A ) Mitochondrial fractions isolated from heads of flies expressing Control - R were divided in half . One of two samples was treated with 0 . 5 µg / ml Proteinase K both samples were incubated at 4 ° C for 20 minutes . Samples were then subjected to western blot using antibodies to outer Mitofusin ( Mfn ) , inner - associated matrix V β ( Comp V β ) , matrix luminal pyruvate dehydrogenase ( PDH ) . elav - GAL4 was used to drive expression of RNAi constructs used in this following experiments . ( B ) Densitometry of Mfn , Comp V β , PDH bands from experiments represented by panel A were performed using Fiji software , ratios of band intensities in sample containing ProK to sample lacking ProK are shown .

O O B-SUBCELLULAR O O O B-TISSUE O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O

A) Mitochondrial fractions isolated from the heads of flies expressing Control-R were divided in half. One of the two samples was treated with 0.5 µg/ml Proteinase K and both samples were incubated at 4°C for 20 minutes. Samples were then subjected to western blot analysis using antibodies to the outer membrane protein Mitofusin (Mfn), the inner membrane-associated matrix protein Complex V β (Comp V β), and the matrix luminal protein pyruvate dehydrogenase (PDH). elav-GAL4 was used to drive expression of all the RNAi constructs used in this and the following experiments. (B) Densitometry of Mfn, Comp V β, and PDH bands from experiments represented by panel A were performed using Fiji software, and the ratios of the band intensities in the sample containing ProK to the sample lacking ProK are shown.


( C ) Mitochondrial fractions isolated from heads of flies expressing Control - R PINK1 - Myc were divided in half . Each sample was treated with indicated concentrations of ProK . Following incubation , samples were subjected to western blot using antibodies to indicated . ( D ) Densitometry of PINK1 - Myc , Comp V β , PDH bands from experiments represented by panel C were performed as described in B . Mfn data from panel B is also included for comparison .

O O O B-SUBCELLULAR O O O B-TISSUE O B-ORGANISM O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O

(C) Mitochondrial fractions isolated from the heads of flies expressing Control-R and PINK1-Myc were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (D) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel C were performed as described in B. The Mfn quantification data from panel B is also included for comparison.


( E ) Mitochondrial fractions isolated from heads of Lon - R2 PINK1 - Myc expressing flies were divided in half . Each sample was treated with indicated concentrations of ProK . Following incubation , samples were subjected to western blot using antibodies to indicated . ( F ) Densitometry of PINK1 - Myc , Comp V β , PDH bands from experiments represented by panel E were performed as described in B . experiments described were repeated at least three times . Error bars represent s . e . m .

O O O B-SUBCELLULAR O O O B-TISSUE O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-ORGANISM O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O 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) Mitochondrial fractions isolated from the heads of Lon-R2 and PINK1-Myc expressing flies were divided in half. Each sample was treated with the indicated concentrations of ProK. Following incubation, samples were subjected to western blot analysis using antibodies to the indicated proteins. (F) Densitometry of the PINK1-Myc, Comp V β, and PDH bands from experiments represented by panel E were performed as described in B. All experiments described were repeated at least three times. Error bars represent s.e.m.


PINK1 accumulates in mitochondrialmatrix upon knockdown of Lon . Immunofluorescent of thoracic expressing matrix - targeted YFP ( Mito - YFP ) under control of sqh genomic promoter , together with either ( A ) Myc - tagged MitochondrialRho ( Miro - Myc ) , ( B ) FLAG - tagged Optic atrophy 1 ( Opa1 - FLAG ) , ( C ) endogenous Cytochrome C ( Cyto C ) , ( D ) Myc - tagged PINK1 ( PINK1 - Myc ) , or ( E ) Lon - RNAi1 ( Lon - R1 ) PINK1 - Myc . muscle - specific Mhc - GAL4 driver was used to drive expression of Miro - Myc in panel A Opa1 - FLAG in panel B , muscle - specific Dmef2 - GAL4 driver was used to drive expression of Lon - R1 in panel E . ( F ) We determined volume shared between green objects ( representing matrix marker Mito - YFP ) red objects ( representing mitochondrial analyzed in A - E ) . Red objects that shared at least 60 % of their volume with Mito - YFP were considered to co - localize . Bar represents 2 µm .

B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD O B-EXP_ASSAY O B-TISSUE O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O

PINK1 accumulates in the mitochondrialmatrix upon knockdown of Lon.Immunofluorescent staining of thoracic muscles expressing matrix-targeted YFP (Mito-YFP) under control of the sqh genomic promoter, together with either (A) Myc-tagged MitochondrialRho (Miro-Myc), (B) FLAG-tagged Optic atrophy 1 (Opa1-FLAG), (C) endogenous Cytochrome C (Cyto C), (D) Myc-tagged PINK1 (PINK1-Myc), or (E) Lon-RNAi1 (Lon-R1) and PINK1-Myc. The muscle-specific Mhc-GAL4 driver was used to drive expression of Miro-Myc in panel A and Opa1-FLAG in panel B, and the muscle-specific Dmef2-GAL4 driver was used to drive expression of Lon-R1 in panel E. (F) We determined the percent volume shared between green objects (representing the matrix marker Mito-YFP) and red objects (representing the mitochondrial proteins analyzed in A-E). Red objects that shared at least 60% of their volume with Mito-YFP were considered to co-localize. Bar represents 2 µm.


A ) eye severity score of flies co - overexpressing PINK1 with Control - R ( n = 74 ) , Lon - R1 ( n = 163 ) , or Lon - R2 ( n = 90 ) using ey - GAL4 driver .

O O B-TISSUE O O O B-ORGANISM O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O

A) The eye severity score of flies co-overexpressing PINK1 with Control-R (n = 74), Lon-R1 (n = 163), or Lon-R2 (n = 90) using the ey-GAL4 driver.


( B ) Comparison of frequency of thoracic indentations in flies expressing PINK1 - RNAi construct ( PINK1 - R ) in an otherwise wild - type background to frequency in that also bear heterozygous mutations in Lon : PINK1 - RNAi + / + ( n = 306 ) , PINK1 - RNAi LonEP / + ( n = 74 ) , PINK1 - RNAi LonDf / + ( n = 83 ) . LonEP refers to LonG3998 P - element allele of Lon , LonDf refers to Df ( 3L ) Exel9011 deletion that removes Lon gene along with several other genes . Dmef2 - GAL4 driver was used to drive expression of PINK1 - R construct in experiments described in panel B . Error bars represent s . e . m . * p < 0 . 05 , * * p < 0 . 005 by Student t .

O O O O O O O B-TISSUE O O B-ORGANISM O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O 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) Comparison of the frequency of thoracic indentations in flies expressing the PINK1-RNAi construct (PINK1-R) in an otherwise wild-type background to the frequency in animals that also bear heterozygous mutations in Lon: PINK1-RNAi +/+ (n = 306), PINK1-RNAi LonEP/+ (n = 74), and PINK1-RNAi LonDf/+ (n = 83). LonEP refers to the LonG3998 P-element allele of Lon, and LonDf refers to the Df(3L)Exel9011 deletion that removes the Lon gene along with several other genes. The Dmef2-GAL4 driver was used to drive expression of the PINK1-R construct in experiments described in panel B. Error bars represent s.e.m. *p<0.05, **p<0.005 by Student t test.


A . Viability of human PMNs following treatment with purified recombinant bi - component leukocidins using viability dye CellTiter . Bars indicate Mean ± , with n = 5 donors .

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A. Viability of primary human PMNs following treatment with purified recombinant bi-component leukocidins using the viability dye CellTiter. Bars indicate Mean ± SEM, with n = 5 donors.


B . Half maximal effective concentrations ( EC50 ) of native heterologous toxin pairings on human PMNs using viability dye CellTiter . E = LukE , D = LukD , A = HlgA , B = HlgB , C = HlgC , S = LukS - PV , F = LukF - PV . Bars indicate Mean ± , with n = 6 donors .

O O O O O O O O O O O O O O O B-ORGANISM B-CELL_TYPE O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

B. Half maximal effective concentrations (EC50) of native and heterologous toxin pairings on primary human PMNs using the viability dye CellTiter. E = LukE, D = LukD, A = HlgA, B = HlgB, C = HlgC, S = LukS-PV, F = LukF-PV. Bars indicate Mean ± SEM, with n = 6 donors.


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

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

O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD 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 human PMNs following incubation with mutant toxin pairings using viability dye CellTiter . Bars indicate Mean ± , with n = 3 donors .

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

O O O O B-EXP_ASSAY O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O 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 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 ) indicated dominant - negative toxins using viability dye CellTiter . Open 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 ± , with n = 5 donors .

O O O O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 human PMNs following incubation with filtered culture supernatants collected after 8 hours from CA - MRSA USA300 LAC using viability dye CellTiter . Bars indicate Mean ± , with n = 6 donors .

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

O O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 human PMNs during a 3 - hour ex vivo infection with S . aureus Newman toxinless overproducing indicated toxins using CytoTox - One Homogenous Integrity to measure lactate dehydrogenase ( LDH ) release . Bars indicate Mean ± , with n = 3 donors .

O O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD 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 human PMNs during a 4 - hr ex vivo infection with a MOI of 50 of S . aureus Newman toxinless overproducing indicated toxins in presence of a dose response of DN toxins using CytoTox - One Homogenous Integrity to measure lactate dehydrogenase ( LDH ) release . Emut1Dmut = LukEmut1LukDmut , Amut1Bmut = HlgAmut1HlgBmut , Cmut1Bmut = HlgCmut1HlgBmut , Smut1Fmut = LukS - PVmut1LukF - PVmut . Bars indicate Mean ± , with n = 3 donors .

O O B-EXP_ASSAY O B-ORGANISM B-CELL_TYPE O O O O O O O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD 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 of LukAB mutants produced by S . aureus Newman in a toxinless background . Samples were electrophoresed on a 12 % SDS - PAGE gel visualized by with Coomassie blue . A representative of two experiments is shown .

O O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY I-EXP_ASSAY 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 immunoblot analyses of wildtype S . aureus Newman transformed with empty plasmid ( p - ) or with plasmids containing mutated lukAB loci . A representative of two independent experiments is shown .

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

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

O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-ORGANISM B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O O O O B-CELL_TYPE 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 using GFP - LukD ( 156 . 3 nM ) a dose response of unlabeled dominant - negative ( DN ) toxin subunits evaluated by flow cytometry on surface of human PMNs . Emut1 = LukEmut1 , Amut1 = HlgAmut1 . Data is presented as total emitted from GFP - positive PMNs . A representative of three independent experiments each done with hPMNs isolated from different donors is shown .

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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 oligomer formation by purified polyhistidine ( 6xHis ) - tagged WT or dominant - negative LukED toxins in 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 B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O B-GENEPROD 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 ) 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 B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O B-SMALL_MOLECULE 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 , Buffer vs Cmut1Bmut . p = 0 . 1050 ( not significant , ns ) for Buffer vs Smut1Fmut . Statistics performed using Log - rank ( Mantel - Cox ) . A representative of two independent experiments is shown .

O O B-EXP_ASSAY O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 ) . A representative of two independent experiments is shown .

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

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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 / + RagAGTP / GTP genotypes were deprived of for 1 h re - stimulated for 10 min . Whole - lysates were immunoblotted for indicated .

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

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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 ) RagAGTP / GTP ( n = 22 ) mice at birth ( data are scatter dots , mean ) .

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


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

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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 extracts from liver heart of RagA + / + ( + / + ) , RagAGTP / + ( G / + ) RagAGTP / GTP ( G / G ) neonates immediately after Caesarean section ( 0 h ) or after 1 h of fasting ( 1 h fast ) .

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

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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 resuscitated were fasted 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 resuscitated were fasted their survival monitored ( + / + : n = 13 ; G / + : n = 13 ; G / G : n = 6 ) . Numbers indicate median survival for each curve . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 005 .

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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 / + RagAGTP / GTP recovery in fasted RagA + / + 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 10 h , respectively ; mean ± s . e . m . ) .

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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 10 h ( mean ± s . e . m . ) . NS , not significant .

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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 ) .

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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 showing abundant glycogen content in RagA + / + RagAGTP / GTP hepatocytes before fasting ( 0 h , upper panels ) more pronounced glycogen depletion after 10 h of fasting ( lower panels ) in RagAGTP / GTP neonates . Right : 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 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 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 , of neonatal plasma amounts of branched - chain ( BCAA ) essential at birth ( left ) , after 10 h fasting ( middle ) after 10 h fasting with rapamycin treatment ( right ) ( n for + / + , G / + G / G , respectively : n = 4 , 5 4 for 0 h ; n = 4 , 4 3 for 10 h ; n = 2 , 5 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 ( a . a . ) in fasted RagAGTP / GTP neonates ( untreated : n = 10 ; : 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 of autophagosomes autophagolysosomes in hepatocytes from RagA + / + neonates fasted for 1 h . Typical autophagosome with a double limiting ( arrows ) ; autophagosome several autolysosomes ( arrowheads ) . Scale bars , 5 µm . Bottom : frequency of two types of organelle ( early : autophagosomes ; late : autophagolysosomes ) detected in profiles of hepatocytes skeletal myocytes from RagA + / + 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 , extracts from livers of neonates at Caesarean section ( 0 h ) fasted for 10 h were immunoblotted for autophagy markers LC3B 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 , extracts from skeletal muscle heart from neonates at Caesarean section ( 0 h ) , fasted for 1 2 h , were immunoblotted for indicated .

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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 - acid withdrawal in MEFs . MEFs were deprived of for indicated time points , whole - extracts were obtained mTORC1 activity 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 + / + RagAGTP / GTPMEFs LC3B localization , in presence absence of , monitored by microscopy . LC3B - GFP ( green ) clustering , indicative of autophagy , was observed in 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 . ( active ) TFEB was observed in RagA + / + MEFs upon - 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 ) wild - type ( WT ) MEFs were deprived of glucose for 1 h re - stimulated for 10 min . Wholecell extracts were immunoblotted for indicated .

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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 indicated genotypes were deprived of growth factors , glucose , or glucose for 1 h re - stimulated with glucose / or for 10 min . Whole - lysates were immunoblotted for indicated .

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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 + / + RagAGTP / GTP immortalized MEFs were deprived of glucose or surviving quantified in triplicate after 48 h . number is indicated relative to number at start of treatment ; mean ± s . d . ; * * * P 0 .

O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O 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, 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 , glucose deprivation causes mTOR to localize to diffuse small throughout cytoplasm . Re - addition of glucose leads to mTOR shuttling to lysosomal surface , co - localizing with lysosomal Lamp2 . HEK - 293T - RagBGTP show mTOR localized at lysosomal surface , regardless of glucose concentrations . Scale bars , 10 μm .

O O B-GENEPROD O O O O B-EXP_ASSAY O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O

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 affect binding of v - ATPase to Ragulator . HEK - 293T expressing Flag - p14 was deprived of glucose or for 90 min re - stimulated for 20 min . extracts immunoprecipitates were immunoblotted for indicated .

O O B-SMALL_MOLECULE O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-SUBCELLULAR O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O B-EXP_ASSAY O O O

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 + / + RagAGTP / GTP MEFs were cultured for 1 h in media with glucose - acid concentrations measured in neonates at birth ( 0 h ) or after fasting for 1 h ( 1 h ) whole - extracts were analysed by immunoblotting .

O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-ORGANISM O 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-EXP_ASSAY O

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 left panel ( scale bar - 100 µm ) . middle right panels show read normalized Pol II ChIP - seq tracks ( RPM ) from six tissues at genes . For each gene , grey red tracks indicate non - expressing tissues expressing tissue , respectively . middle panel shows Pol II profile at two non - paused genes , which have Pol II only in expressing tissues . expression is limited to specific tissues as shown in in situ from BDGP . right panel shows Pol II profile at a paused gene , which has Pol II in observed tissues , although expression is limited to specific tissues as shown in in situ from BDGP . Paused Pol II is generally highest in tissue with 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 .

O O O B-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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-SUBCELLULAR I-SUBCELLULAR B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O 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) 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 ( read normalized for each , log2 ) in different tissues , from scRNA - seq experiment , for a TATA group gene , a highly paused group gene , are shown . ( C ) TATA gene , Ccp84Aa , shows noisy expression in epidermis , without detectable background expression in non - expressing tissues . ( D ) highly paused gene , Gip , shows very robust expression in crystal , but has high background expression in non - expressing tissues .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE O O O O O O O O O O O O

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

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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

O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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

O O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-GENEPROD O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O B-GENEPROD O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

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 with indicated antibodies in BT - 549 upon glucose deprivation 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 + from 3 experiments . Significance determined by Student ' s t - comparing feedback activation in MASTL - depleted versus control ( ns , not significant ) . F . Immunoblot with indicated antibodies in MCF - 7 upon glucose deprivation 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 + from 3 experiments . Significance determined by Student ' s t - comparing feedback activation in MASTL - depleted versus control ( ns , not significant ; * P < 0 . 05 ) .

O O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O 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-GENEPROD O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O O O O O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

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

O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-TISSUE O O O B-GENEPROD O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-SMALL_MOLECULE O 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. 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 using specific ( + ) or Scrambled ( − ) shRNAs . were starved for glucose for 1 h . plot shows of phospho - AKT T308 . Data are mean + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

O O B-GENEPROD O O O O O O B-GENEPROD O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O 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. 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 ( + ) were treated with 100 nM rapamycin or vehicle as control . were starved for glucose for 1 h re - stimulated with glucose for 15 min ; rapamycin was added 15 min before glucose stimulation . plot shows of phospho - AKT T308 . Data are mean + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O B-GENEPROD O O O O 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. 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 in conditions of serum starvation insulin stimulation . DAPI ( ) is in cyan a mask is in yellow . Arrowheads indicate positive for GLUT4 enrichment at plasma ( PM ) . Representative magnifications are also shown . Scale bars , 10 μm plot shows of of positive for GLUT4 translocation at PM . Data are mean fold increase vs serum deprivation in scramble condition + from 3 independent experiments . ns , not significant ; * P < 0 . 05 , 1 - way ANOVA . Control inmunoblotting in conditions of insulin stimulation is also shown for indicated antibodies .

O O B-EXP_ASSAY O B-GENEPROD O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O B-TISSUE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O B-TISSUE O O O O 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-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O

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

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

O O B-EXP_ASSAY O O O O B-TISSUE O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-ORGANISM O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O

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 ARPP19 in MDA - MB - 231 using specific siRNAs or control siRNAs against luciferase ( Luc ) . 72 h after transfection were starved for glucose for 1 h re - stimulated with glucose for 15 min . Total extracts were collected analyzed for indicated antibodies .

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

O O O O O O O B-ORGANISM O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O O O O O O O O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O

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 − ) MASTL knockout ( Dox + ) 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 indicated antibodies P - T308 - AKT signal was quantified . β - Actin was used as a loading control . plot represents mean + from 3 independent experiments , except for fostriecin treatment in which only data from two experiments were obtained no error bars are shown . * P < 0 . 05 ; Student ' s t - .

O O O O B-SMALL_MOLECULE O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O 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 were co - transduced with specific siRNAs against PPP2R2A ( B55α ) PPP2R2D ( B55δ ) transcripts ( B55αδ ) or against luciferase ( Luc ) as a control . Asynchronous were collected blotted for indicated antibodies . histograms represent quantifications of specified phospho - residues . Plots show mean + from 3 independent experiments . * P < 0 . 05 , Student ' s t - .

O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-EXP_ASSAY O 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 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 indicated antigens in control ( − ) MASTL knockout ( + ) starved from glucose re - stimulated with glucose for 15 min .

O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O

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 delta subunits ( B55αδ ) in control ( Dox − ) MASTL - null ( Dox + ) . phosphorylation status of different was scored in conditions of glucose re - addition . n = 3 independent experiments . plot shows mean + from 3 independent experiments . * P < 0 . 05 , 1 - way ANOVA .

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

O O B-EXP_ASSAY O O O O B-TISSUE I-TISSUE O B-TISSUE O O O B-ORGANISM O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-EXP_ASSAY O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O 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 were starved for 1 h re - stimulated with glucose for 15 min T194 phosphorylation was tested with a specific antibody . plot shows of phospho - MASTL T194 ( n = 2 independent experiments ) . Data are mean + from 3 independent experiments . ns , not significant ; Student ' s t - .

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

O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD 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 using purified GST - human MASTL as a substrate , recombinant mTORC1 S6K1 kinases . Red arrowheads show phosphorylation of MASTL in presence of mTORC1 or S6K1 . Note appearance of other phosporylation signals in mTORC1 S6K1 reactions , independently of presence of MASTL , which correspond to autophosphorylation of these kinases . Imperial shows amount of GST - MASTL used as a substrate in each reaction .

O O O O B-EXP_ASSAY O O B-GENEPROD O B-ORGANISM B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O B-EXP_ASSAY O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-GENEPROD O B-GENEPROD O O O O O O O O

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

O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O B-GENEPROD O O B-ORGANISM B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O O O O O O 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. 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 were transduced with indicated Mastl mutants or GFP alone as a control , treated with doxycycline ( Dox ) to induce Mastl deletion . were collected after glucose stimulation , total lysates blotted for indicated antibodies .

O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O

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 unstimulated ( left ) or IFN - γ stimulated ( right ) . Enriched sgRNAs ( dashed ) are highlighted ; hit genes ( black , red ) controls ( green , yellow or purple ) are shown labeled with target gene names .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O 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 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 purple ) .

O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O

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 surface presentation ( ± SD ) in RKO mock control ( black ) or monoclonal MLLT6 knockout ( grey ) ( Student ' s t - ; * * 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 ( left ) showing ± SD of PD - L1 surface presentation in mock control ( black ) , MLLT6 polyclonal knockout ( grey ) MLLT6 knockout + BAC ( grey ) ( Student ' s t - ; * p < 0 . 05 ; n = 3 biological replicates ; df = 2 ) . Representative flow cytometry plots ( right ) of PD - L1 surface presentation after with anti - PD - L1 antibody ( top right , bottom left , bottom right ) isotype control ( top left ) . Percentages of in PD - L1 + gate as indicated .

O B-GENEPROD O O O O B-CELL_LINE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O

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

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O O B-CELL_LINE B-GENEPROD O O O O O B-GENEPROD O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O

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

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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 surface presentation in presence absence of IFN - γ in mock , polyclonal MLLT6 knockout PD - L1 knockout . Percentages of in PD - L1 + gate after with anti - PD - L1 antibody or isotype control as indicated .

O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD I-GENEPROD I-GENEPROD B-SUBCELLULAR O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

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

O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O O O B-CELL_LINE O B-GENEPROD O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O

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

O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O 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 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 ( ) of mock ( grey ) MLLT6 knockout ( red ) treated with CTLs EpCAM - CD3 BiTE over a course of 9 . 5 hours . Representative ( right ) of time - lapse microscopy following U2OS mock ( eGFP tagged , green ) MLLT6 knockout ( mCherry tagged , red ) immediately ( top left bottom left ) after adding CTLs EpCAM - CD3 BiTE 9 . 5 hours later ( top right bottom right ) ( scale bars = 100 μm ) .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O O O O O O B-CELL_TYPE B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_LINE O O B-GENEPROD O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O

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

O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O O 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-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-CELL_LINE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O

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

O O O B-EXP_ASSAY O O O O O O O O O O O O B-CELL_LINE O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O

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

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O 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 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 sequencing ( x axis ) Nanostring ( y axis ) measurements in mock + / - IFN - γ ( left ) MLLT6 knockout with IFN - γ ( right ) . Transcripts previously described to have a role in immunity ( red ) PD - L1 ( blue ) are represented on plots as indicated .

O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O

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 treated with or without IFN - γ total measured by immunoblotting ( right ) . Numbers indicate band intensities normalized to GAPDH .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-CELL_LINE O O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

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 of implicated in IFNγ signal transduction in U2OS mock or polyclonal MLLT6 knockout 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 of STAT1 isoforms ( α β ) , IDO1 , GBP5 CD74 in wild type U2OS transfected with esiRNAs targeting renilla luciferase ( Rluc , negative control ) , total STAT1 , STAT1α STAT1β treated with 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 of calyx synapses from littermate control Nrxn123 TKO mice . Brainstem sections were labeled with antibodies to vGluT1 ( red ) Syt2 ( green ) . Scale bar , 10 μm . D . Summary graphs of synaptic vGluT1 Syt2 immunostaining ( 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 ) , summary graphs of sEPSC frequency ( F ) , amplitude ( G ) , kinetics ( H ) recorded from littermate control Nrxn123 TKO mice .

O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-ORGANISM O

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

O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O

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 cumulative EPSC amplitude during a high - frequency stimulus train ( 100 Hz for 0 . 5 sec ) . Left , representative ESPC traces ; right , cumulative summary plot of EPSC amplitudes during train ( dotted lines show linear regression fits for estimating cumulative EPSC amplitude by back - extrapolation to zero time , which is used to correct for vesicle replenishment during train ) .

O O O O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O

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 readily - releasable pool of vesicles , but lowers initial release probability during a high - frequency stimulus train . Summary graphs show cumulative EPSC amplitudes extrapolated to time zero as an estimate of readily - releasable pool ( left ) , of first EPSC amplitude divided by cumulative EPSC amplitudes extrapolated to time zero as an estimate of initial release probability ( right ) .

O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

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 estimated physiological 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 slice were incubated with 0 . 2 mM EGTA - AM for 30 mins before recording , which introduces EGTA into 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.