability of primary humanPMNs following simultaneous incubation with a lethal dose of WT LukED (78.12 nM per subunit) and a dose response of WT or dominant-negative (DN) toxin subunits using the viability dye LukE. E = LukE, D = LukD, Emut1 = LukEmut1, Emut2 = LukEmut2, Emut1Dmut = LukEmut1LukDmut, Emut2Dmut = LukEmut2LukDmut . Bars indicate Mean ± SEM, with n = 6 donors.


C . Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 3 donors .

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

C. Viability of primary human PMNs following incubation with mutant toxin pairings using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 3 donors.


D - F . Viability of primary humanPMNs following incubation with WT or mutant toxin pairings . Insert : 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12 % SDS - PAGE gel . Amut1 = HlgAmut1 , Bmut = HlgBmut , Cmut1 = HlgCmut1 , Smut1 = LukS - PVmut1 , Fmut = LukF - PVmut . Bars indicate Mean ± SEM , with n = 4 donors ( D ) , n = 4 donors ( E ) , and n = 5 donors ( F ) .

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

D-F. Viability of primary humanPMNs following incubation with WT or mutant toxin pairings. Insert: 2 μg per lane of purified recombinant toxins visualized by Coomassie blue staining using a 12% SDS-PAGE gel. Amut1 = HlgAmut1, Bmut = HlgBmut, Cmut1 = HlgCmut1, Smut1 = LukS-PVmut1, Fmut = LukF-PVmut. Bars indicate Mean ± SEM, with n = 4 donors (D), n = 4 donors (E), and n = 5 donors (F).


A - D . Viability of primary human PMNs following incubation with a LD90 of LukED ( 78 . 12 nM ) , HlgAB ( 78 . 12 nM ) , HlgCB ( 39 . 1 nM ) , or LukSF - PV ( 19 . 53 nM ) and the indicated dominant - negative toxins using the viability dye CellTiter . Open white symbols indicate no treatment . Filled black symbols indicate treatment with dominant - negative toxins . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 5 donors .

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

A-D. Viability of primary human PMNs following incubation with a LD90 of LukED (78.12 nM), HlgAB (78.12 nM), HlgCB (39.1 nM), or LukSF-PV (19.53 nM) and the indicated dominant-negative toxins using the viability dye CellTiter. Open white symbols indicate no treatment. Filled black symbols indicate treatment with dominant-negative toxins. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 5 donors.


E . Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA - MRSA USA300 strain LAC using the viability dye CellTiter . Bars indicate Mean ± SEM , with n = 6 donors .

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

E. Viability of primary human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA-MRSA USA300 strain LAC using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 6 donors.


F . Viability of primary human PMNs following incubation with 1 . 25 % filtered culture supernatants and a dose response of the indicated dominant - negative ( DN ) toxins using the viability dye CellTiter . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

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

F. Viability of primary human PMNs following incubation with 1.25% filtered culture supernatants and a dose response of the indicated dominant-negative (DN) toxins using the viability dye CellTiter. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


G . Viability of primary human PMNs during a 3 - hour ex vivo infection with the S . aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 donors .

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

G. Viability of primary human PMNs during a 3-hour ex vivo infection with the S. aureus Newman toxinless strain overproducing the indicated toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3 donors.


H . Viability of primary human PMNs during a 4 - hr ex vivo infection with a MOI of 50 of S . aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± SEM , with n = 3 donors .

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

H. Viability of primary human PMNs during a 4-hr ex vivo infection with a MOI of 50 of S. aureus Newman toxinless strain overproducing the indicated toxins in the presence of a dose response of the DN toxins using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. Bars indicate Mean ± SEM, with n = 3 donors.


B . Exoproteome analysis of LukAB mutants produced by S . aureus strain Newman in a toxinless background . Samples were electrophoresed on a 12 % SDS - PAGE gel and visualized by staining with Coomassie blue . A representative of two experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O 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. Exoproteome analysis of LukAB mutants produced by S. aureus strain Newman in a toxinless background. Samples were electrophoresed on a 12% SDS-PAGE gel and visualized by staining with Coomassie blue. A representative of two experiments is shown.


C . Exoproteome and immunoblot analyses of wildtype S . aureus strain Newman transformed with empty plasmid ( p - ) or with plasmids containing the mutated lukAB loci . A representative of two independent experiments is shown .

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

C. Exoproteome and immunoblot analyses of wildtype S. aureus strain Newman transformed with empty plasmid (p-) or with plasmids containing the mutated lukAB loci. A representative of two independent experiments is shown.


D . Viability of primary human PMNs during a 2 - hour ex vivo infection with the indicated S . aureus strains using the CytoTox - One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± SEM , with n = 3 . Statistical significance is indicated as follows : MOI of 100 : * * p = 0 . 0026 for p - vs LukABmut1 and * * * p = 0 . 0003 for p - vs LukABmut2 ; MOI of 50 : * * * * p < 0 . 0001 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 ; MOI of 25 : * * * p = 0 . 0007 for p - vs LukABmut1 and * * * * p < 0 . 0001 for p - vs LukABmut2 using two - way ANOVA with Tukey ' s post hoc test correction for multiple comparisons .

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

D. Viability of primary human PMNs during a 2-hour ex vivo infection with the indicated S. aureus strains using the CytoTox-One Homogenous Membrane Integrity Assay to measure lactate dehydrogenase (LDH) release. Bars indicate Mean ± SEM, with n = 3. Statistical significance is indicated as follows: MOI of 100: **p = 0.0026 for p- vs LukABmut1 and ***p = 0.0003 for p- vs LukABmut2; MOI of 50: ****p <0.0001 for p- vs LukABmut1 and ****p <0.0001 for p- vs LukABmut2; MOI of 25: ***p = 0.0007 for p- vs LukABmut1 and ****p < 0.0001 for p- vs LukABmut2 using two-way ANOVA with Tukey's post hoc test correction for multiple comparisons.


B . Competition assay using GFP - LukE ( 312 . 5 nM ) and a dose response of unlabeled WT or dominant - negative ( DN ) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs . E = LukE , Emut1 = LukEmut1 , A = HlgA , Amut1 = HlgAmut1 , S = LukS - PV , C = HlgC . Data is presented as total fluorescence emitted from PMNs . A representative of three independent experiments each done with hPMNs isolated from different donors is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Competition assay using GFP-LukE (312.5 nM) and a dose response of unlabeled WT or dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. E = LukE, Emut1 = LukEmut1, A = HlgA, Amut1 = HlgAmut1, S = LukS-PV, C = HlgC. Data is presented as total fluorescence emitted from PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.


D . Recruitment assay using GFP - LukD ( 156 . 3 nM ) and a dose response of unlabeled dominant - negative ( DN ) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs . Emut1 = LukEmut1 , Amut1 = HlgAmut1 . Data is presented as total fluorescence emitted from GFP - positive PMNs . A representative of three independent experiments each done with hPMNs isolated from different donors is shown .

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

D. Recruitment assay using GFP-LukD (156.3 nM) and a dose response of unlabeled dominant-negative (DN) toxin subunits evaluated by flow cytometry on the surface of primary human PMNs. Emut1 = LukEmut1, Amut1 = HlgAmut1. Data is presented as total fluorescence emitted from GFP-positive PMNs. A representative of three independent experiments each done with hPMNs isolated from different donors is shown.


E . Immunoblot using anti - His antisera to detect dimer and oligomer formation by purified polyhistidine ( 6xHis ) - tagged WT or dominant - negative LukED toxins in the absence or presence of 2 - methyl - 2 , 4 - pentanediol ( MPD , 40 % v / v ) . Emut1 = LukEmut1 , Dmut = LukDmut . A representative of two independent experiments is shown .

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

E. Immunoblot using anti-His antisera to detect dimer and oligomer formation by purified polyhistidine (6xHis)-tagged WT or dominant-negative LukED toxins in the absence or presence of 2-methyl-2,4-pentanediol (MPD, 40% v/v). Emut1 = LukEmut1, Dmut = LukDmut. A representative of two independent experiments is shown.


A . Survival of Swiss - Webster mice following intravenous administration of purified recombinant toxins : LukED ( 10 μg per subunit ) , HlgAB ( 5 μg per subunit ) , HlgCB ( 35 μg per subunit ) and LukSF - PV / PVL ( 35 μg per subunit ) . Buffer : n = 2 mice ; LukED : n = 3 mice ; HlgAB : n = 3 mice ; HlgCB : n = 2 mice ; PVL : n = 3 . A representative of two independent experiments is shown .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

A. Survival of Swiss-Webster mice following intravenous administration of purified recombinant toxins: LukED (10 μg per subunit), HlgAB (5 μg per subunit), HlgCB (35 μg per subunit) and LukSF-PV/PVL (35 μg per subunit). Buffer: n = 2 mice; LukED: n = 3 mice; HlgAB: n = 3 mice; HlgCB: n = 2 mice; PVL: n = 3. A representative of two independent experiments is shown.


B . Survival of Swiss - Webster mice following prophylactic intravenous administration of dominant - negative mutants , followed by intravenous challenge with a lethal dose of WT LukED . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . n = 6 mice per treatment . * * * p = 0 . 0005 for Buffer vs Emut1Dmut , Buffer vs Amut1Bmut , and Buffer vs Cmut1Bmut . p = 0 . 1050 ( not significant , ns ) for Buffer vs Smut1Fmut . Statistics performed using Log - rank ( Mantel - Cox ) test . A representative of two independent experiments is shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT LukED. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0005 for Buffer vs Emut1Dmut, Buffer vs Amut1Bmut, and Buffer vs Cmut1Bmut. p = 0.1050 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.


C . Survival of Swiss - Webster mice following prophylactic intravenous administration of dominant - negative mutants , followed by intravenous challenge with a lethal dose of WT HlgAB . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . n = 6 mice per treatment . * * * p = 0 . 0004 for Buffer vs Emut1Dmut and Buffer vs Amut1Bmut . * * p = 0 . 0015 for Buffer vs Cmut1Bmut . p = 0 . 7955 ( not significant , ns ) for Buffer vs Smut1Fmut . Statistics performed using Log - rank ( Mantel - Cox ) test . A representative of two independent experiments is shown .

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

C. Survival of Swiss-Webster mice following prophylactic intravenous administration of dominant-negative mutants, followed by intravenous challenge with a lethal dose of WT HlgAB. Emut1Dmut = LukEmut1LukDmut, Amut1Bmut = HlgAmut1HlgBmut, Cmut1Bmut = HlgCmut1HlgBmut, Smut1Fmut = LukS-PVmut1LukF-PVmut. n = 6 mice per treatment. ***p = 0.0004 for Buffer vs Emut1Dmut and Buffer vs Amut1Bmut. **p = 0.0015 for Buffer vs Cmut1Bmut. p = 0.7955 (not significant, ns) for Buffer vs Smut1Fmut. Statistics performed using Log-rank (Mantel-Cox) test. A representative of two independent experiments is shown.


D . Bacterial burden 96 hours post infection in the liver of mice infected systemically with ~ 1 . 5 x 107 CFU of S . aureus strain Newman and intravenously treated with buffer or LukEmut1LukDmut ( Emut1Dmut , 10 μg per subunit ) at t = 0 , 24 , and 48 h . p . i . Bars indicate Mean ± SEM , with n = 20 mice ( Buffer ) and n = 19 mice ( Emut1Dmut2 ) combined from two independent experiments . * p = 0 . 0303 by unpaired , parametric , two - tailed Student ' s t - test .

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

D. Bacterial burden 96 hours post infection in the liver of mice infected systemically with ~1.5 x 107 CFU of S. aureus strain Newman and intravenously treated with buffer or LukEmut1LukDmut (Emut1Dmut, 10 μg per subunit) at t = 0, 24, and 48 h.p.i. Bars indicate Mean ± SEM, with n = 20 mice (Buffer) and n = 19 mice (Emut1Dmut2) combined from two independent experiments. *p = 0.0303 by unpaired, parametric, two-tailed Student's t-test.


a , MEFs of RagA + / + , RagAGTP / + and RagAGTP / GTP genotypes were deprived of amino acids for 1 h and re - stimulated for 10 min . Whole - cell protein lysates were immunoblotted for indicated proteins .

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

a, MEFs of RagA+/+, RagAGTP/+ and RagAGTP/GTP genotypes were deprived of amino acids for 1 h and re-stimulated for 10 min. Whole-cell protein lysates were immunoblotted for indicated proteins.


b , Total RNA was extracted from RagA + / + ( n = 3 ) , RagAGTP / + ( n = 3 ) and RagAGTP / GTP ( n = 2 ) MEFs and RagA mRNA expression determined by quantitative PCR ( mean ± s . e . m . ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Total RNA was extracted from RagA+/+ (n = 3), RagAGTP/+ (n = 3) and RagAGTP/GTP (n = 2) MEFs and RagA mRNA expression determined by quantitative PCR (mean ± s.e.m.).


c , Weights of RagA + / + ( n = 24 ) , RagAGTP / + ( n = 52 ) and RagAGTP / GTP ( n = 22 ) mice at birth ( data are scatter dots , mean ) .

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

c, Weights of RagA+/+ (n = 24), RagAGTP/+ (n = 52) and RagAGTP/GTP (n = 22) mice at birth (data are scatter dots, mean).


d , Representative photographs of RagA + / + , RagAGTP / + and RagAGTP / GTP neonates . Scale bar , 1 cm .

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

d, Representative photographs of RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates. Scale bar, 1 cm.


e , Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA + / + ( + / + ) , RagAGTP / + ( G / + ) and RagAGTP / GTP ( G / G ) neonates immediately after Caesarean section ( 0 h ) or after 1 h of fasting ( 1 h fast ) .

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

e, Early suppression of mTORC1 signalling after birth was determined by immunoblotting of protein extracts from liver and heart of RagA+/+ (+/+), RagAGTP/+ (G/+) and RagAGTP/GTP (G/G) neonates immediately after Caesarean section (0 h) or after 1 h of fasting (1 h fast).


f , Liver and heart extracts from RagA + / + , RagAGTP / + and RagAGTP / GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins .

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

f, Liver and heart extracts from RagA+/+, RagAGTP/+ and RagAGTP/GTP neonates fasted for 10 h were analysed by immunoblotting for the indicated proteins.


g , Survival curve of fasted neonates . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 26 ; G / G : n = 10 ) . h , Survival curve of fasted neonates treated with rapamycin . Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored ( + / + : n = 13 ; G / + : n = 13 ; G / G : n = 6 ) . Numbers indicate the median survival for each curve . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 005 .

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

g, Survival curve of fasted neonates. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 26; G/G: n = 10). h, Survival curve of fasted neonates treated with rapamycin. Neonates obtained by Caesarean section and resuscitated were fasted and their survival monitored (+/+: n = 13; G/+: n = 13; G/G: n = 6). Numbers indicate the median survival for each curve. *P<0.05; **P<0.01; ***P<0.005.


a , Glycaemia drop in RagA + / + , RagAGTP / + and RagAGTP / GTP and recovery in fasted RagA + / + and RagAGTP / + but not in RagAGTP / GTP neonates ( + / + : n = 5 , 18 , 4 , 5 , 9 , 8 ; G / + : n = 10 , 26 , 10 , 13 , 26 , 21 ; G / G : n = 7 , 20 , 9 , 10 , 16 , 11 , for 0 , 1 , 2 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ) .

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

a, Glycaemia drop in RagA+/+, RagAGTP/+ and RagAGTP/GTP and recovery in fasted RagA+/+ and RagAGTP/+ but not in RagAGTP/GTP neonates (+/+: n = 5, 18, 4, 5, 9, 8; G/+: n = 10, 26, 10, 13, 26, 21; G/G: n = 7, 20, 9, 10, 16, 11, for 0, 1, 2, 3, 6 and 10 h, respectively; mean ± s.e.m.).


b , Rapamycin significantly increases glycaemia in RagAGTP / GTP fasted for 6 and 10 h ( mean ± s . e . m . ) . NS , not significant .

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

b, Rapamycin significantly increases glycaemia in RagAGTP/GTP fasted for 6 and 10 h (mean ± s.e.m.). NS, not significant.


c , Extension of survival by glucose injections in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; glucose : n = 5 ) .

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

c, Extension of survival by glucose injections in fasted RagAGTP/GTP neonates (untreated: n = 10; glucose: n = 5).


d , Normal expression of genes involved in glucose metabolism in neonatal liver ( + / + : n = 2 , G / + : n = 5 ; G / G : n = 4 ; mean ± s . e . m . ) .

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d, Normal expression of genes involved in glucose metabolism in neonatal liver (+/+: n = 2, G/+: n = 5; G/G: n = 4; mean ± s.e.m.).


e , Left : representative electron microscopy images showing abundant glycogen content in RagA + / + and RagAGTP / GTP hepatocytes before fasting ( 0 h , upper panels ) and more pronounced glycogen depletion after 10 h of fasting ( lower panels ) in RagAGTP / GTP neonates . Right : quantification of hepatic glycogen content ( + / + : n = 5 , 3 , 4 , 4 ; G / + : n = 11 , 7 , 14 , 15 ; G / G : n = 6 , 4 , 4 , 6 ; for 0 , 3 , 6 and 10 h , respectively ; mean ± s . e . m . ; AU , arbitrary units ) .

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e, Left: representative electron microscopy images showing abundant glycogen content in RagA+/+ and RagAGTP/GTP hepatocytes before fasting (0 h, upper panels) and more pronounced glycogen depletion after 10 h of fasting (lower panels) in RagAGTP/GTP neonates. Right: quantification of hepatic glycogen content (+/+: n = 5, 3, 4, 4; G/+: n = 11, 7, 14, 15; G/G: n = 6, 4, 4, 6; for 0, 3, 6 and 10 h, respectively; mean ± s.e.m.; AU, arbitrary units).


. f , Partial rescue of hepatic glycogen content in RagAGTP / GTP neonates fasted for 10 h and treated with rapamycin ( rapa ) ( mean ± s . e . m . ) .

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. f, Partial rescue of hepatic glycogen content in RagAGTP/GTP neonates fasted for 10 h and treated with rapamycin (rapa) (mean ± s.e.m.).


g , Quantification of neonatal plasma amounts of branched - chain ( BCAA ) and essential amino acids at birth ( left ) , after 10 h fasting ( middle ) and after 10 h fasting with rapamycin treatment ( right ) ( n for + / + , G / + and G / G , respectively : n = 4 , 5 and 4 for 0 h ; n = 4 , 4 and 3 for 10 h ; n = 2 , 5 and 3 for rapamycin ; mean ± s . d . ) . Values are expressed relative to RagA + / + amounts at each time point .

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g, Quantification of neonatal plasma amounts of branched-chain (BCAA) and essential amino acids at birth (left), after 10 h fasting (middle) and after 10 h fasting with rapamycin treatment (right) (n for +/+, G/+ and G/G, respectively: n = 4, 5 and 4 for 0 h; n = 4, 4 and 3 for 10 h; n = 2, 5 and 3 for rapamycin; mean ± s.d.). Values are expressed relative to RagA+/+ amounts at each time point.


h , Extension of survival by injection of a combination of gluconeogenic amino acids ( a . a . ) in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; amino acids : n = 8 ) . * P 0 . 05 ; * * P 0 . 01 ; ns , P > 0 .

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h, Extension of survival by injection of a combination of gluconeogenic amino acids (a.a.) in fasted RagAGTP/GTP neonates (untreated: n = 10; amino acids: n = 8). *P  0.05; **P  0.01; ns, P > 0.0


a , Top : representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA + / + neonates fasted for 1 h . Typical autophagosome with a double limiting membrane ( arrows ) ; autophagosome and several autolysosomes ( arrowheads ) . Scale bars , 5 µm . Bottom : frequency of the two types of organelle ( early : autophagosomes ; late : autophagolysosomes ) detected in cell profiles of hepatocytes and skeletal myocytes from RagA + / + and RagAGTP / GTPneonates .

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a, Top: representative micrographs of autophagosomes and autophagolysosomes in hepatocytes from RagA+/+ neonates fasted for 1 h. Typical autophagosome with a double limiting membrane (arrows); autophagosome and several autolysosomes (arrowheads). Scale bars, 5 µm. Bottom: frequency of the two types of organelle (early: autophagosomes; late: autophagolysosomes) detected in cell profiles of hepatocytes and skeletal myocytes from RagA+/+ and RagAGTP/GTPneonates.


b , Protein extracts from livers of neonates at Caesarean section ( 0 h ) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62 .

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b, Protein extracts from livers of neonates at Caesarean section (0 h) and fasted for 10 h were immunoblotted for autophagy markers LC3B and p62.


c , Protein extracts from skeletal muscle and heart from neonates at Caesarean section ( 0 h ) , fasted for 1 and 2 h , were immunoblotted for indicated proteins .

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c, Protein extracts from skeletal muscle and heart from neonates at Caesarean section (0 h), fasted for 1 and 2 h, were immunoblotted for indicated proteins.


d , Triggering of autophagy by amino - acid withdrawal in MEFs . MEFs were deprived of amino acids for the indicated time points , whole - cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting .

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d, Triggering of autophagy by amino-acid withdrawal in MEFs. MEFs were deprived of amino acids for the indicated time points, whole-cell protein extracts were obtained and mTORC1 activity and autophagic activity determined by immunoblotting.


e , Recombinant LC3B - GFP was expressed in RagA + / + and RagAGTP / GTPMEFs and LC3B localization , in the presence and absence of amino acids , monitored by fluorescence microscopy . LC3B - GFP ( green fluorescent protein ) clustering , indicative of autophagy , was observed in amino acid - starved RagA + / + but not RagAGTP / GTPMEFs . Scale bar , 10 µm .

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e, Recombinant LC3B-GFP was expressed in RagA+/+ and RagAGTP/GTPMEFs and LC3B localization, in the presence and absence of amino acids, monitored by fluorescence microscopy. LC3B-GFP (green fluorescent protein) clustering, indicative of autophagy, was observed in amino acid-starved RagA+/+ but not RagAGTP/GTPMEFs. Scale bar, 10 µm.


f , Localization of recombinant TFEB - GFP was determined in MEFs as in e . Nuclear ( active ) TFEB was observed in RagA + / + MEFs upon amino - acid withdrawal .

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f, Localization of recombinant TFEB-GFP was determined in MEFs as in e. Nuclear (active) TFEB was observed in RagA+/+ MEFs upon amino-acid withdrawal.


a ) AMPKa1 / a2 double knockout ( DKO ) and wild - type ( WT ) MEFs were deprived of glucose for 1 h and re - stimulated for 10 min . Wholecell extracts were immunoblotted for the indicated proteins .

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a) AMPKa1/a2 double knockout (DKO) and wild-type (WT) MEFs were deprived of glucose for 1 h and re-stimulated for 10 min. Wholecell extracts were immunoblotted for the indicated proteins.


b , Immortalized MEFs of the indicated genotypes were deprived of growth factors , glucose , amino acids or glucose and amino acids for 1 h and re - stimulated with glucose and / or amino acids for 10 min . Whole - cell lysates were immunoblotted for the indicated proteins .

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b, Immortalized MEFs of the indicated genotypes were deprived of growth factors, glucose, amino acids or glucose and amino acids for 1 h and re-stimulated with glucose and/or amino acids for 10 min. Whole-cell lysates were immunoblotted for the indicated proteins.


c , RagA + / + and RagAGTP / GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h . Cell number is indicated relative to cell number at the start of the treatment ; mean ± s . d . ; * * * P 0 .

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c, RagA+/+ and RagAGTP/GTP immortalized MEFs were deprived of glucose or amino acids and surviving cells quantified in triplicate after 48 h. Cell number is indicated relative to cell number at the start of the treatment; mean ± s.d.; ***P  0.005


d , mTOR localization as detected by immunofluorescence . In HEK - 293T cells , glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm . Re - addition of glucose leads to mTOR shuttling to the lysosomal surface , co - localizing with the lysosomal protein Lamp2 . HEK - 293T - RagBGTP cells show mTOR localized at the lysosomal surface , regardless of glucose concentrations . Scale bars , 10 μm .

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d, mTOR localization as detected by immunofluorescence. In HEK-293T cells, glucose deprivation causes mTOR to localize to diffuse small puncta throughout the cytoplasm. Re-addition of glucose leads to mTOR shuttling to the lysosomal surface, co-localizing with the lysosomal protein Lamp2. HEK-293T-RagBGTP cells show mTOR localized at the lysosomal surface, regardless of glucose concentrations. Scale bars, 10 μm.


e , Glucose and amino acids affect the binding of the v - ATPase to the Ragulator complex . HEK - 293T expressing Flag - p14 was deprived of glucose or amino acids for 90 min and re - stimulated for 20 min . Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins .

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e, Glucose and amino acids affect the binding of the v-ATPase to the Ragulator complex. HEK-293T expressing Flag-p14 was deprived of glucose or amino acids for 90 min and re-stimulated for 20 min. Protein extracts and immunoprecipitates were immunoblotted for the indicated proteins.


f , RagA + / + and RagAGTP / GTP primary MEFs were cultured for 1 h in media with the glucose and amino - acid concentrations measured in neonates at birth ( 0 h ) or after fasting for 1 h ( 1 h ) and whole - cell protein extracts were analysed by immunoblotting .

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f, RagA+/+ and RagAGTP/GTP primary MEFs were cultured for 1 h in media with the glucose and amino-acid concentrations measured in neonates at birth (0 h) or after fasting for 1 h (1 h) and whole-cell protein extracts were analysed by immunoblotting.


( B ) Pol II ChIP - seq was performed in six different tissues shown in the left panel ( scale bar - 100 µm ) . The middle and the right panels show the read count normalized Pol II ChIP - seq tracks ( RPM ) from the six tissues at individual genes . For each gene , grey and red tracks indicate non - expressing tissues and expressing tissue , respectively . The middle panel shows the Pol II profile at two non - paused genes , which have Pol II only in the expressing tissues . The expression is limited to specific tissues as shown in the in situ images from BDGP . The right panel shows the Pol II profile at a paused gene , which has Pol II in all observed tissues , although the expression is limited to specific tissues as shown in the in situ images from BDGP . Paused Pol II is generally highest in the tissue with the highest expression . We also found systematic differences between samples , thus some tissues have generally higher enrichments than others , presumably because they are easier to crosslink .

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(B) Pol II ChIP-seq was performed in six different tissues shown in the left panel (scale bar - 100 µm). The middle and the right panels show the read count normalized Pol II ChIP-seq tracks (RPM) from the six tissues at individual genes. For each gene, grey and red tracks indicate non-expressing tissues and expressing tissue, respectively. The middle panel shows the Pol II profile at two non-paused genes, which have Pol II only in the expressing tissues. The expression is limited to specific tissues as shown in the in situ images from BDGP. The right panel shows the Pol II profile at a paused gene, which has Pol II in all observed tissues, although the expression is limited to specific tissues as shown in the in situ images from BDGP. Paused Pol II is generally highest in the tissue with the highest expression. We also found systematic differences between samples, thus some tissues have generally higher enrichments than others, presumably because they are easier to crosslink.


( C , D ) Normalized gene expression levels ( read count normalized for each cell , log2 ) in different tissues , from the scRNA - seq experiment , for a TATA group gene , and a highly paused group gene , are shown . ( C ) The TATA gene , Ccp84Aa , shows noisy expression in the epidermis , without detectable background expression in non - expressing tissues . ( D ) The highly paused gene , Gip , shows very robust expression in crystal cells , but has high background expression in the non - expressing tissues .

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(C,D) Normalized gene expression levels (read count normalized for each cell, log2) in different tissues, from the scRNA-seq experiment, for a TATA group gene, and a highly paused group gene, are shown. (C) The TATA gene, Ccp84Aa, shows noisy expression in the epidermis, without detectable background expression in non-expressing tissues. (D) The highly paused gene, Gip, shows very robust expression in crystal cells, but has high background expression in the non-expressing tissues.


C , D ) ATAC - seq chromatin accessibility was measured in different tissues isolated from 14 - 17 h embryos using INTACT . ( C ) Read - count normalized ATAC ( RPM ) signal at individual genes from the TATA group ( Ccp84Aa ) and highly paused gene group ( ect ) are shown . ( D ) Read - count normalized ATAC signals ( RPM ) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS . Paused genes show higher average accessibility across all tissues compared to the TATA genes ( Wilcoxon two - sided test , * P < 2 . 2 * 10 - 16 ) .

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C, D) ATAC-seq chromatin accessibility was measured in different tissues isolated from 14-17 h embryos using INTACT. (C) Read-count normalized ATAC (RPM) signal at individual genes from the TATA group (Ccp84Aa) and highly paused gene group (ect) are shown. (D) Read-count normalized ATAC signals (RPM) from different tissues were calculated for each gene from 150 bp upstream of the TSS to the TSS. Paused genes show higher average accessibility across all tissues compared to the TATA genes (Wilcoxon two-sided test, *P <2.2*10-16).


B . MASTL was ablated in MDA - MB - 231 cells using the isgMASTL system . 72 h after Dox , cells were starved of glucose in the media in presence of 10 % dFBS for 1 h ( - ) and then re - stimulated with 25 mM of glucose for 10 min before recovery ( + ) . Whole - cell lysates were blotted with the indicated antibodies . * in indicates unspecific band . β - Actin was used as a loading control .

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B. MASTL was ablated in MDA-MB-231 cells using the isgMASTL system. 72 h after Dox, cells were starved of glucose in the media in presence of 10% dFBS for 1 h (-) and then re-stimulated with 25 mM of glucose for 10 min before recovery (+). Whole-cell lysates were blotted with the indicated antibodies. * in indicates unspecific band. β-Actin was used as a loading control.


C . MASTL was depleted in MDA - MB - 231 cells using shRNAs against MASTL ( + ) or scrambled shRNAs ( − ) as control . Cells were starved from glucose for 2 h and re - stimulated with 5 mM glucose for 15 min and 1 h . Total extracts were recovered and tested for the indicated antibodies . β - Actin was used as a loading control . D . Quantification of phospho - AKT T308 levels in glucose deprivation or glucose stimulation ( 15 min ) conditions in MDA - MB - 231 control cells and MASTL - depleted cells either using shRNA or the inducible sgRNA . Bars displays mean data + SEM from three independent experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ; * P < 0 . 05 ) .

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C. MASTL was depleted in MDA-MB-231 cells using shRNAs against MASTL (+) or scrambled shRNAs (−) as control. Cells were starved from glucose for 2 h and re-stimulated with 5 mM glucose for 15 min and 1 h. Total extracts were recovered and tested for the indicated antibodies. β-Actin was used as a loading control. D. Quantification of phospho-AKT T308 levels in glucose deprivation or glucose stimulation (15 min) conditions in MDA-MB-231 control cells and MASTL-depleted cells either using shRNA or the inducible sgRNA. Bars displays mean data + SEM from three independent experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).


E . Immunoblot analysis with the indicated antibodies in BT - 549 cells upon glucose deprivation and 15 min glucose stimulation . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . β - Actin was used as a loading control . Bar chart displays mean data + SEM from 3 experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ) . F . Immunoblot analysis with the indicated antibodies in MCF - 7 cells upon glucose deprivation and glucose stimulation for 15 min . MASTL knockdown was performed by infection with a shRNA for MASTL ( + ) or a Scramble shRNA ( − ) as a control . Bar chart displays mean data + SEM from 3 experiments . Significance determined by Student ' s t - test comparing feedback activation in MASTL - depleted cells versus control cells ( ns , not significant ; * P < 0 . 05 ) .

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E. Immunoblot analysis with the indicated antibodies in BT-549 cells upon glucose deprivation and 15 min glucose stimulation. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. β-Actin was used as a loading control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant). F. Immunoblot analysis with the indicated antibodies in MCF-7 cells upon glucose deprivation and glucose stimulation for 15 min. MASTL knockdown was performed by infection with a shRNA for MASTL (+) or a Scramble shRNA (−) as a control. Bar chart displays mean data + SEM from 3 experiments. Significance determined by Student's t-test comparing feedback activation in MASTL-depleted cells versus control cells (ns, not significant; *P<0.05).


A . Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum - starved control and MASTL knockout MDA - MB - 231 cells using the isgMASTL system . The plot shows the quantification of phospho - AKT T308 after 30 min of insulin stimulation . Plots displays mean data + SEM from 5 independent experiments . * P < 0 . 05 , Student ' s t - test .

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A. Time course experiment at the indicated time points after 100 nM insulin stimulation in 6 h serum-starved control and MASTL knockout MDA-MB-231 cells using the isgMASTL system. The plot shows the quantification of phospho-AKT T308 after 30 min of insulin stimulation. Plots displays mean data + SEM from 5 independent experiments. *P<0.05, Student's t-test.


B . TSC2 was knocked down in control or MASTL - null MDA - MB - 231 cells using specific ( + ) or Scrambled ( − ) shRNAs . Cells were starved for glucose for 1 h . The plot shows the quantification of phospho - AKT T308 . Data are mean + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

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B. TSC2 was knocked down in control or MASTL-null MDA-MB-231 cells using specific (+) or Scrambled (−) shRNAs. Cells were starved for glucose for 1 h. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.


C . MDA - MB - 231 control ( − ) or MASTL null ( + ) cells were treated with 100 nM rapamycin or vehicle as control . Cells were starved for glucose for 1 h and re - stimulated with glucose for 15 min ; rapamycin was added 15 min before glucose stimulation . The plot shows the quantification of phospho - AKT T308 . Data are mean + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

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C. MDA-MB-231 control (−) or MASTL null (+) cells were treated with 100 nM rapamycin or vehicle as control. Cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min; rapamycin was added 15 min before glucose stimulation. The plot shows the quantification of phospho-AKT T308. Data are mean + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA.


A . Immunofluorescence for GLUT4 ( magenta ) in MDA - MB - 231 cells in conditions of serum starvation and insulin stimulation . DAPI ( DNA ) is in cyan and a cell mask is in yellow . Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane ( PM ) . Representative magnifications are also shown . Scale bars , 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM . Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA . Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies .

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A. Immunofluorescence for GLUT4 (magenta) in MDA-MB-231 cells in conditions of serum starvation and insulin stimulation. DAPI (DNA) is in cyan and a cell mask is in yellow. Arrowheads indicate positive cells for GLUT4 enrichment at the plasma membrane (PM). Representative magnifications are also shown. Scale bars, 10 μm The plot shows the quantification of the percentage of cells positive for GLUT4 translocation at the PM. Data are mean fold increase vs serum deprivation in scramble condition + SEM from 3 independent experiments. ns, not significant; *P<0.05, 1-way ANOVA. Control inmunoblotting in conditions of insulin stimulation is also shown for the indicated antibodies.


E . GLUT4 staining ( magenta ) in skeletal muscle sections . Vinculin ( membrane ) and DAPI ( DNA ) staining is shown in yellow and cyan , respectively . Scale bars , 50 µm . Mice were fasted overnight for 16 h , and re - fed for 2 h before sample collection . Quantification of the mean intensity [ relative units x103 ; n = 3 Mastl ( + / + ) and 3 Mastl ( ∆ / ∆ ) ] is shown . Data points reflect the mean + SEM ; * P < 0 . 05 ; * * * P < 0 . 001 ; unpaired Student ' s t - test .

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E. GLUT4 staining (magenta) in skeletal muscle sections. Vinculin (membrane) and DAPI (DNA) staining is shown in yellow and cyan, respectively. Scale bars, 50 µm. Mice were fasted overnight for 16 h, and re-fed for 2 h before sample collection. Quantification of the mean intensity [relative units x103; n=3 Mastl(+/+) and 3 Mastl(∆/∆)] is shown. Data points reflect the mean + SEM; *P<0.05; ***P <0.001; unpaired Student's t-test.


G . Immunoblot with the indicated antibodies in liver tissues from Mastl ( + / + ) ( n = 6 ) and Mastl ( Δ / Δ ) ( n = 7 ) mice . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg body ) , and sacrificed 30 min later for sample collection . Quantification of the relative fold change signal of phospho - AKT T308 ( normalized to total AKT level ) . n = 6 Mastl ( + / + ) and 7 Mastl ( Δ / Δ ) mice . Charts reflect the mean + SEM ; * P < 0 . 05 ; unpaired Student ' s t - test .

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G. Immunoblot with the indicated antibodies in liver tissues from Mastl(+/+) (n=6) and Mastl(Δ/Δ) (n=7) mice. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of phospho-AKT T308 (normalized to total AKT level). n=6 Mastl(+/+) and 7 Mastl(Δ/Δ) mice. Charts reflect the mean + SEM; *P<0.05; unpaired Student's t-test.


A . Co - depletion of ENSA and ARPP19 proteins in the MDA - MB - 231 cell line using specific siRNAs or control siRNAs against luciferase ( Luc ) . 72 h after transfection cells were starved for glucose for 1 h and re - stimulated with glucose for 15 min . Total cell extracts were collected and analyzed for the indicated antibodies .

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

A. Co-depletion of ENSA and ARPP19 proteins in the MDA-MB-231 cell line using specific siRNAs or control siRNAs against luciferase (Luc). 72 h after transfection cells were starved for glucose for 1 h and re-stimulated with glucose for 15 min. Total cell extracts were collected and analyzed for the indicated antibodies.


B . isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG - and HA - tagged versions of ENSA and ARPP19 phospho - mimetic mutants ( S67D and S62D ) or GFP as a control . Cells were treated with Dox to induce MASTL CRISPR / Cas9 - dependent knock - out , and 72 h later cells were treated as specified in A . The plot displays mean + SEM from 3 independent experiments . Significance determined by Student ' s t - test comparing ENSA / ARPP19 overexpression versus control cells ( ns , not significant ; * P < 0 . 05 ) .

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B. isgMASTL cells were previously infected with lentiviral supernatants expressing FLAG- and HA-tagged versions of ENSA and ARPP19 phospho-mimetic mutants (S67D and S62D) or GFP as a control. Cells were treated with Dox to induce MASTL CRISPR/Cas9-dependent knock-out, and 72 h later cells were treated as specified in A. The plot displays mean + SEM from 3 independent experiments. Significance determined by Student's t-test comparing ENSA/ARPP19 overexpression versus control cells (ns, not significant; *P<0.05).


C . Control ( Dox − ) and MASTL knockout ( Dox + ) cells were treated with 5 µM fostriecin , 50 nM okadaic acid ( OA ) or vehicle ( Veh ) 10 min before insulin stimulation for 30 min . Total lysates were analyzed for the indicated antibodies and P - T308 - AKT signal was quantified . β - Actin was used as a loading control . The plot represents mean + SEM from 3 independent experiments , except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown . * P < 0 . 05 ; Student ' s t - test .

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

C. Control (Dox −) and MASTL knockout (Dox +) cells were treated with 5 µM fostriecin, 50 nM okadaic acid (OA) or vehicle (Veh) 10 min before insulin stimulation for 30 min. Total lysates were analyzed for the indicated antibodies and P-T308-AKT signal was quantified. β-Actin was used as a loading control. The plot represents mean + SEM from 3 independent experiments, except for fostriecin treatment in which only data from two experiments were obtained and no error bars are shown. *P<0.05; Student's t-test.


A . MDA - MB - 231 cells were co - transduced with specific siRNAs against the PPP2R2A ( B55α ) and PPP2R2D ( B55δ ) transcripts ( B55αδ ) or against luciferase ( Luc ) as a control . Asynchronous cells were collected and blotted for the indicated antibodies . The histograms represent the quantifications of the specified phospho - residues . Plots show mean + SEM from 3 independent experiments . * P < 0 . 05 , Student ' s t - test .

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A. MDA-MB-231 cells were co-transduced with specific siRNAs against the PPP2R2A (B55α) and PPP2R2D (B55δ) transcripts (B55αδ) or against luciferase (Luc) as a control. Asynchronous cells were collected and blotted for the indicated antibodies. The histograms represent the quantifications of the specified phospho-residues. Plots show mean + SEM from 3 independent experiments. *P<0.05, Student's t-test.


B . Immunodetection of the indicated antigens in control ( − ) and MASTL knockout ( + ) cells starved from glucose and re - stimulated with glucose for 15 min .

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B. Immunodetection of the indicated antigens in control (−) and MASTL knockout (+) cells starved from glucose and re-stimulated with glucose for 15 min.


C . siRNA - mediated genetic depletion of B55alpha and delta subunits ( B55αδ ) in control ( Dox − ) and MASTL - null ( Dox + ) cells . The phosphorylation status of the different proteins was scored in conditions of glucose re - addition . n = 3 independent experiments . The plot shows mean + SEM from 3 independent experiments . * P < 0 . 05 , 1 - way ANOVA .

C. siRNA-mediated genetic depletion of B55alpha and delta subunits (B55αδ) in control (Dox −) and MASTL-null (Dox +) cells. The phosphorylation status of the different proteins was scored in conditions of glucose re-addition. n=3 independent experiments. The plot shows mean + SEM from 3 independent experiments. *P<0.05, 1-way ANOVA.


D . Immunoblot with the indicated antibodies in skeletal muscle ( gastrocnemius ) extracts . Mice were fasted overnight for 16 h , injected intraperitoneally with glucose ( 2 g / kg body ) , and sacrificed 30 min later for sample collection . Quantification of the relative fold change signal of total IRS1 , and phospho - S6K1 T389 ( normalized to total S6K1 level ; n = 6 Mastl ( + / + ) and 7 Mastl ( ∆ / ∆ ) ) . vinculin were used as a loading control . Data are mean + SEM ; * P < 0 . 05 , unpaired Student ' s t - test .

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

D. Immunoblot with the indicated antibodies in skeletal muscle (gastrocnemius) extracts. Mice were fasted overnight for 16 h, injected intraperitoneally with glucose (2 g/kg body), and sacrificed 30 min later for sample collection. Quantification of the relative fold change signal of total IRS1, and phospho-S6K1 T389 (normalized to total S6K1 level; n=6 Mastl(+/+) and 7 Mastl(∆/∆)). vinculin were used as a loading control. Data are mean + SEM; *P<0.05, unpaired Student's t-test.


A . MDA - MB - 231 cells were starved for 1 h and re - stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody . The plot shows the quantification of phospho - MASTL T194 ( n = 2 independent experiments ) . Data are mean + SEM from 3 independent experiments . ns , not significant ; Student ' s t - test .

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

A. MDA-MB-231 cells were starved for 1 h and re-stimulated with glucose for 15 min and T194 phosphorylation was tested with a specific antibody. The plot shows the quantification of phospho-MASTL T194 (n=2 independent experiments). Data are mean + SEM from 3 independent experiments. ns, not significant; Student's t-test.


B . Cas9 - expressing control ( − ) cells or cells with CRIPSR / Cas9 - mediated TSC2 knockout ( sgTSC2 ) were treated with rapamycin ( Rap ) , Torin1 ( Tor ) , or vehicle as control ( V ) . MASTL - knockdown cells ( shMASTL ) were used as an additional control of the specificity of the antibody . Inhibitors were added 15 min before glucose re - stimulation for additional 15 min and total cell lysates were harvested for immunoblot . The plot shows the quantification of phospho - MASTL T194 as mean from 2 independent experiments .

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

B. Cas9-expressing control (−) cells or cells with CRIPSR/Cas9-mediated TSC2 knockout (sgTSC2) were treated with rapamycin (Rap), Torin1 (Tor), or vehicle as control (V). MASTL-knockdown cells (shMASTL) were used as an additional control of the specificity of the antibody. Inhibitors were added 15 min before glucose re-stimulation for additional 15 min and total cell lysates were harvested for immunoblot. The plot shows the quantification of phospho-MASTL T194 as mean from 2 independent experiments.


C . In vitro kinase assay using purified GST - human MASTL as a substrate , and recombinant mTORC1 and S6K1 kinases . Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1 . Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions , independently of the presence of MASTL , which correspond to autophosphorylation of these kinases . Imperial staining shows the amount of GST - MASTL protein used as a substrate in each reaction .

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C. In vitro kinase assay using purified GST-human MASTL as a substrate, and recombinant mTORC1 and S6K1 kinases. Red arrowheads show the phosphorylation of MASTL in the presence of mTORC1 or S6K1. Note the appearance of other phosporylation signals in mTORC1 and S6K1 reactions, independently of the presence of MASTL, which correspond to autophosphorylation of these kinases. Imperial staining shows the amount of GST-MASTL protein used as a substrate in each reaction.


F . In vitro kinase activity of Mastl mutant isoforms . HEK - 293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants . Anti - GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 ( white circles ) or ENSA ( filled circles ) as a substrate . The graph shows the relative catalytic activity of the indicated mutants . Kinase activity of GFP - mMastl wild - type was set as 1 ( n = 3 independent experiments ) . Data are mean + SEM from 3 independent experiments . * * * P < 0 . 001 ; 1 - way ANOVA .

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F. In vitro kinase activity of Mastl mutant isoforms. HEK-293T cells were transiently transfected with the indicated GFP fusions of mouse Mastl mutants. Anti-GFP immunocomplexes were subjected to a kinase reaction using recombinant ARPP19 (white circles) or ENSA (filled circles) as a substrate. The graph shows the relative catalytic activity of the indicated mutants. Kinase activity of GFP-mMastl wild-type was set as 1 (n=3 independent experiments). Data are mean + SEM from 3 independent experiments. ***P <0.001; 1-way ANOVA.


G . isgMASTL MDA - MB - 231 cells were transduced with the indicated Mastl mutants or GFP alone as a control , and treated with doxycycline ( Dox ) to induce Mastl deletion . Cells were collected after glucose stimulation , and total lysates blotted for the indicated antibodies .

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G. isgMASTL MDA-MB-231 cells were transduced with the indicated Mastl mutants or GFP alone as a control, and treated with doxycycline (Dox) to induce Mastl deletion. Cells were collected after glucose stimulation, and total lysates blotted for the indicated antibodies.


B sgRNA frequencies ( log2 of fold change ) in PD - L1low cells unstimulated ( left ) or IFN - γ stimulated ( right ) . Enriched sgRNAs ( dashed line ) are highlighted ; hit genes ( black , red ) and controls ( green , yellow or purple ) are shown and labeled with target gene names .

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B sgRNA frequencies (log2 of fold change) in PD-L1low cells unstimulated (left) or IFN-γ stimulated (right). Enriched sgRNAs (dashed line) are highlighted; hit genes (black, red) and controls (green, yellow or purple) are shown and labeled with target gene names.


C sgRNA frequencies ( log2 of fold change ) in viability screen . sgRNAs targeting controls are highlighted ; essential ( orange ) , non - essential ( blue ) . Genes attributed to PD - L1 modulation are shown in different colors ( red , yellow and purple ) .

C sgRNA frequencies (log2 of fold change) in viability screen. sgRNAs targeting controls are highlighted; essential (orange), non-essential (blue). Genes attributed to PD-L1 modulation are shown in different colors (red, yellow and purple).


A PD - L1 cell surface presentation ( percentage ± SD ) in RKO mock control ( black ) or monoclonal MLLT6 knockout cells ( grey ) ( Student ' s t - test ; * * p < 0 . 01 ; n = 3 biological replicates ; degrees of freedom ( df ) = 2 ) .

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A PD-L1 cell surface presentation (percentage ± SD) in RKO mock control (black) or monoclonal MLLT6 knockout cells (grey) (Student's t-test; **p < 0.01; n=3 biological replicates; degrees of freedom (df)=2).


B MLLT6 BAC mediated rescue in U2OS cells ( left ) showing percentage ± SD of PD - L1 cell surface presentation in mock control ( black ) , MLLT6 polyclonal knockout ( light grey ) and MLLT6 knockout + BAC cells ( dark grey ) ( Student ' s t - test ; * p < 0 . 05 ; n = 3 biological replicates ; df = 2 ) . Representative flow cytometry plots ( right ) of PD - L1 cell surface presentation after staining with anti - PD - L1 antibody ( top right , bottom left , bottom right ) and isotype control ( top left ) . Percentages of cells in the PD - L1 + gate as indicated .

B MLLT6 BAC mediated rescue in U2OS cells (left) showing percentage ± SD of PD-L1 cell surface presentation in mock control (black), MLLT6 polyclonal knockout (light grey) and MLLT6 knockout + BAC cells (dark grey) (Student's t-test; *p < 0.05; n=3 biological replicates; df=2). Representative flow cytometry plots (right) of PD-L1 cell surface presentation after staining with anti-PD-L1 antibody (top right, bottom left, bottom right) and isotype control (top left). Percentages of cells in the PD-L1+ gate as indicated.


C Total cellular protein levels of PD - L1 ( top ) and GAPDH ( bottom ) in monoclonal RKO MLLT6 knockout , mock control or MLLT6 knockout + BAC cells visualized by immunoblotting . Numbers indicate relative band intensities of PD - L1 protein normalized to GAPDH .

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C Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in monoclonal RKO MLLT6 knockout, mock control or MLLT6 knockout + BAC cells visualized by immunoblotting. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


D PD - L1 transcript levels in U2OS mock ( black ) and polyclonal MLLT6 knockout lines ( grey ) treated with and without IFN - γ normalized to expression in mock U2OS cells without IFN - γ treatment ( ANOVA ; * * p < 0 . 01 ; n = 4 biological replicates ; df = 12 ) .

D PD-L1 transcript levels in U2OS mock (black) and polyclonal MLLT6 knockout lines (grey) treated with and without IFN-γ normalized to expression in mock U2OS cells without IFN-γ treatment (ANOVA; **p < 0.01; n=4 biological replicates; df=12).


E Representative flow cytometry plots of PD - L1 cell surface presentation in the presence and absence of IFN - γ in mock , polyclonal MLLT6 knockout and PD - L1 knockout cells . Percentages of cells in the PD - L1 + gate after staining with anti - PD - L1 antibody or isotype control as indicated .

E Representative flow cytometry plots of PD-L1 cell surface presentation in the presence and absence of IFN-γ in mock, polyclonal MLLT6 knockout and PD-L1 knockout cells. Percentages of cells in the PD-L1+ gate after staining with anti-PD-L1 antibody or isotype control as indicated.


F Total cellular protein levels of PD - L1 ( top ) and GAPDH ( bottom ) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN - γ stimulation . Numbers indicate relative band intensities of PD - L1 protein normalized to GAPDH .

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F Total cellular protein levels of PD-L1 (top) and GAPDH (bottom) in U2OS polyclonal MLLT6 knockout or mock control cells with and without IFN-γ stimulation. Numbers indicate relative band intensities of PD-L1 protein normalized to GAPDH.


B Relative cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( black ) or polyclonal MLLT6 knockout ( grey ) cells treated with varying amounts of EpCAM - CD3 BiTE and CTLs ( Student ' s t - test ; * * p < 0 . 01 ; degrees of freedom ( df ) = 4 ) .

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B Relative cell numbers (± SD; n=3; biological replicates) of U2OS mock (black) or polyclonal MLLT6 knockout (grey) cells treated with varying amounts of EpCAM-CD3 BiTE and CTLs (Student's t-test; **p < 0.01; degrees of freedom (df)=4).


C Kaplan Meier survival plot ( left ) showing changes in survival ( percent ) of mock ( grey ) and MLLT6 knockout ( red ) cells treated with CTLs and EpCAM - CD3 BiTE over a course of 9 . 5 hours . Representative images ( right ) of time - lapse fluorescence microscopy following U2OS mock ( eGFP tagged , green ) and MLLT6 knockout ( mCherry tagged , red ) cells immediately ( top left and bottom left ) after adding CTLs and EpCAM - CD3 BiTE and 9 . 5 hours later ( top right and bottom right ) ( scale bars = 100 μm ) .

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C Kaplan Meier survival plot (left) showing changes in survival (percent) of mock (grey) and MLLT6 knockout (red) cells treated with CTLs and EpCAM-CD3 BiTE over a course of 9.5 hours. Representative images (right) of time-lapse fluorescence microscopy following U2OS mock (eGFP tagged, green) and MLLT6 knockout (mCherry tagged, red) cells immediately (top left and bottom left) after adding CTLs and EpCAM-CD3 BiTE and 9.5 hours later (top right and bottom right) (scale bars = 100 μm).


D Relative cell numbers ( left ) ( ± SD ; n = 3 ; biological replicates ) of mock ( black ) , PD - L1 ( dark grey ) or MLLT6 ( light grey ) knockout cells treated with BiTE or bi - specific antibodies and CTLs as indicated ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) . Representative images ( right ) from bright field microscopy of polyclonal U2OS mock ( top ) , PD - L1 ( middle ) or MLLT6 ( bottom ) knockout cells treated without or with EpCAM - CD3 BiTE or bispecific antibodies .

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D Relative cell numbers (left) (± SD; n=3; biological replicates) of mock (black), PD-L1 (dark grey) or MLLT6 (light grey) knockout cells treated with BiTE or bi-specific antibodies and CTLs as indicated (Student's t-test; **p < 0.01; df=4). Representative images (right) from bright field microscopy of polyclonal U2OS mock (top), PD-L1 (middle) or MLLT6 (bottom) knockout cells treated without or with EpCAM-CD3 BiTE or bispecific antibodies.


E Relative normalized cell numbers ( ± SD ; n = 3 ; biological replicates ) of U2OS mock ( left ) , MLLT6 ( middle ) or PD - L1 ( right ) knockout cells treated with EpCAM - CD3 BiTE and CTLs in the presence ( grey ) or absence ( black ) of IFN - γ ( Student ' s t - test ; * * p < 0 . 01 ; df = 4 ) .

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E Relative normalized cell numbers (± SD; n=3; biological replicates) of U2OS mock (left), MLLT6 (middle) or PD-L1 (right) knockout cells treated with EpCAM-CD3 BiTE and CTLs in the presence (grey) or absence (black) of IFN-γ (Student's t-test; **p < 0.01; df=4 ).


A Scatter plots of pairwise comparisons of transcript expression in U2OS cells . Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN - γ as indicated . Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance ( log10 of p - value , n = 3 ) with candidates nominated for further validation ( red ) .

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A Scatter plots of pairwise comparisons of transcript expression in U2OS cells. Plots describe log2 fold change in transcript expression in mock and polyclonal MLLT6 knockout cells treated with and without IFN-γ as indicated. Horizontal axis shows log2 fold change in transcript expression and vertical axis represents statistical significance (log10 of p-value, n=3) with candidates nominated for further validation (red).


B Correlation plots describing log2 fold change of transcripts in RNA sequencing ( x axis ) and Nanostring ( y axis ) measurements in mock cells + / - IFN - γ ( left ) and MLLT6 knockout cells with IFN - γ ( right ) . Transcripts previously described to have a role in tumor immunity ( red ) and PD - L1 ( blue ) are represented on the plots as indicated .

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B Correlation plots describing log2 fold change of transcripts in RNA sequencing (x axis) and Nanostring (y axis) measurements in mock cells +/- IFN-γ (left) and MLLT6 knockout cells with IFN-γ (right). Transcripts previously described to have a role in tumor immunity (red) and PD-L1 (blue) are represented on the plots as indicated.


C Changes ( log2 fold ) in transcript expression ( left ) of select immune - related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN - γ and total protein levels measured by immunoblotting ( right ) . Numbers indicate band intensities normalized to GAPDH .

C Changes (log2 fold) in transcript expression (left) of select immune-related genes in U2OS mock or MLLT6 knockout cells treated with or without IFN-γ and total protein levels measured by immunoblotting (right). Numbers indicate band intensities normalized to GAPDH.


A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN - γ . Numbers indicate band intensities normalized to GAPDH .

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A Immunoblot expression analysis of proteins implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout cells treated with or without IFN-γ. Numbers indicate band intensities normalized to GAPDH.


B Immunoblot showing total protein levels of STAT1 isoforms ( α and β ) , IDO1 , GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase ( Rluc , negative control ) , total STAT1 , STAT1α and STAT1β treated with and without IFN - γ .

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B Immunoblot showing total protein levels of STAT1 isoforms (α and β), IDO1, GBP5 and CD74 in wild type U2OS cells transfected with esiRNAs targeting renilla luciferase (Rluc, negative control), total STAT1, STAT1α and STAT1β treated with and without IFN-γ.


C . Representative images of calyx synapses from littermate control and Nrxn123 TKO mice . Brainstem sections were labeled with antibodies to vGluT1 ( red ) and Syt2 ( green ) . Scale bar , 10 μm . D . Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity ( normalized to control ) .

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C. Representative images of calyx synapses from littermate control and Nrxn123 TKO mice. Brainstem sections were labeled with antibodies to vGluT1 (red) and Syt2 (green). Scale bar, 10 μm. D. Summary graphs of the synaptic vGluT1 and Syt2 immunostaining intensity (normalized to control).


E - H . Representative traces of spontaneous EPSCs ( sEPSCs ) ( E ) , and summary graphs of the sEPSC frequency ( F ) , amplitude ( G ) , and kinetics ( H ) recorded from littermate control and Nrxn123 TKO mice .

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E-H. Representative traces of spontaneous EPSCs (sEPSCs)(E), and summary graphs of the sEPSC frequency (F), amplitude (G), and kinetics (H) recorded from littermate control and Nrxn123 TKO mice.


A . Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s , recorded in a standard bath solution containing 2 mM Ca2 + . B . Pan - neurexin deletion suppresses synaptic transmission . Summary graphs show the amplitude ( left ) and charge transfer ( right ) of the first EPSC recorded in response to the paired stimuli . C . Pan - neurexin deletion more than doubles the paired - pulse ratio ( PPR ) , suggesting a large decrease in release probability . D . Pan - neurexin deletion decelerates the EPSC time course . Summary graphs show the rise time ( left ) and decay time constants ( right ) of the first EPSC in response to the paired stimuli .

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A. Representative traces of EPSCs evoked by paired stimuli separated by 10 ms and repeated every 20 s, recorded in a standard bath solution containing 2 mM Ca2+. B. Pan-neurexin deletion suppresses synaptic transmission. Summary graphs show the amplitude (left) and charge transfer (right) of the first EPSC recorded in response to the paired stimuli. C. Pan-neurexin deletion more than doubles the paired-pulse ratio (PPR), suggesting a large decrease in release probability. D. Pan-neurexin deletion decelerates the EPSC time course. Summary graphs show the rise time (left) and decay time constants (right) of the first EPSC in response to the paired stimuli.


E - F . Pan - neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high - frequency stimulus train ( 100 Hz for 0 . 5 sec ) . Left , representative ESPC traces ; right , cumulative summary plot of the EPSC amplitudes during the train ( dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back - extrapolation to zero time , which is used to correct for vesicle replenishment during the train ) .

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E-F. Pan-neurexin deletion does not significantly alter the cumulative EPSC amplitude during a high-frequency stimulus train (100 Hz for 0.5 sec). Left, representative ESPC traces; right, cumulative summary plot of the EPSC amplitudes during the train (dotted lines show linear regression fits for estimating the cumulative EPSC amplitude by back-extrapolation to zero time, which is used to correct for vesicle replenishment during the train).


G . Pan - neurexin deletion does not decrease the readily - releasable pool of vesicles , but lowers the initial release probability during a high - frequency stimulus train . Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily - releasable pool size ( left ) , and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability ( right ) .

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G. Pan-neurexin deletion does not decrease the readily-releasable pool of vesicles, but lowers the initial release probability during a high-frequency stimulus train. Summary graphs show the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the readily-releasable pool size (left), and the ratio of the first EPSC amplitude divided by the cumulative EPSC amplitudes extrapolated to time zero as an estimate of the initial release probability (right).


A - D . Same as Fig 2A - D , except that ACSF contained only 1 mM Ca2 + , which is close to the estimated physiological concentration of extracellular Ca2 + ( Forsberg et al . , 2019 ) .

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A-D. Same as Fig 2A-D, except that ACSF contained only 1 mM Ca2+, which is close to the estimated physiological concentration of extracellular Ca2+ (Forsberg et al., 2019).


E - H . Same as Fig 2A - D , except that the slice were incubated with 0 . 2 mM EGTA - AM for 30 mins before recording , which introduces EGTA into the presynaptic terminals .

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E-H. Same as Fig 2A-D, except that the slice were incubated with 0.2 mM EGTA-AM for 30 mins before recording, which introduces EGTA into the presynaptic terminals.


A . Example traces of presynaptic Ca2 + - currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV . B . Summary graphs of the I - V relationship for the peak Ca2 + - current amplitude ( left ) and of the peak Ca2 + - current density ( right ) .

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A. Example traces of presynaptic Ca2+-currents recorded in patched calyx terminals as induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV. B. Summary graphs of the I-V relationship for the peak Ca2+-current amplitude (left) and of the peak Ca2+-current density (right).


C . Example traces ( left ) and summary graphs ( right ) of the Ca2 + - current activation and deactivation kinetics . Currents were induced by a 50 ms step depolarization from - 80 mV to + 10 mV .

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C. Example traces (left) and summary graphs (right) of the Ca2+-current activation and deactivation kinetics. Currents were induced by a 50 ms step depolarization from -80 mV to +10 mV.


D . Example traces of Ca2 + - currents evoked by an action potential - equivalent depolarization ( APe , from - 80 mV to + 17 mV for 1 ms ; left ) , and summary graph of the APe - evoked Ca2 + - current peak amplitude and charge ( right ) .

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D. Example traces of Ca2+-currents evoked by an action potential-equivalent depolarization (APe, from -80 mV to +17 mV for 1 ms; left), and summary graph of the APe-evoked Ca2+-current peak amplitude and charge (right).


E . Example traces of presynaptic Ca2 + - currents induced by a 50 ms step depolarization from − 50 mV to + 40 mV in 10 mV increments at a holding potential of − 80 mV before and after perfusion of 200 nM agatoxin ( Agtx ) , a selective P / Q - type Ca2 + channel blocker .

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E. Example traces of presynaptic Ca2+-currents induced by a 50 ms step depolarization from −50 mV to +40 mV in 10 mV increments at a holding potential of −80 mV before and after perfusion of 200 nM agatoxin (Agtx), a selective P/Q-type Ca2+ channel blocker.


F . Summary graph of the relative contribution of P / Q - type Ca2 + channels to the total presynaptic Ca2 + currents , as quantified by the relative reduction in Ca2 + currents evoked by step depolarization to + 10 mV , in control and Nrxn123 TKO synapses .

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F. Summary graph of the relative contribution of P/Q-type Ca2+ channels to the total presynaptic Ca2+ currents, as quantified by the relative reduction in Ca2+ currents evoked by step depolarization to +10 mV, in control and Nrxn123 TKO synapses.


B . Example traces of the presynaptic capacitance and Ca2 + - currents at the calyx terminals induced by a 20 ms step depolarization from − 80 mV to + 10 mV with an extracellular solution containing 2 mM Ca2 + , and an internal pipette solution containing a standard 0 . 05 mM BAPTA . C . Summary graphs of the depolarization - induced capacitance jump that monitors synaptic vesicle exocytosis ( left ) , and the peak Ca2 + - current density ( right ) . D , E . Same as ( B , C ) , except that 0 . 5 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . F , G . Same as ( B , C ) , except that 1 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution . H , I . Same as ( B , C ) , except that 10 . 0 mM EGTA was added in addition to 0 . 05 mM BAPTA to the patch pipette internal solution .

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B. Example traces of the presynaptic capacitance and Ca2+-currents at the calyx terminals induced by a 20 ms step depolarization from −80 mV to +10 mV with an extracellular solution containing 2 mM Ca2+, and an internal pipette solution containing a standard 0.05 mM BAPTA. C. Summary graphs of the depolarization-induced capacitance jump that monitors synaptic vesicle exocytosis (left), and the peak Ca2+-current density (right). D, E. Same as (B, C), except that 0.5 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. F, G. Same as (B, C), except that 1.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution. H, I. Same as (B, C), except that 10.0 mM EGTA was added in addition to 0.05 mM BAPTA to the patch pipette internal solution.


J , K . Summary plots of the relationship between the total capacitance jump ( J ) or between the Ca2 + - current density ( K ) and the concentration of EGTA in the patch pipette .

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J, K. Summary plots of the relationship between the total capacitance jump (J) or between the Ca2+-current density (K) and the concentration of EGTA in the patch pipette.


A . Representative traces of Ca2 + - currents and 4 - AP insensitive K + - currents evoked by step depolarizations ( from − 50 mV to + 10 mV in 10 mV increments ) , recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12 - P14 . B . Same as ( A ) but after addition of 200 nM iberiotoxin ( IbTx ) by perfusion . C . Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in ( B ) from currents shown in ( A ) . D . Summary graphs of the capacitance , Ca2 + - current density , and BK - current density .

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A. Representative traces of Ca2+-currents and 4-AP insensitive K+-currents evoked by step depolarizations (from −50 mV to +10 mV in 10 mV increments), recorded from the calyx terminals in acute slices from littermate control and Nrxn123 TKO mice at P12-P14. B. Same as (A) but after addition of 200 nM iberiotoxin (IbTx) by perfusion. C. Representative traces of presynaptic BK currents , which are calculated by subtracting currents shown in (B) from currents shown in (A). D. Summary graphs of the capacitance, Ca2+-current density, and BK-current density.


A . Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 ( green ) , CaV2 . 1 - type Ca2 + - channels ( red ) , and the active zone protein Bassoon ( purple ) . Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12 - 14 . Scale bar , 10 μm .

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A. Representative images of brainstem cryosections stained by triple immunofluorescence labeling for vGluT1 (green), CaV2.1-type Ca2+-channels (red), and the active zone protein Bassoon (purple). Cryosections were obtained from littermate control and Nrxn123 TKO mice at P12-14. Scale bar, 10 μm.


B . Summary graphs of the vGluT1 , CaV2 . 1 , and Bassoon immunostaining intensity ( normalized to control ; CaV2 . 1 and Bassoon signals were quantified over the vGluT1 - positive area to measure active zone - localized proteins ) .

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B. Summary graphs of the vGluT1, CaV2.1, and Bassoon immunostaining intensity (normalized to control; CaV2.1 and Bassoon signals were quantified over the vGluT1-positive area to measure active zone-localized proteins).


C . Summary graphs of the diameter and total size of the vGluT1 - positive presynaptic area of the calyx of Held .

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C. Summary graphs of the diameter and total size of the vGluT1-positive presynaptic area of the calyx of Held.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of Ki - 67 + cells ( A ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of Ki-67+ cells (A) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


100 nM paclitaxel treatment of human hair follicles ( HFs ) in organ culture for 24h does not significantly affect the total number of EdU + cells ( B ) ( S - phase ) in the hair matrix . Unpaired t - test performed using N of 9 - 12 HFs from 3 patients .

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100 nM paclitaxel treatment of human hair follicles (HFs) in organ culture for 24h does not significantly affect the total number of EdU+ cells (B) (S-phase) in the hair matrix. Unpaired t-test performed using N of 9-12 HFs from 3 patients.


C ) 100 nM paclitaxel treatment ( 24h ) significantly ( p = < 0 . 0001 [ * * * * ] ) increased the number of mitotic phospho - histone H3 ( pH3 ) + cells in the hair matrix . Welch ' s t - test performed using N of 9 HFs from 3 patients .

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C) 100 nM paclitaxel treatment (24h) significantly (p= <0.0001 [****]) increased the number of mitotic phospho-histone H3 (pH3)+ cells in the hair matrix. Welch's t-test performed using N of 9 HFs from 3 patients.


D ) 100 nM docetaxel treatment ( 24h ) significantly ( p = 0 . 0004 [ * * * ] ) increases the number of pH3 + cells in the hair matrix . Unpaired t - test performed using N of 8 - 9 HFs from 3 patients .

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D) 100 nM docetaxel treatment (24h) significantly (p = 0.0004 [***]) increases the number of pH3+ cells in the hair matrix. Unpaired t-test performed using N of 8-9 HFs from 3 patients.


E ) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on ( i ) Ki - 67 expression [ paclitaxel ] ; ( ii ) EdU incorporation and pH3 immunoreactivity [ paclitaxel ] ; ( iii ) pH3 immunoreactivity [ docetaxel ] . 20 µm scale . Values plotted represent the mean number of positive cells counted per HF analysed . Error bars are standard error .

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E) Representative immunofluorescence images highlight the effects of 24h 100 nM taxane treatment on (i) Ki-67 expression [paclitaxel]; (ii) EdU incorporation and pH3 immunoreactivity [paclitaxel]; (iii) pH3 immunoreactivity [docetaxel]. 20 µm scale. Values plotted represent the mean number of positive cells counted per HF analysed. Error bars are standard error.


The presence of micronucleated cells in the hair matrix in paclitaxel ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 12 - 13 HFs ( paclitaxel ) from 3 patients .

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The presence of micronucleated cells in the hair matrix in paclitaxel (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 12-13 HFs (paclitaxel) from 3 patients.


The presence of micronucleated cells in the hair matrix in docetaxel treated ( 100 nM , 24h ) hair follicles ( HF ) compared to vehicle is significant ( p = < 0 . 0001 [ * * * * ] ) . Mann Whitney U test performed using N of 8 HFs ( docetaxel ) from 3 patients .

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The presence of micronucleated cells in the hair matrix in docetaxel treated (100 nM, 24h) hair follicles (HF) compared to vehicle is significant (p = <0.0001 [****]). Mann Whitney U test performed using N of 8 HFs (docetaxel) from 3 patients.


C ) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix ( lined ) and dermal papilla in untreated ( vehicle ) human HFs . 20 µm scale .

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C) Hoechst 33342 staining of healthy cell nuclei comprising the hair matrix (lined) and dermal papilla in untreated (vehicle) human HFs. 20 µm scale.


D ) Paclitaxel treatment ( 100 nM , 24h ) induces the formation of micronucleated bodies , as visualised by Hoechst 33342 staining ( arrows ) , localising to the proliferative region of the hair matrix . i - 20 µm scale ; ii - 10 µm scale .

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D) Paclitaxel treatment (100 nM, 24h) induces the formation of micronucleated bodies, as visualised by Hoechst 33342 staining (arrows), localising to the proliferative region of the hair matrix. i - 20 µm scale; ii - 10 µm scale.


E ) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies ( arrows ) . 10 µm scale . Error bars are standard error .

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E) 100 nM docetaxel treatment was also seen to promote the formation of micronucleated bodies (arrows). 10 µm scale. Error bars are standard error.


A ) Nascent RNA synthesis , as detected by ethynyl uridine ( EU ) incorporation , is blocked within clusters of nuclei in the hair matrix ( arrows ) following paclitaxel treatment ( 100 nM , 24h ) . 20 µm scale .

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A) Nascent RNA synthesis, as detected by ethynyl uridine (EU) incorporation, is blocked within clusters of nuclei in the hair matrix (arrows) following paclitaxel treatment (100 nM, 24h). 20 µm scale.