Representative confocal z - stack of microglia ( IBA1 , green , upper ) inhibitory synapses ( Gephyrin : green , VGAT : red , lower ) in IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO Cdc50a cKO ; PLX3397 mice . dashed represents where high magnified insert was taken from . Scale bar , 20 μm . Bar graphs showing relative of IBA1 - positive in IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , control ; DMSO versus control ; PLX3397 , * P = 0 . 0428 , control ; DMSO versus Cdc50a cKO ; DMSO , * * * P = 0 . 0002 , Cdc50a cKO ; DMSO versus Cdc50a cKO ; PLX3397 , * * * P = 0 . 0005 ) . Bar graphs showing number of inhibitory ( Gephyrin - VGAT - positive ) synapses in IC of control ; DMSO , control ; PLX3397 , Cdc50a cKO ; DMSO Cdc50a cKO ; PLX3397 mice ( n = 3 for each group , P * * * * < 0 . 0001 ) .

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

Representative confocal z-stack images of microglia (IBA1, green, upper) and inhibitory synapses (Gephyrin: green, VGAT: red, lower) in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm. Bar graphs showing the relative area of IBA1-positive cells in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, control;DMSO versus control;PLX3397, *P = 0.0428, control;DMSO versus Cdc50a cKO;DMSO, ***P = 0.0002, Cdc50a cKO;DMSO versus Cdc50a cKO;PLX3397, ***P = 0.0005). Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the IC of control;DMSO, control;PLX3397, Cdc50a cKO;DMSO and Cdc50a cKO;PLX3397 mice (n = 3 for each group, P**** < 0.0001).


Representative confocal z - stack of Gephyrin ( green ) - VGAT ( red ) - positive inhibitory synapses in IC ( inferior colliculus ) of control ; C3 + / + , control ; C3 - / - , Cdc50a cKO ; C3 + / + Cdc50a cKO ; C3 - / - mice . dashed represents where high magnified insert was taken from . Scale bar , 10 μm .

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Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC (inferior colliculus) of control;C3+/+, control;C3-/-, Cdc50a cKO;C3+/+ and Cdc50a cKO;C3-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 10 μm.


Representative confocal z - stack of Gephyrin ( green ) - VGAT ( red ) - positive inhibitory synapses in IC of control ; Mertk + / + , control ; Mertk - / - , Cdc50a cKO ; Mertk + / + Cdc50a cKO ; Mertk - / - mice . dashed represents where high magnified insert was taken from . Scale bar , 20 μm .

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Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the IC of control;Mertk+/+, control;Mertk-/-, Cdc50a cKO;Mertk+/+ and Cdc50a cKO;Mertk-/- mice. The dashed line represents where the high magnified insert was taken from. Scale bar, 20 μm.


Schematic diagram of experiment in which PS exposure was measured in brains of 1 - month - 3 - month - old wild - type mice by injecting AAV9 - GFAP - secA5 - mCherry in neonatal stage ( P0 ) . Representative confocal z - stack colocalization of Gephyrin ( green ) , VGAT ( blue ) , secA5 ( red ) in HP ( hippocampus ) of 1 - month - 3 - month - old wild - type mice . circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing expression of secA5 ( B ) IBA1 ( C ) in inhibitory post - synapses ( Gephyrin - positive , green ) pre - synapses ( VGAT - positive , blue ) in HP of 1 - month - 3 - month - old wild - type mice ( n = 4 for each group , * * * P = 0 . 0009 , * P = 0 . 0248 , n . s . , not significant ) .

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Schematic diagram of the experiment in which PS exposure was measured in the brains of 1-month- and 3-month-old wild-type mice by injecting AAV9-GFAP-secA5-mCherry in the neonatal stage (P0). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue), and secA5 (red) in the HP (hippocampus) of 1-month- and 3-month-old wild-type mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the expression of secA5 (B) and IBA1 (C) in inhibitory post-synapses (Gephyrin-positive, green) and pre-synapses (VGAT-positive, blue) in the HP of 1-month- and 3-month-old wild-type mice (n = 4 for each group, ***P = 0.0009, *P = 0.0248, n.s., not significant).


Schematic diagram of experiment in which Annexin V was injected intravenously ( every two days ) to block PS in brains of 1 - month - 3 - month - old wild - type mice . Representative confocal z - stack of Gephyrin ( green ) - VGAT ( red ) - positive inhibitory synapses in HP of 1 - month - old wild - type mice . circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing number of inhibitory ( Gephyrin - VGAT - positive ) synapses in HP of 1 - month - old mice after PBS or Annexin V injection ( n = 4 for each group , * * * P = 0 . 0013 , * * P = 0 . 0090 ) .

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Schematic diagram of the experiment in which Annexin V was injected intravenously (every two days) to block PS in the brains of 1-month- and 3-month-old wild-type mice. Representative confocal z-stack images of Gephyrin (green)- and VGAT (red)-positive inhibitory synapses in the HP of 1-month-old wild-type mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of 1-month-old mice after PBS or Annexin V injection (n = 4 for each group, ***P = 0.0013, **P = 0.0090).


Schematic diagram of experiment in which microglial Mertk KO ( CX3CR1 - CreERT2 ; Mertkfl / fl ) control ( Mertkfl / fl ) mice were generated . Both 1 - month - old microglial Mertk KO mice 1 - month - old control mice were injected with 4 - OHT for 5 consecutive days . Representative confocal z - stack of Gephyrin ( green ) - VGAT ( magenta ) - positive inhibitory synapses in HP of Mertkfl / fl CX3CR1 - CreERT2 ; Mertkfl / fl mice . circles indicate inhibitory synapses . Scale bar , 10 μm . Bar graphs showing number of inhibitory ( Gephyrin - VGAT - positive ) synapses in HP of Mertkfl / fl CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * * P = 0 . 0027 , * P = 0 . 0062 ) . Representative confocal z - stack colocalization of Gephyrin ( green ) , VGAT ( blue ) secA5 ( red ) in HP of Mertkfl / fl CX3CR1 - CreERT2 ; Mertkfl / fl mice . circles indicate secA5 - positive inhibitory synapses . Scale bar , 10 μm . Bar graphs showing relative expression of secA5 in inhibitory synapses in HP of Mertkfl / fl CX3CR1 - CreERT2 ; Mertkfl / fl mice ( n = 4 for each group , * P = 0 . 0015 ) .

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Schematic diagram of the experiment in which microglial Mertk KO (CX3CR1-CreERT2;Mertkfl/fl) and control (Mertkfl/fl) mice were generated. Both 1-month-old microglial Mertk KO mice and 1-month-old control mice were injected with 4-OHT for 5 consecutive days. Representative confocal z-stack images of Gephyrin (green)- and VGAT (magenta)-positive inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the number of inhibitory (Gephyrin- and VGAT-positive) synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, **P = 0.0027, *P = 0.0062). Representative confocal z-stack colocalization images of Gephyrin (green), VGAT (blue) and secA5 (red) in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice. The white circles indicate secA5-positive inhibitory synapses. Scale bar, 10 μm. Bar graphs showing the relative expression of secA5 in inhibitory synapses in the HP of Mertkfl/fl and CX3CR1-CreERT2;Mertkfl/fl mice (n = 4 for each group, *P = 0.0015).


( A ) 293T were transfected with Flag - ULK1 or control empty vector subjected to immunoprecipitation with anti - Flag antibody , followed by immunoblot with indicated antibodies .

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(A) 293T cells were transfected with Flag-ULK1 or control empty vector and subjected to immunoprecipitation with anti-Flag antibody, followed by immunoblot analysis with the indicated antibodies.


( B ) 293T were co - transfected with Flag - AMPKα2 HA - ULK1 . lysates were subjected to immunoprecipitation using anti - Flag antibody followed by SDS - PAGE / immunoblot with anti - HA anti - Flag antibodies .

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(B) 293T cells were co-transfected with Flag-AMPKα2 and HA-ULK1. Cell lysates were subjected to immunoprecipitation using anti-Flag antibody followed by SDS-PAGE/immunoblot analysis with anti-HA and anti-Flag antibodies.


( C ) 293T were transiently transfected with Flag - AMPKα2 or Flag - AMPKβ1 . After 24 h , immunoprecipitation was performed using anti - Flag or control anti - HA antibodies analyzed by immunoblotting with anti - ULK1 anti - Flag polyclonal antibodies .

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(C) 293T cells were transiently transfected with Flag-AMPKα2 or Flag-AMPKβ1. After 24 h, immunoprecipitation was performed using anti-Flag or control anti-HA antibodies and analyzed by immunoblotting with anti-ULK1 and anti-Flag polyclonal antibodies.


( D ) 293T lysates were subjected to immunoprecipitation with anti - ULK1 polyclonal antibody or control preimmune rabbit serum ( NRS ) , followed by immunoblot with anti - ULK1 anti - AMPKα antibodies .

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(D) 293T cell lysates were subjected to immunoprecipitation with anti-ULK1 polyclonal antibody or control preimmune rabbit serum (NRS), followed by immunoblot analysis with anti-ULK1 and anti-AMPKα antibodies.


( E ) 293T were infected with ULK1 shRNA or control shRNA lentiviruses subjected to immunoprecipitation with control rabbit IgG or anti - AMPKα antibody followed by immunoblotting with anti - ULK1 anti - AMPKα antibodies .

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(E) 293T cells were infected with ULK1 shRNA or control shRNA lentiviruses and subjected to immunoprecipitation with control rabbit IgG or anti-AMPKα antibody followed by immunoblotting with anti-ULK1 and anti-AMPKα antibodies.


( F ) Purified His6 - AMPKα1 / β1 / γ1 fusion ( 250 ng ) were mixed with purified His6 - tagged ULK1 ( 1 µg ) or Bcl - XL ( 200 ng ) in 1 % Triton X - 100 lysis buffer containing protease inhibitors subjected to immunoprecipitation with anti - ULK1 antibody . resulting complexes 10 % of input were analyzed by immunoblotting with anti - His - Tag polyclonal antibody .

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(F) Purified His6-AMPKα1/β1/γ1 fusion proteins (250 ng) were mixed with purified His6-tagged ULK1 (1 µg) or Bcl-XL (200 ng) proteins in 1% Triton X-100 lysis buffer containing protease inhibitors and subjected to immunoprecipitation with anti-ULK1 antibody. The resulting protein complexes and 10% of the input proteins were analyzed by immunoblotting with anti-His-Tag polyclonal antibody.


( B , C ) 293T were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutants of ULK1 subjected to immunoprecipitation with anti - HA antibody . resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR , raptor , AMPKα or HA - tag .

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(B, C) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutants of ULK1 and subjected to immunoprecipitation with anti-HA antibody. The resulting immune complexes were analyzed by immunoblotting with antibodies specific for mTOR, raptor, AMPKα or HA-tag.


( A ) 293T were transfected with empty vector or HA - tagged wild type ( WT ) or deletion mutant ( Δ654 - 828 ) ULK1 subjected to immunoprecipitation with anti - HA antibody followed by immunoblotting with indicated antibodies .

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(A) 293T cells were transfected with empty vector or HA-tagged wild type (WT) or deletion mutant (Δ654-828) ULK1 and subjected to immunoprecipitation with anti-HA antibody followed by immunoblotting with the indicated antibodies.


( B ) U - 2OS stably expressing GFP - LC3 were infected with lentivirus expressing human Ulk1 shRNA ( shhUlk1 ) or scrambled shRNA ( shScr ) subjected to selection with 1 µg / ml puromycin for 5 days . were then transfected with wild type mouse Ulk1 ( mUlk1 ) , mUlk1 Δ654 - 828 or control empty vector for 24 h , incubated in serum - free DMEM overnight , cultured in complete medium with or without 10 mM metformin for 20 h . were obtained using a microscope . scale bars represent 10 µm .

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(B) U-2OS cells stably expressing GFP-LC3 were infected with lentivirus expressing human Ulk1 shRNA (shhUlk1) or scrambled shRNA (shScr) and subjected to selection with 1 µg/ml puromycin for 5 days. The cells were then transfected with wild type mouse Ulk1 (mUlk1), mUlk1 Δ654-828 or control empty vector for 24 h, incubated in serum-free DMEM overnight, and cultured in complete medium with or without 10 mM metformin for 20 h. The images were obtained using a fluorescence microscope. The scale bars represent 10 µm.


( C ) number of GFP - LC3 dots per GFP - positive in ( B ) was quantified ( mean ± s . d . ; n = 81 ) .

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(C) The number of GFP-LC3 dots per GFP-positive cell in (B) was quantified (mean ± s.d.; n = 81).


( D ) U - 2OS stably expressing GFP - LC3 were transfected with 0 , 2 or 4 µg of pcDNA3 encoding HA - tagged ULK1 deletion mutant ( 554 - 828 ) . total amount of plasmid used for each transfection was normalized at 4 µg with empty vector . After 24 h transfection , were starved in nutrient - free medium for 1 . 5 h , fixed in 4 % paraformaldehyde , analyzed by microscopy . number of GFP - LC3 dots per GFP - positive was quantified ( mean ± s . d . ; n = 100 ) . Statistical significance was determined by Student ' s t - asterisks indicate P < 0 . 0001 .

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(D) U-2OS cells stably expressing GFP-LC3 were transfected with 0, 2 or 4 µg of pcDNA3 encoding HA-tagged ULK1 deletion mutant (554-828). The total amount of plasmid DNA used for each transfection was normalized at 4 µg with empty vector. After 24 h transfection, the cells were starved in nutrient-free medium for 1.5 h, fixed in 4% paraformaldehyde, and analyzed by fluorescence microscopy. The number of GFP-LC3 dots per GFP-positive cell was quantified (mean ± s.d.; n = 100). Statistical significance was determined by Student's t-test and the asterisks indicate P<0.0001.


293 stably expressing HA - ULK1 were treated with 1 mM AICAR for indicated times subjected to immunoprecipitation with anti - HA or control anti - FLAG antibodies . resulting immune complexes were analyzed by immunoblotting with indicated antibodies .

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293 cells stably expressing HA-ULK1 were treated with 1 mM AICAR for the indicated times and subjected to immunoprecipitation with anti-HA or control anti-FLAG antibodies. The resulting immune complexes were analyzed by immunoblotting with the indicated antibodies.


( A ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were transfected with GFP - LC3 , treated with 2 mM AICAR or control vehicle for 4 h , then analyzed by microscopy . number of GFP - LC3 dots per GFP - positive was counted ( mean ± s . d . ; n = 60 ) . scale bars represent 10 µm .

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(A) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were transfected with GFP-LC3, treated with 2 mM AICAR or control vehicle for 4 h, and then analyzed by fluorescent microscopy. The number of GFP-LC3 dots per GFP-positive cell was counted (mean ± s.d.; n = 60). The scale bars represent 10 µm.


( B ) TSC2 − / − , p53 − / − MEFs stably reconstituted with wild - type raptor or S722A / S792A mutant raptor were treated with 2 mM AICAR for indicated times . lysates were prepared in RIPA buffer analyzed by immunoblotting with indicated antibodies . of p62 are listed relative to those of untreated WT raptor , which were set as 1 .

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(B) TSC2−/−, p53−/− MEFs stably reconstituted with wild-type raptor or S722A/S792A mutant raptor were treated with 2 mM AICAR for the indicated times. Cell lysates were prepared in RIPA buffer and analyzed by immunoblotting with the indicated antibodies. The levels of p62 are listed relative to those of untreated WT raptor cells, which were set as 1.


Representative for TIF shown in ( C ) . Shown are POT1Y223C / - hESCs an unedited wild - type hESC clone ( WT / WT ) isolated in parallel , as well as a POT1 WT / Q623H hESC a contemporaneously isolated hECS clones ( WT / WT ) . Scale bars = 10µm . Box whisker plot ( showing min , max average ) quantifying telomere - dysfunction induced foci ( TIF ) in two POT1 Y223C / - hESC lines , one POT1 Q623H / Q623H , one POT1WT / Q623H hESC lines compared to wild - type unedited control hESCs . were analyzed 3 months post targeting . were stained for TRF1 γ - H2Ax . POT1Y223C / - have increased TIFs compared to an unedited wild - type clone , isolated cultured in parallel ( P < 0 . 01 , two - Mann - Whitney , one biological replicate ) .

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Representative images for the TIF analysis shown in (C). Shown are POT1Y223C/- hESCs and an unedited wild-type hESC clone (WT/WT) isolated in parallel, as well as a POT1 WT/Q623H hESC and a contemporaneously isolated hECS clones (WT/WT). Scale bars = 10µm.   Box and whisker plot (showing min, max and average) quantifying telomere-dysfunction induced foci (TIF) in two POT1 Y223C/- hESC lines, one POT1 Q623H/Q623H, and one POT1WT/Q623H hESC lines compared to wild-type unedited control hESCs. Cells were analyzed 3 months post targeting. Cells were stained for TRF1 and γ-H2Ax. POT1Y223C/- cells have increased TIFs compared to an unedited wild-type cell clone, isolated and cultured in parallel (P<0.01, two-tailed Mann-Whitney test, one biological replicate).


of telomere centromere FISH on metaphase spreads of lines as in ( B ) . Cy3 Tel - C PNA probes were used . Arrows indicate chromosomal abnormalities . E ) of chromosomal abnormalities as determined by FISH in ( D ) .

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Images of telomere and centromere FISH on metaphase spreads of cell lines as in (B). Cy3 Tel-C PNA probes were used. Arrows indicate chromosomal abnormalities. E) Quantification of chromosomal abnormalities as determined by FISH analysis in (D).


B ) of telomere data from two STELA experiments using same genomic , shown in ( A ) ( Appendix Figure 2C ) . Indicated in red is mean telomere error bars indicate standard deviation .

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B) Quantification of telomere length data from two STELA experiments using the same genomic DNA, shown in (A) and (Appendix Figure 2C). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


B ) TIF of wild - type , POT1WT / Y223C hESCs , indicated controls using antibodies against TRF1 γ - H2AX . Scale bars = 10µm . Lentiviral expression of TPP1 ∆ PBD served as a positive control to demonstrate that have an intact telomere damage response . TPP1 ∆ PBD infected were analyzed 72h post infection without selection .

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B) TIF analysis of wild-type, POT1WT/Y223C hESCs, and the indicated controls using antibodies against TRF1 and γ-H2AX. Scale bars = 10µm. Lentiviral expression of TPP1∆PBD served as a positive control to demonstrate that cells have an intact telomere DNA damage response. TPP1∆PBD infected cells were analyzed 72h post infection and without selection.


E ) of two STELA experiments , including that shown in ( D ) . Indicated in red is mean telomere error bars indicate standard deviation .

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E) Quantification of two STELA experiments, including that shown in (D). Indicated in red is the mean telomere length and the error bars indicate the standard deviation.


D ) of edited or unedited colonies isolated in in vitro differentiation described in ( C ) WT : wild - type , MT : mutant .

D) Quantification of edited or unedited colonies isolated in the in vitro differentiation described in (C) WT: wild-type, MT: mutant.


C ) Representative genotyping outcomes of hCD34 + nucleofected sgRNA - 1 , xenografted isolated from bone marrow 16 after transplantation into mice determined by NGS . Data information : vertical dashed shows site of CAS9 cut base pairs noted in red show insertion . A dash inside a gray box indicates a deleted base pair .

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C) Representative genotyping outcomes of hCD34+ cells nucleofected sgRNA-1, xenografted and isolated from the bone marrow 16 weeks after transplantation into mice determined by NGS. Data information: The vertical dashed line shows the site of CAS9 cut and the base pairs noted in red show insertion. A dash inside a gray box indicates a deleted base pair.


B ) Telomere restriction fragment for five subjects shown in ( A ) . Mean telomere is indicated .

B) Telomere restriction fragment assay for the five subjects shown in (A). Mean telomere length is indicated.


( A ) Histogram ( mean , 95 % CI ) of fold induction of PXR activity reporter ( luc , luciferase ) in LS180 transiently transfected with wild - type PXR p3A4 - luc reporter plasmids . Chemical structures of FKK5 , FKK6 FKK999 compounds are overlaid . bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc . * significant over vehicle ( DMSO ) control .

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

(A) Histogram (mean, 95 %CI) of fold induction of PXR activity reporter assay (luc, luciferase) in LS180 cells transiently transfected with wild-type PXR and p3A4-luc reporter plasmids. Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.


( B ) , same as ( A ) , in HepG2 stably expressing pCYP1A1 luciferase plasmid ( AhR reporter ) . Chemical structures of FKK5 , FKK6 FKK999 compounds are overlaid . Indole is colored blue . bar graph ( s ) depicts one representative experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of . * p < 0 . 05 , one - way ANOVA with Dunnett ' s post hoc . * significant over vehicle ( DMSO ) control .

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(B), same as (A), in HepG2 cells stably expressing pCYP1A1 luciferase plasmid (AhR reporter). Chemical structures of FKK5, FKK6 and FKK999 compounds are overlaid. Indole is colored blue. The bar graph(s) depicts one representative experiment of a series of experiments (n > 3) performed in four consecutive passages of cells. *p < 0.05, one-way ANOVA with Dunnett's post hoc test. *significant over vehicle (DMSO) control.


C , Histogram ( mean , 95 % CI ) of fold mRNA expression , CYP1A1 ( top panel ) , CYP3A4 ( middle panel ) MDR1 ( bottom panel ) in LS180 with or without ( mock ) transfected PXR plasmid , HepaRG hepatic progenitor ( PXR - knockout , PXR - KO ; AhR - knockout , AhR - KO ; parental control 5F clone ) human hepatocytes ( HEP ) from four donors is shown . bar graph represents one experiment of a series of experiments ( n > 3 ) performed in four consecutive passages of LS180 ; n = 2 independent experiments with one well / compound RT - PCR performed in triplicate for each HepaRG genotype ; for each donor hepatocyte , n = 1 well / compound RT - PCR performed in triplicate . * , # p < 0 . 05 , two - way ANOVA with Tukey ' s post hoc . * significant over vehicle control . # significant over same treatment in corresponding mock transfected or knock - out .

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

C, Histogram (mean, 95 %CI) of fold mRNA expression, CYP1A1 (top panel), CYP3A4 (middle panel) and MDR1 (bottom panel) in LS180 cells with or without (mock) transfected PXR plasmid, HepaRG hepatic progenitor cells (PXR-knockout, PXR-KO; AhR-knockout, AhR-KO; parental control 5F clone) and primary human hepatocytes (HEP) from four donors is shown. The bar graph represents one experiment of a series of experiments (n > 3) performed in four consecutive passages of LS180 cells; n = 2 independent experiments with one well/compound and RT-PCR performed in triplicate for each HepaRG genotype; for each donor hepatocyte, n = 1 well/compound and RT-PCR performed in triplicate. *, #p < 0.05, two-way ANOVA with Tukey's post hoc test. *significant over vehicle control. #significant over the same treatment in corresponding mock transfected or knock-out cells.


D , Immunoprecipitation ( ChIP ) in LS174T . Top panel , PCR products exposed to vehicle or FKK6 run on a 2 % agarose gel . 100 base pair marker ; Vehicle , 10 % DMSO ; FKK6 ( 10 μM ) ; 1 / 10 Input - 0 . 2 million before IP ; IgG - IP with polyclonal rabbit IgG ; PXR - IP with PXR antibody . Bottom panel , quantitative PCR from ChIP for compounds tested with gene specific PCR amplicon normalized to GAPDH ( fold expression ) . Dash , 2 - fold expression . data is one representative experiment of two independent experiments ( each n = 3 biological replicates , n = 4 technical replicates ) . Mean fold expression is expressed above each histogram ( mean + SD ) .

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

D, Chromatin Immunoprecipitation (ChIP) assay in LS174T cells. Top panel, PCR products cells exposed to vehicle or FKK6 and run on a 2% agarose gel. DNA 100 base pair marker; Vehicle,10% DMSO; FKK6 (10 μM); 1/10 Input - 0.2 million cells before IP; IgG - IP with polyclonal rabbit IgG; PXR - IP with PXR antibody. Bottom panel, quantitative PCR from the ChIP assay for compounds tested with the gene specific PCR amplicon normalized to GAPDH (fold expression). Dash line, 2-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biological replicates, n = 4 technical replicates). Mean fold expression is expressed above each histogram (mean + SD).


E , PXR TR - FRET . TR - FRET 520 / 495 nm are plotted against of compound ( s ) . Half - maximal inhibitory concentrations IC50 were obtained from interpolated standard curves ( sigmoidal , 4PL , variable slope ) ; error bars show standard deviation of n = 2 independent experiments each with four technical replicates .

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E, PXR TR-FRET assay. TR-FRET ratio 520/495 nm are plotted against concentration of compound(s). Half-maximal inhibitory concentrations IC50 were obtained from interpolated standard curves (sigmoidal, 4PL, variable slope); error bars show standard deviation of n = 2 independent experiments each with four technical replicates.


F , Microcalorimetric titrations of PXR ligand binding domain ( LBD ) with FKK5 FKK6 . A single - site binding model was used for fitting data . upper panel shows output signal , dQ / dt , as a function of time . middle panel shows integrated heats as a function of ligand / PXR molar in . solid represents best non - linear least - squares fit of data . lower panel shows respective thermodynamic signatures of binding to PXR ligand binding domain .

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F, Microcalorimetric titrations of PXR ligand binding domain (LBD) with FKK5 and FKK6. A single-site binding model was used for fitting the data. The upper panel shows the output signal, dQ/dt, as a function of time. The middle panel shows the integrated heats as a function of the ligand/PXR molar ratio in the cell. The solid line represents the best non-linear least-squares fit of the data. The lower panel shows respective thermodynamic signatures of binding to the PXR ligand binding domain.


A , PXR ( luciferase ) reporter in HEK293T transiently transfected with PXR plasmids ( wild - type ligand binding domain mutant C285I / C301A ) . RLU , relative units are shown normalized to beta - galactosidase ( β - Gal ) expression . histogram represents one experimental data ( of n > 2 independent experiments each performed in quadruplicate ) mean ( 95 % CI ) ; n . s . not significant ; * p < 0 . 05 , two - way ANOVA .

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

A, PXR (luciferase) reporter assay in HEK293T cells transiently transfected with PXR plasmids (wild-type and ligand binding domain mutant C285I/C301A). RLU, relative light units are shown normalized to beta-galactosidase (β-Gal) expression. The histogram represents one experimental data (of n > 2 independent experiments each performed in quadruplicate) mean (95 %CI); n.s. not significant; * p < 0.05, two-way ANOVA.


B , Histogram represents fold change in mRNA expression , normalized to GAPDH , by RT - qPCR from Caco - 2 exposed to compounds . DMSO , 0 . 1 % DMSO vehicle ; 9 . 3 is 9 . 3 - fold expression . data is one representative experiment of two independent experiments ( each n = 3 biologic replicates , n = 4 technical replicates ) .

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B, Histogram represents fold change in mRNA expression, normalized to GAPDH, by RT-qPCR from Caco-2 cells exposed to compounds. DMSO, 0.1% DMSO vehicle; 9.3 is 9.3-fold expression. The data is one representative experiment of two independent experiments (each n = 3 biologic replicates, n = 4 technical replicates).


Points error represent mean ( 95 % CI ) values for control ( TNFα 5 ng / ml ) normalized NF - κB ( luciferase ) reporter activity in LS180 after C , Passage 13 ( P13 ) D , Passage 8 ( P8 ) C , D , graph depicts one representative experiment of a series of experiments ( n > 4 ) performed in four consecutive passages of . Data was expressed as mean with 95 % CI of four technical replicates .

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Points and error represent mean (95 %CI) values for control (TNFα 5 ng/ml) normalized NF-κB (luciferase) reporter activity in LS180 cells after C, Passage 13 (P13) and D, Passage 8 (P8) and C, D, Line graph depicts one representative experiment of a series of experiments (n > 4) performed in four consecutive passages of cells. Data was expressed as mean with 95% CI of four technical replicates.


E , LS174T PXR - KO ( knockout ) transiently transfected with pNL3 . 2 . NF - κB - RE vector . mean ( + SD ) . * , * * p < 0 . 05 , two way ANOVA with Tukey ' s multiple comparisons .

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E, LS174T PXR-KO (knockout) cells transiently transfected with pNL3.2.NF-κB-RE vector. mean (+ SD). *, ** p < 0.05, two way ANOVA with Tukey's multiple comparisons test.


A In Caco - 2 monolayer , Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , cytokine cocktail plus FKK drugs ( 10 25 μM ) with 2 h or 12 h incubations .

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A In Caco-2 monolayer cells, Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations.


B , Salmonella invasion in colony forming units per ml ( CFU / ml ) at different time periods of incubation .

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B, Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation.


C , confocal of NFĸB ( red ) nucleus ( blue ) . Scale bars = 80 µm . Arrows represent localization of NFĸB ( red , ; purple , ) . Confocal shown are representative from ( n = 3 ) biological replicates .

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C, confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. Arrows represent localization of NFĸB (red, cytoplasmic; purple, nuclear). Confocal images shown are representative images from (n = 3) biological replicates.


Log10 relative expression of IL8 mRNA with or without cytokines ( 50 ng / ml IL - 1 , 10 ng / ml IFNγ , 10 ng / ml TNF α ) , cytokine cocktail plus FKK drugs ( 10 25 μM ) with 2 h or 12 h incubations . In human intestinal organoids ( HIO ) ,

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

Log10 relative expression of IL8 mRNA with or without cytokines (50 ng/ml IL-1, 10 ng/ml IFNγ, 10 ng/ml TNF α), and cytokine cocktail plus FKK drugs (10 and 25 μM) with 2 h or 12 h incubations. In human intestinal organoids (HIO),


Salmonella invasion in colony forming units per ml ( CFU / ml ) at different time periods of incubation . In human intestinal organoids ( HIO ) ,

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Salmonella invasion in colony forming units per ml (CFU/ml) at the different time periods of incubation. In human intestinal organoids (HIO),


confocal of NFĸB ( red ) nucleus ( blue ) . Scale bars = 80 µm . n human intestinal organoids ( HIO ) ,

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confocal images of NFĸB (red) and the nucleus (blue). Scale bars = 80 µm. n human intestinal organoids (HIO),


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK5 ( n = 9 paired samples ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc .

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

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK5 (n = 9 paired samples). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK6 ( n = 3 paired samples ; one removed due to poor inflammatory response ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc .

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

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK6 (n = 3 paired samples; one removed due to poor inflammatory response). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Quantitative gene expression by RT - qPCR of IL8 mRNA from human colonic enteroids ( over 40 enteroids per sample per well ) exposed to control ( vehicle ) or TNF α . with or without FKK9 ( n = 5 ; 3 removed due to poor response tissue integrity ) . Data shown as mean fold change relative to vehicle control ( con ) for each paired sample ( n = 3 replicates ) . Each paired sample data set represents biopsies from an . * , # # p < 0 . 05 , Pair - wise one way ANOVA with Tukey ' s post hoc .

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

Quantitative gene expression by RT-qPCR of IL8 mRNA from human colonic enteroids (over 40 enteroids per individual sample per well) exposed to control (vehicle) or TNF α. with or without FKK9 (n = 5; 3 removed due to poor response and tissue integrity). Data shown as mean fold change relative to vehicle control (con) for each paired sample (n = 3 replicates). Each paired sample data set represents biopsies from an individual patient. *, ## p < 0.05, Pair-wise one way ANOVA with Tukey's post hoc test.


Fold induction ( mRNA ) of genes in C57BL / 6 mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . entire experiment was repeated two independent times one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays normalized to internal control , GAPDH . histograms show mean ( 95 % CI ) values for gene expression . dotted marks basal fold

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Fold induction (mRNA) of genes in C57BL/6 mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression


Fold induction ( mRNA ) of genes in pxr - / - mice gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . entire experiment was repeated two independent times one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays normalized to internal control , GAPDH . histograms show mean ( 95 % CI ) values for gene expression . dotted marks basal fold

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Fold induction (mRNA) of genes in pxr -/- mice gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks basal fold expression


Fold induction ( mRNA ) of genes in hPXR mice ( mice expressing human PXR gene ) gavaged with vehicle ( 10 % DMSO ; n = 3 ) or FKK6 ( 500 μM in 10 % DMSO ; n = 3 ) every 12h for 3 total doses . entire experiment was repeated two independent times one representative experiment is shown . Each mouse ( each organ ) was studied in quadruplicate assays normalized to internal control , GAPDH . histograms show mean ( 95 % CI ) values for gene expression . dotted marks 2 - fold gene induction .

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Fold induction (mRNA) of genes in hPXR mice (mice expressing the human PXR gene) gavaged with vehicle (10% DMSO; n = 3) or FKK6 (500 μM in 10% DMSO; n = 3) every 12h for 3 total doses. The entire experiment was repeated two independent times and one representative experiment is shown. Each mouse (each organ) was studied in quadruplicate assays and normalized to internal control, GAPDH. The histograms show mean (95% CI) values for gene expression. The dotted line marks 2-fold gene induction.


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . A , % loss from baseline ( day 1 vs day 10 )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. A, % weight loss from baseline (day 1 vs day 10)


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . B , colon ( cm )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. B, colon length (cm)


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . C , Inflammation

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. C, Inflammation score


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . D , Fecal lipocalin 2 ( pg / ml )

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. D, Fecal lipocalin 2 (pg/ml)


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . E , serum FITC - dextran ( μg / ml ) .

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. E, serum FITC-dextran (μg/ml).


After coin - toss randomization , hPXR mice ( mice expressing human PXR gene ) were allocated to treatment with vehicle ( 0 . 8 % DMSO ) ( n = 3 / genotype ) or FKK6 ( 200 micromolar ) ( n = 3 / genotype ) , by simultaneous oral gavage intra - rectal delivery starting day 1 - 10 of DSS administration . F , fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS ( n = 3 / group ; each PCR performed in quadruplicate ) .

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After coin-toss randomization, hPXR mice (mice expressing the human PXR gene) were allocated to treatment with vehicle (0.8% DMSO) (n = 3/genotype) or FKK6 (200 micromolar) (n = 3/genotype), by simultaneous oral gavage and intra-rectal delivery starting day 1 - 10 of DSS administration. F, fold expression of mRNA as illustrated in hPXR mouse colon tissue exposed to DSS (n = 3/group; each PCR performed in quadruplicate).


As in Fig 5 , pxr - / - mice underwent identical experimental procedure . entire experiment was repeated three independent times one representative experiment is shown .

As in Fig 5, pxr -/- mice underwent the identical experimental procedure and analysis. The entire experiment was repeated three independent times and one representative experiment is shown.


( C - E ) Western blots showing increased polyubiquitylation on phagosomes compared to Total Lysate ( TCL ) further increases by IFN - γ activation ( C , E ) . Equal amounts were loaded for phagosomal TCL samples . Vimentin serves as loading control ; EEA1 as purity marker . ( D ) Phagosomal ubiquitylation is not changing substantially over maturation of phagosome ( 30 min , 0 , 30 , 180 min chase , respectively ) . LAMP1 serves as purity marker . Representative of three replicates .

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(C-E) Western blots showing increased polyubiquitylation on phagosomes compared to Total Cell Lysate (TCL) and further increases by IFN-γ activation (C, E). Equal protein amounts were loaded for phagosomal and TCL samples. Vimentin serves as loading control; EEA1 as purity marker. (D) Phagosomal ubiquitylation is not changing substantially over the maturation of the phagosome (30 min pulse, 0, 30, and 180 min chase, respectively). LAMP1 serves as purity marker. Representative images of three replicates.


( H ) Ubiquitin Chain Restriction ( UbiCRest ) experiment removing polyubiquitin chains from phagosomal extracts using specific unspecific DUBs . UT = untreated .

(H) Ubiquitin Chain Restriction (UbiCRest) experiment removing polyubiquitin chains from phagosomal extracts using specific and unspecific DUBs. UT = untreated.


( B ) Western blot of phagosomal TCL extracts showing enrichment of RNF115 on phagosomes of RAW264 . 7 its increased abundance in response to IFN - γ activation . Rab7 serves as purity control , iNOS as activation control vimentin as loading control . Representative image of three replicates . Relative mobilities of reference ( masses in kDa ) are shown on right of each blot .

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(B) Western blot of phagosomal and TCL extracts showing enrichment of RNF115 on phagosomes of RAW264.7 cells and its increased abundance in response to IFN-γ activation. Rab7 serves as purity control, iNOS as activation control and vimentin as loading control. Representative image of three replicates. Relative mobilities of reference proteins (masses in kDa) are shown on the right of each blot.


( A - B ) Intraphagosomal proteolysis acidification assays show that loss of RNF115 increases phagosome maturation in BMA . ( C ) Complementing RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes , shows that RNF115 ligase activity is required for increase of phagosome maturation .

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(A-B) Intraphagosomal proteolysis and acidification assays show that loss of RNF115 increases phagosome maturation in BMA cells. (C) Complementing the RNF115 KO with WT RNF115 or a W259A mutant which is unable to bind to E2 enzymes, shows that RNF115 ligase activity is required for the increase of phagosome maturation.


( C ) Immunofluorescence of VAMP8 ( green ) Syntaxin - 7 ( STX7 ; red ) show strong colocation around 3 µm silica bead phagosomes ( ) in BMA macrophages . Nuclei are stained with Dapi . Scale bar is 5 µm . To account for people with red - green colour - blindness , ( D ) Colocation of VAMP8 ( green ) STX7 ( red ) on phagosomes between WT RNF115 KO represented by intersection , Manders ' M1 overlap coefficients . Data is represented as a box whisker plot of total values across biological duplicate experiments , where whiskers represent minimum to maximum values central band indicates median .

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(C) Immunofluorescence micrographs of VAMP8 (green) and Syntaxin-7 (STX7; red) show strong colocation around 3 µm silica bead phagosomes (white) in BMA macrophages. Nuclei are stained with Dapi. Scale bar is 5 µm. To account for people with red-green colour-blindness, (D) Colocation of VAMP8 (green) and STX7 (red) on individual phagosomes between WT and RNF115 KO cells represented by intersection, Manders' M1 and overlap coefficients. Data is represented as a box and whisker plot of total values across biological duplicate experiments, where the whiskers represent the minimum to maximum values and the central band indicates the median.


( D ) Box whisker plot represents of ( % ) of hepatic lesion in livers from six wild - type ( WT ) six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Haematoxylin eosin ( H & E ) - stained liver sections of one representative per genotype are shown on right , dotted depicts damaged .

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(D) Box and whisker plot represents the quantification of the percentage (%) of the hepatic lesion area in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Haematoxylin and eosin (H&E)-stained liver sections of one representative animal per genotype are shown on the right, dotted line depicts the damaged area.


( E - F ) Box whisker plots represent average per high power field ( HPF ) of CD3 positive T - ( E ) , CD68 positive macrophages ( F ) in livers from six wild - type ( WT ) six RNF115 KO ( KO ) mice after 48 h infection with S . aureus . Representative immunohistochemistry sections are shown on right .

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(E-F) Box and whisker plots represent the average cell counts per high power field (HPF) of CD3 positive T-cells (E), and CD68 positive macrophages (F) in the livers from six wild-type (WT) and six RNF115 KO (KO) mice after 48 h infection with S. aureus. Representative immunohistochemistry sections are shown on the right.


C Expression of F9 mRNA in hepatic tissue of indicated mouse strains . Data represent means ± SE . M : Marker ; arrowhead : FIX ; Note : bands of FIX in F9383STOP lanes are faint with small molecular smears .

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C Expression of F9 mRNA and protein in hepatic tissue of indicated mouse strains. Data represent means ± SE. M: Marker; arrowhead: FIX protein; Note: the bands of FIX in F9383STOP lanes are faint with small molecular weight smears.


D Measurement of coagulation activity by aPTT in mice at 8 of . n = 5 for each group , Data represent means ± SE .

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D Measurement of coagulation activity by aPTT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


E Measurement of coagulation activity by PT in mice at 8 of . n = 5 for each group , Data represent means ± SE .

E Measurement of coagulation activity by PT in mice at 8 weeks of age. n=5 for each group, Data represent means ± SE.


F Measurement of blood loss over a 5 - minute period after tail transection in mice at 8 of . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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F Measurement of blood loss over a 5-minute period after tail transection in mice at 8 weeks of age. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


G Survival rate of mice after tail - clip challenge . mice were monitored for two days after tail clipping . Wild - type , n = 5 ; F9Y381S , n = 6 ; F9Y381D , n = 13 ; F9383STOP , n = 5 .

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G Survival rate of mice after the tail-clip challenge. The mice were monitored for two days after tail clipping. Wild-type, n=5; F9Y381S, n=6; F9Y381D, n=13; F9383STOP, n=5.


B of clotting activity by aPTT PT at 8 following hydrodynamic tail vein injection of 120 µg pX458 120 µg donor plasmids ( or 120 µg ssODN ) per mouse .

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B Test of clotting activity by aPTT and PT at 8 weeks following hydrodynamic tail vein injection of 120 µg pX458 and 120 µg donor plasmids (or 120 µg ssODN) per mouse.


C Survival rate of mice after tail - clip challenge . F9Y381D mice were treated with or without Cas9 / sgRNA / donor for 3 months . After tail - clip challenge , survival rate of each group over 2 days was determined . Wild - type , n = 9 ; untreated F9Y381D , n = 13 ; treated F9Y381D , n = 14 .

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C Survival rate of mice after the tail-clip challenge. F9Y381D mice were treated with or without Cas9/sgRNA/donor DNA for 3 months. After tail-clip challenge, the survival rate of each group over 2 days was determined. Wild-type, n=9; untreated F9Y381D, n=13; DNA treated F9Y381D, n=14.


D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing ( Cas9 / donor vector treated group ) or by TA - clone sequencing ( Cas9 / ssODN group ) .

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D Frequency of genetic modification in F9Y381D hepatocytes was determined either by deep sequencing (Cas9/donor vector treated group) or by TA-clone sequencing (Cas9/ssODN group).


F genome editing efficiency of mice is presented .

F The genome editing efficiency of individual mice is presented.


G Plasma aspartate aminotransferase ( AST ) alanine aminotransferase ( ALT ) level was determined in F9Y381D mice 8 after HTV injection ( n = 5 ) .

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G Plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level was determined in F9Y381D mice 8 weeks after HTV injection (n=5).


H mRNA of inflammatory cytokines from liver tissue of naked treated mice were determined by real - time PCR . Data represent means ± SE .

H mRNA levels of inflammatory cytokines from liver tissue of naked DNA treated mice were determined by real-time PCR. Data represent means ± SE.


B In vivo infection efficiency 4 days after Adv injection . Inserts : negative control from untreated mouse liver tissue . Scale bar : 100 μm .

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B In vivo infection efficiency 4 days after Adv injection. Inserts: negative control from untreated mouse liver tissue. Scale bar: 100 μm.


C T7E1 was performed on liver obtained from samples in ( B ) , frequency of Cas9 - induced indels is indicated as ' % ' below lane .

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C T7E1 assay was performed on liver DNA obtained from samples in (B), and the frequency of Cas9-induced indels is indicated as '%' below the lane.


D Measurement of clotting activity by aPTTs PTs 8 after tail vein injection with indicated vectors . Data represent means ± SE .

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D Measurement of clotting activity by aPTTs and PTs 8 weeks after tail vein injection with indicated vectors. Data represent means ± SE.


E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv ( Cas9 + G / T ) . ( Left ) HDR donor used for correction of Y381D mutation , Illumina sequencing reads ( rn ) are presented . Red text indicates correction of F9 mutation , whereas blue text indicates intended synonymous mutations . ( Right ) of indel or HDR corrections obtained through sequencing of 235 TA - clones ( low dose ) or Illumina sequencing ( high dose ) of treated mouseliver tissues .

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E In vivo genome editing efficiency of whole liver tissues from mice treated with Adv(Cas9+G/T). (Left) HDR donor used for correction of Y381D mutation, and Illumina sequencing reads (rn) are presented. Red text indicates the correction of the F9 mutation, whereas blue text indicates the intended synonymous mutations. (Right) The percentage of indel or HDR corrections obtained through sequencing of 235 individual TA-clones (low dose) or Illumina sequencing (high dose) of treated mouseliver tissues.


Immunofluorescence of early pluripotent marker Sall4 in DMSO - R406 - treated on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of early pluripotent marker Sall4 in DMSO- and R406- treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR of Sall4 , Cdh1 , Epcam Esrrb gene expression in MEFs , intermediate on d12 treated with DMSO R406 , R1 ( mESCs ) . n = 3 .

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RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in MEFs, intermediate cells on d12 treated with DMSO and R406, and R1 (mESCs). n = 3.


Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC shSyk . n = 3 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates infected with shNC and shSyk. n = 3. Scale bar, 100 μm.


ciPSCs express key pluripotent markers Oct4 , Sox2 Nanog . Scale bar , 100 μm .

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ciPSCs express key pluripotent markers Oct4, Sox2 and Nanog. Scale bar, 100 μm.


Immunofluorescence of Sall4 in WT Ppp3ca knockdown on d12 . n = 6 . Scale bar , 100 μm . RT - qPCR of pluripotent genes expression in treated with shNC shPpp3ca . n = 3 .

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Immunofluorescence of Sall4 in WT cells and Ppp3ca knockdown cells on d12. n = 6. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shPpp3ca. n = 3.


Immunofluorescence of Sall4 in WT Nfatc1 knockdown on d12 . n = 3 . Scale bar , 100 μm . RT - qPCR of pluripotent genes expression in treated with shNC shNfatc1 . n = 3 .

Immunofluorescence of Sall4 in WT cells and Nfatc1 knockdown cells on d12. n = 3. Scale bar, 100 μm. RT-qPCR analysis of pluripotent genes expression in cells treated with shNC and shNfatc1. n = 3.


RT - qPCR of Gldc , Cbs Tdh expression in R406 - shSyk - treated ( E ) FK506 - , shPpp3ca - shNfatc1 - treated ( F ) on d8 . n = 3 .

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RT-qPCR analysis of Gldc, Cbs and Tdh expression in R406- and shSyk-treated cells (E) and FK506-, shPpp3ca- and shNfatc1-treated cells (F) on d8. n = 3.


Metabolite abundance of SAM , SAH in samples treated with DMSO R406 . n = 6 .

Metabolite abundance of SAM, SAH in samples treated with DMSO and R406. n = 6.


Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA R406 + DPBS on d12 . n = 3 . Scale bar , 100 μm . Immunofluorescence of Sall4 in NAC - DPBS - treated on d12 . n = 5 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in reprogramming intermediates treated by R406 + HA and R406 + DPBS on d12. n = 3. Scale bar, 100 μm. Immunofluorescence of Sall4 in NAC- and DPBS-treated cells on d12. n = 5. Scale bar, 100 μm.


RT - qPCR of Sall4 , Cdh1 , Epcam Esrrb gene expression in DPBS - HA - treated on d12 . n = 3 . RT - qPCR of Sall4 , Cdh1 , Epcam Esrrb gene expression in DPBS - NAC - treated on d12 . n = 3 .

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RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and HA-treated cells on d12. n = 3. RT-qPCR analysis of Sall4, Cdh1, Epcam and Esrrb gene expression in DPBS- and NAC-treated cells on d12. n = 3.


Immunofluorescence of Sall4 in WT Cbs overexpressing on d12 . n = 4 . Scale bar , 100 μm .

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Immunofluorescence of Sall4 in WT cells and Cbs overexpressing cells on d12. n = 4. Scale bar, 100 μm.


of oxygen consumption rate ( OCR ) of reprogramming intermediates treated with small molecules ( A , C ) indicated OCR parameters ( B , D ) . n = 9 .

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Quantification of oxygen consumption rate (OCR) of reprogramming intermediates treated with small molecules (A, C) and the indicated OCR parameters (B, D). n = 9.


of DCFH - DA for ROS in treated with DMSO , HA , NAC , R406 on d12 ( E ) of ROS ( F ) . n = 4 . Scale bar , 100 μm . MDA in treated with DMSO , HA , NAC , R406 on d12 . n = 9 .

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Fluorescence images of DCFH-DA staining for ROS levels in cells treated with DMSO, HA, NAC, and R406 on d12 (E) and the quantification of the ROS levels (F). n = 4. Scale bar, 100 μm. MDA quantification in cells treated with DMSO, HA, NAC, and R406 on d12. n = 9.


( a , b ) Immunoblot of LAMP - 2A in total lysates ( a ) quantitative PCR of Lamp2a mRNA ( b ) of naive CD4 + T TH1 TH2 following activation with anti - CD3 anti - CD28 . β - actin ( a ) serves as a loading control throughout ; mRNA results ( b ) are presented relative to those of resting .

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(a,b) Immunoblot analysis of LAMP-2A in total lysates (a) and quantitative PCR analysis of Lamp2a mRNA (b) of naive CD4+ T cells and TH1 and TH2 cells following activation with anti-CD3 and anti-CD28. β-actin (a) serves as a loading control throughout; mRNA results (b) are presented relative to those of resting cells.


( c ) Immunofluorescence microscopy of LAMP - 2A ( green ) nuclei ( stained with - binding dye DAPI ; blue ) in resting TH1 ( ) TH1 activated for 24 h as in a , b ( Act ) ( left ) , of lysosomes in as at left ( right ) . Original magnification , × 630 ; scale bar , 10 μm . * P = 0 . 013 ( t - ) .

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(c) Immunofluorescence microscopy of LAMP-2A (green) and nuclei (stained with the DNA-binding dye DAPI; blue) in resting TH1 cells (Rest) and TH1 cells activated for 24 h as in a,b (Act) (left), and quantification of lysosomes in cells as at left (right). Original magnification, ×630; scale bar, 10 μm. *P = 0.013 (t-test).


( d ) Electron microscopy ( left ) of resting TH1 TH1 activated for 24 h as in a , b ( top row ) ; below , enlargement of areas outlined above , showing lysosomes ( arrows ) . Original magnification ( top row ) , × 12 , 000 ; scale bars ( bottom row ) , 1 μm . Right , morphometric of as at left . * P = 0 . 037 * * P = 0 . 034 ( t - ) .

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(d) Electron microscopy (left) of resting TH1 cells and TH1 cells activated for 24 h as in a,b (top row); below, enlargement of areas outlined above, showing individual lysosomes (arrows). Original magnification (top row), ×12,000; scale bars (bottom row), 1 μm. Right, morphometric analysis of cells as at left. *P = 0.037 and **P = 0.034 (t-test).


( e ) Distribution of KFERQ - PA - mCherry - 1 in TH1 transiently transfected with a CMA reporter vector , photoactivated , left unstimulated ( ) or stimulated with anti - CD3 anti - CD28 in presence ( 3 - MA ) or absence ( vehicle ( veh ) ) of 3 - methyladenine ( left ) ; arrows indicate mCherry + . Original magnification , × 630 . Right , of results for at left . * P 0 . 05 ( of variance with Tukey ' s post - ) .

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(e) Distribution of KFERQ-PA-mCherry-1 in TH1 cells transiently transfected with a CMA reporter vector, photoactivated, and left unstimulated (Rest) or stimulated with anti-CD3 and anti-CD28 in the presence (3-MA) or absence (vehicle (veh)) of 3-methyladenine (left); arrows indicate mCherry+ puncta. Original magnification, ×630. Right, quantification of the results for the cells at left. *P 0.05 (analysis of variance with Tukey's post-test).


( f ) Immunoblot of hsc70 in lysosomal fractions of resting ( Lys - R ) stimulated ( Lys - A ) TH1 in fractions of TH1 activated as in a , b ( Cyto ) ; LAMP - 2A LAMP - 1 serve as controls .

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(f) Immunoblot analysis of hsc70 in lysosomal fractions of resting (Lys-R) and stimulated (Lys-A) TH1 cells and in cytosolic fractions of TH1 cells activated as in a,b (Cyto); LAMP-2A and LAMP-1 serve as controls.


( g ) Immunoblot of LAMP - 2A in resting TH1 TH1 activated in presence ( NAc ) or absence ( Veh ) of N - acetylcysteine . ( h ) Quantitative PCR of Lamp2a mRNA in as in g ( results presented as in b ) . * P = 0 . 007 ( t ) .

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(g) Immunoblot analysis of LAMP-2A in resting TH1 cells and TH1 cells activated in the presence (NAc) or absence (Veh) of N-acetylcysteine. (h) Quantitative PCR analysis of Lamp2a mRNA in cells as in g (results presented as in b). *P = 0.007 (t test).


( i ) Luciferase activity in TH1 transiently transfected with a vector containing a luciferase reporter under control of Lamp2 proximal promoter stimulated for 0 , 12 or 24 h with plate - bound anti - CD3 anti - CD28 ; results are presented relative to those of resting ( time 0 ) .

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(i) Luciferase activity in TH1 cells transiently transfected with a vector containing a luciferase reporter under the control of the Lamp2 proximal promoter and stimulated for 0, 12 or 24 h with plate-bound anti-CD3 and anti-CD28; results are presented relative to those of resting cells (time 0).


( j ) Luciferase activity in TH1 transiently transfected as in i left unstimulated ( control ( Ctrl ) ) or stimulated for 16 h with 50 μM paraquat ( Pq ) or 1 mM H2O2 ; results are presented in relative units ( RLU ) .

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(j) Luciferase activity in TH1 cells transiently transfected as in i and left unstimulated (control (Ctrl)) or stimulated for 16 h with 50 μM paraquat (Pq) or 1 mM H2O2; results are presented in relative light units (RLU).


( k ) Immunoblot of LAMP - 2A in total lysates of resting TH1 ( − ) TH1 activated for 24 h as in a , b ( + ) , transfected with a control nontargeting siRNA ( with a scrambled ( Scr ) ) or siRNA specific for mRNA encoding Duox1 ( either of two siRNAs : Dx ( 1 ) Dx ( 2 ) ) or RISP ( Uq ( 1 ) ) .

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(k) Immunoblot analysis of LAMP-2A in total lysates of resting TH1 cells (−) and TH1 cells activated for 24 h as in a,b (+), transfected with a control nontargeting siRNA (with a scrambled sequence (Scr)) or siRNA specific for mRNA encoding Duox1 (either of two siRNAs: Dx(1) and Dx(2)) or RISP (Uq(1)).


( l ) Immunoblot of LAMP - 2A in lysates of resting or activated TH1 in presence ( right half ) or absence ( left half ) of cyclosporine A ( CsA ) .

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(l) Immunoblot analysis of LAMP-2A in lysates of resting or activated TH1 cells in the presence (right half) or absence (left half) of cyclosporine A (CsA).


( m ) - immunoprecipitation of recruitment of NFAT1 to promoter of Lamp2 or Adad1 ( negative control ) ( left ) or to Il2 ( positive control ) ( right ) in resting activated TH1 ; results are presented relative to input precipitated with anti - NFAT1 .

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(m) Chromatin-immunoprecipitation analysis of the recruitment of NFAT1 to the promoter of Lamp2 or Adad1 (negative control) (left) or to Il2 (positive control) (right) in resting and activated TH1 cells; results are presented relative to input chromatin precipitated with anti-NFAT1.


( a ) Flow cytometry of T transduced with lentivirus expressing GFP nontargeting control shRNA ( Scr ) or Lamp2a mRNA - specific shRNA ( shL2A ) ( left ) , immunoblot of LAMP - 2A in sorted GFP + T ( right ) . Numbers above bracketed lines ( left ) indicate GFP + .

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(a) Flow cytometry of T cells transduced with lentivirus expressing GFP and nontargeting control shRNA (Scr) or Lamp2a mRNA-specific shRNA (shL2A) (left), and immunoblot analysis of LAMP-2A in sorted GFP+ T cells (right). Numbers above bracketed lines (left) indicate percent GFP+ cells.


( b ) Enzyme - linked immunosorbent of IL - 2 interferon - γ ( IFN - γ ) in TH1 transduced as in a then left unstimulated ( ) or stimulated for 24 h with anti - CD3 anti - CD28 ( Act ) . * P = 0 . 038 * * P = 0 . 031 ( t - ) .

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(b) Enzyme-linked immunosorbent assay of IL-2 and interferon-γ (IFN-γ) in TH1 cells transduced as in a and then left unstimulated (Rest) or stimulated for 24 h with anti-CD3 and anti-CD28 (Act). *P = 0.038 and **P = 0.031 (t-test).


( c ) Proliferation in TH1 transduced as in a left unstimulated or stimulated for 24 h as in b , assessed by incorporation of thymidine analog BrdU presented as absorbance at 495 nm ( A495 ) . * P = 0 . 013 ( t - ) .

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(c) Proliferation in TH1 cells transduced as in a and left unstimulated or stimulated for 24 h as in b, assessed by incorporation of the thymidine analog BrdU and presented as absorbance at 495 nm (A495). *P = 0.013 (t-test).


( d ) of annexin V - positive ( AnnV + ) among resting or stimulated TH1 transduced as in a .

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(d) Quantification of annexin V-positive (AnnV+) cells among resting or stimulated TH1 cells transduced as in a.


( a ) Immunoblot of Itch RCAN1 in lysosomal fractions isolated from resting TH1 TH1 activated for 24 h with anti - CD3 anti - CD28 ; LAMP - 1 serves as a control .

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(a) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions isolated from resting cells TH1 cells and TH1 cells activated for 24 h with anti-CD3 and anti-CD28; LAMP-1 serves as a control.


( b ) Immunoblot of Itch RCAN1 in lysosomal fractions of TH1 activated for 24 h as in a with ( + ) or without ( − ) inhibition of lysosomal proteases during final 3 h with NH4Cl leupeptin ( Lys inh ) ; LAMP - 1 serves as a control .

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(b) Immunoblot analysis of Itch and RCAN1 in lysosomal fractions of TH1 cells activated for 24 h as in a with (+) or without (−) inhibition of lysosomal proteases during the final 3 h with NH4Cl and leupeptin (Lys inh); LAMP-1 serves as a control.


( c ) Immunoblot of Itch RCAN1 in resting TH1 TH1 activated for 24 h as in a in presence ( NAc ) or absence ( Veh ) of N - acetylcysteine .

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(c) Immunoblot analysis of Itch and RCAN1 in resting TH1 cells and TH1 cells activated for 24 h as in a in the presence (NAc) or absence (Veh) of N-acetylcysteine.


( d ) Immunoblot of lysates of TH1 transfected with GFP - Itch or GFP - mu - Itch ( top ) or with Myc - RCAN1 or Myc - mu - RCAN1 ( bottom ) cotransfected with a GFP expression plasmid ( to assess transfection efficiency ) , then activated for 24 h as in a .

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(d) Immunoblot analysis of lysates of TH1 cells transfected with GFP-Itch or GFP-mu-Itch (top) or with Myc-RCAN1 or Myc-mu-RCAN1 (bottom) and cotransfected with a GFP expression plasmid (to assess transfection efficiency), then activated for 24 h as in a.


( e , f ) Proliferation ( e ) IL - 2 production ( f ) of TH1 transfected with plasmid expressing GFP ( Ctrl ) or plasmids as in c left unstimulated ( ) or activated for 48 h ( e ) or 24 h ( f ) as in a ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 019 * * P = 0 . 0034 ( e ) or * P = 0 . 046 * * P = 0 . 044 ( f ) ( t ) . Data are representative of four ( a , b ) , two ( c ) or three ( d ) independent experiments or are from three independent experiments ( e , f ; mean s . e . m . ) .

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(e,f) Proliferation (e) and IL-2 production (f) of TH1 cells transfected with plasmid expressing GFP (Ctrl) or plasmids as in c and left unstimulated (Rest) or activated for 48 h (e) or 24 h (f) as in a (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.019 and **P = 0.0034 (e) or *P = 0.046 and **P = 0.044 (f) (t test). Data are representative of four (a,b), two (c) or three (d) independent experiments or are from three independent experiments (e,f; mean and s.e.m.).


( a ) Immunoblot of LAMP - 2A in CD4 + T ( left ) liver , kidneys , thymus lungs ( right ) of L2A - cKO mice ( KO ) their control ( Ctrl ) .

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(a) Immunoblot analysis of LAMP-2A in CD4+ T cells (left) and liver, kidneys, thymus and lungs (right) of L2A-cKO mice (KO) and their control littermates (Ctrl).


( b ) Quantitative PCR of Lamp2a , Lamp2b Lamp2c mRNA in T from L2A - cKO mice ( right ) ; results are presented relative to those of their control ( left ) , set as 1 .

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(b) Quantitative PCR analysis of Lamp2a, Lamp2b and Lamp2c mRNA in T cells from L2A-cKO mice (right); results are presented relative to those of their control littermates (left), set as 1.


( c ) Immunoblot of autophagy related LC3 in its form ( LC3 - I ) its autophagosome - associated form ( LC3 - II ) in total lysates of CD4 + T obtained from L2A - cKO mice or their control then left unstimulated ( ) or activated for 24 h with anti - CD3 anti - CD218 ( Act ) , cultured for final 4 h in presence ( + ) or absence ( − ) of NH4Cl leupeptin ( NH4Cl + Leup ) .

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(c) Immunoblot analysis of the autophagy related protein LC3 in its cytosolic form (LC3-I) and its autophagosome membrane-associated form (LC3-II) in total lysates of CD4+ T cells obtained from L2A-cKO mice or their control littermates and then left unstimulated (Rest) or activated for 24 h with anti-CD3 and anti-CD218 (Act), cultured for the final 4 h in the presence (+) or absence (−) of NH4Cl and leupeptin (NH4Cl+Leup).


( d ) Spleens from an L2A - cKO mouse its control littermate ( left ) , flow cytometry of peripheral T from such mice ( right ) . Numbers in quadrants ( right ) indicate CD4 − CD8 + T ( top left ) or CD4 + CD8 − T ( bottom right ) .

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(d) Spleens from an L2A-cKO mouse and its control littermate (left), and flow cytometry of peripheral T cells from such mice (right). Numbers in quadrants (right) indicate percent CD4−CD8+ T cells (top left) or CD4+CD8− T cells (bottom right).


( e , f ) Proliferation ( e ) IL - 2 production ( f ) by CD4 + T isolated from spleen of L2A - cKO mice their control stimulated ex vivo for 48 h with anti - CD3 anti - CD28 ( assessed as in Fig . 2c ( e ) or Fig . 2b ( f ) ) . * P = 0 . 0062 ( e ) or 0 . 006 ( f ) ( t - ) .

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(e,f) Proliferation (e) and IL-2 production (f) by CD4+ T cells isolated from the spleen of L2A-cKO mice and their control littermates and stimulated ex vivo for 48 h with anti-CD3 and anti-CD28 (assessed as in Fig. 2c (e) or Fig. 2b (f)). *P = 0.0062 (e) or 0.006 (f) (t-test).


( g ) Immunoblot of LAMP - 2A , Itch RCAN1 in total lysates of CD4 + T obtained from L2A - cKO mice ( n = 3 ( KO1 , KO2 KO3 ) ) their control ( n = 3 ) activated for 24 h as in e , f . * , nonspecific band .

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(g) Immunoblot analysis of LAMP-2A, Itch and RCAN1 in total lysates of CD4+ T cells obtained from L2A-cKO mice (n = 3 (KO1, KO2 and KO3)) and their control littermates (n = 3) and activated for 24 h as in e,f. *, nonspecific band.


( h ) Quantitative PCR of Rcan1 Itch mRNA in CD4 + T isolated from L2A - cKO mice their control activated for 24 h as in e , f ; results are presented relative to those of resting .

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(h) Quantitative PCR analysis of Rcan1 and Itch mRNA in CD4+ T cells isolated from L2A-cKO mice and their control littermates and activated for 24 h as in e,f; results are presented relative to those of resting cells.


( i , j ) Recall responses to OVA ( 323 - 339 ) in draining lymph nodes isolated from L2A - cKO mice their control after immunization with OVA ( 323 - 339 ) , assessed as proliferation ( i ) IL - 2 production ( j ) ( as in e , f ) . * P = 0 . 0054 ( i ) or 0 . 0052 ( j ) ( t - ) .

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(i,j) Recall responses to OVA(323-339) in draining lymph nodes isolated from L2A-cKO mice and their control littermates after immunization with OVA(323-339), assessed as proliferation (i) and IL-2 production (j) (as in e,f). *P = 0.0054 (i) or 0.0052 (j) (t-test).


( k , l ) Recall responses to L . monocytogenes in CD4 + T isolated from L2A - cKO mice their control ( n = 6 per group ) previously uninfected ( UI ) or previously infected with L . monocytogenes ( Inf ) , assessed as IFN - γ production ( k ) proliferation ( l ) ( as in e , f ) . * P = 0 . 032 ( k ) or 0 . 02 ( l ) ( t - ) .

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(k,l) Recall responses to L. monocytogenes in CD4+ T cells isolated from L2A-cKO mice and their control littermates (n = 6 per group) previously uninfected (UI) or previously infected with L. monocytogenes (Inf), assessed as IFN-γ production (k) and proliferation (l) (as in e,f). *P = 0.032 (k) or 0.02 (l) (t-test).


( m ) L . monocytogenes in spleen liver of L2A - cKO mice their control ( n = 3 per genotype ) immunized with L . monocytogenes then reinfected with a high dose of L . monocytogenes . CFU , colony - forming units . * P = 0 . 008 * * P = 0 . 001 ( t - ) .

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(m) L. monocytogenes in spleen and liver of L2A-cKO mice and their control littermates (n = 3 per genotype) immunized with L. monocytogenes and then reinfected with a high dose of L. monocytogenes. CFU, colony-forming units. *P = 0.008 and **P = 0.001 (t-test).


( a ) Immunoblot of LAMP - 2A in CD4 + T obtained from 4 - month - old or 22 - month old mice , then left resting or stimulated for 24 h with anti - CD3 plus anti - CD28 .

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(a) Immunoblot analysis of LAMP-2A in CD4+ T cells obtained from 4-month-old or 22-month old mice, then left resting or stimulated for 24 h with anti-CD3 plus anti-CD28.


( b ) Immunoblot of RCAN1 Itch in CD4 + T obtained from 4 - month - old or 22 - month old mice ( n = 3 per group ) , then stimulated for 24 h as in a ( left ) , of in 22 - month old mice , presented relative to that of 4 - month - old mice , set as 100 % . * P = 0 . 044 ( t - ) .

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(b) Immunoblot analysis of RCAN1 and Itch in CD4+ T cells obtained from 4-month-old or 22-month old mice (n = 3 per group), then stimulated for 24 h as in a (left), and quantification of protein in 22-month old mice, presented relative to that of 4-month-old mice, set as 100%. *P = 0.044 (t-test).


( c ) Immunoblot of LAMP - 2A in total lysates of naive memory CD4 + T left resting or activated for 24 h as in a .

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(c) Immunoblot analysis of LAMP-2A in total lysates of naive and memory CD4+ T cells left resting or activated for 24 h as in a.


( d ) Immunoblot ( bottom ) of LAMP - 2A in total lysates of naive memory CD4 + T obtained from 4 - or 22 - month - old mice left resting ( R ) or activated for 24 h as in a ( A ) ; above , from results below ( n = 3 mice per group ) . * P = 0 . 009 ( t - ) .

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(d) Immunoblot analysis (bottom) of LAMP-2A in total lysates of naive and memory CD4+ T cells obtained from 4- or 22-month-old mice and left resting (R) or activated for 24 h as in a (A); above, quantification from results below (n = 3 mice per group). *P = 0.009 (t-test).


( e ) Electrophoretic mobility - shift of binding of NFAT ( N ) free probe ( FP ) in extracts of CD4 + T isolated from 3 - , 12 - or 22 - month - old mice activated for 4 h as in a .

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(e) Electrophoretic mobility-shift assay of the binding of NFAT (N) and free probe (FP) in nuclear extracts of CD4+ T cells isolated from 3-, 12- or 22-month-old mice and activated for 4 h as in a.


( f ) ROS production in CD4 + T isolated from 4 - or 22 - month - old mice ( n = 3 per group ) left resting or activated for 12 h as in a , assessed by flow cytometry presented as mean ( MFI ) of a fluorogenic probe for oxidative stress ( CellROX Green ) .

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(f) ROS production in CD4+ T cells isolated from 4- or 22-month-old mice (n = 3 per group) and left resting or activated for 12 h as in a, assessed by flow cytometry and presented as the mean fluorescence intensity (MFI) of a fluorogenic probe for oxidative stress (CellROX Green).


( g - i ) Immunoblot of LAMP - 2A ( g ) of proliferation ( h ) IL - 2 production ( i ) of TH1 obtained from 4 - or 22 - month - old mice transduced with retrovirus expressing GFP alone ( RV - GFP ) or human LAMP - 2A GFP ( RV - LAMP2A ) , sorted by GFP expression , cultured for 2 then stimulated for 24 - 48 h as in a ( assessed as in Fig . 2c ( h ) or Fig . 2b ( i ) ) . * P 0 . 05 ( of variance with Tukey ' s post - ) .

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(g-i) Immunoblot analysis of LAMP-2A (g) and analysis of the proliferation (h) and IL-2 production (i) of TH1 cells obtained from 4- or 22-month-old mice and transduced with retrovirus expressing GFP alone (RV-GFP) or human LAMP-2A and GFP (RV-LAMP2A), sorted by GFP expression, cultured for 2 weeks and then stimulated for 24-48 h as in a (assessed as in Fig. 2c (h) or Fig. 2b (i)). *P 0.05 (analysis of variance with Tukey's post-test).


( j ) Immunoblot of LAMP - 2A in total lysates of CD4 + T obtained from two human donors ( A B ) left resting or activated for 24 or 48 h as in a ( above lanes ) .

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(j) Immunoblot analysis of LAMP-2A in total lysates of CD4+ T cells obtained from two human donors (A and B) and left resting or activated for 24 or 48 h as in a (above lanes).


( k ) Real - time PCR of LAMP2A mRNA in naive memory CD4 + T obtained from young human donors ( 26 ± 2 years of ; n = 3 ) or old human donors ( 72 ± 6 years of ; n = 6 ) activated for 24 h as in a ; results were calculated by change - in - cycle - threshold method ( 2 − ΔCt ) relative to those of gene encoding β - actin . * P = 0 . 047 * * P = 0 . 0033 ( Mann - Whitney ) .

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(k) Real-time PCR analysis of LAMP2A mRNA in naive and memory CD4+ T cells obtained from young human donors (26 ± 2 years of age; n = 3) or old human donors (72 ± 6 years of age; n = 6) and activated for 24 h as in a; results were calculated by the change-in-cycle-threshold method (2−ΔCt) relative to those of the gene encoding β-actin. *P = 0.047 and **P = 0.0033 (Mann-Whitney).


( B ) degree of polymerisation of Z α1 - antitrypsin after 72 hours at 37 ° C , as determined by an end - point immunoassay using 2C1 monoclonal antibody , in varying concentrations of compound ( shown in panel A ) . Modification of phenyl indole heterocycle of GSK425 ( pIC50 6 . 5 ) resulted in an ~ 100 fold increase in potency discovery of 2 - oxindole GSK716 ( pIC50 8 . 3 ) . Data presented as mean ± SD , n = 2 ( GSK425 ) n = 25 ( GSK716 ) .

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(B) The degree of polymerisation of Z α1-antitrypsin after 72 hours at 37°C, as determined by an end-point immunoassay using the 2C1 monoclonal antibody, in varying concentrations of compound (shown in panel A). Modification of the phenyl and indole heterocycle of GSK425 (pIC50 6.5) resulted in an ~100 fold increase in potency and the discovery of the 2-oxindole GSK716 (pIC50 8.3). Data presented as mean ± SD, n = 2 (GSK425) and n = 25 (GSK716).