Immunofluorescence images of cGAS in the nucleus ( DAPI ) and cytosol ( phalloidin ) in BMDMos cultured with / without Aphidicolin ( Aphi ) ( C ) . Scale bar : 50 μm . Right : Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells .

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Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.


Immunoblot estimation of cGAS in nuclear / cytosolic subcellular fraction of BMDMos cultured under indicated conditions . Lamin B and α - Tubulin are nuclear and cytosolic markers respectively .

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Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively.


Flow cytometric analysis of cell cycle of BMDMos


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 cells before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . Scale bar : 10 μm ( A ) .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A).


Micronuclei ( indicated by arrow head ) in GFP - NLS - or GFP - hcGAS expressing HEK293 cells before ( 0 h ) or 24 h after γ - irradiation ( IR ; 10 Gy ) . The average MNs / cell . Graphs show Mean ± SEM ( n = 3 independent experiments ) representing six different microscopic fields with over 200 cells .

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Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells.


IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA . Mean ± SEM of n = 3 independent experiments .

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IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments.


Micronuclei ( indicated by arrow head ) and cGAS staining in WT and cGAS - / - BMDMos exposed to γ - irradiation ( 10 Gy ) . Scale bar : 10 μm .

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Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm.


Average MNs / cell in BMDMos . MN graphs show Mean ± SEM ( n = 3 independent experiments ) representing eight different microscopic fields with over 200 cells .

Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells.


Cell death in WT and cGAS - / - BMDMos that were first synchronized at G2 / M , then γ - irradiated ( 10 Gy ) followed by release and analysis at indicated time points . Mean ± SD , x biological triplicates ( n = 3 ) per treatment group are shown .

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Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.


BMDMos from cGAS - / - mice exhibit enhanced DNA repair efficiency than those from WT and Sting - / - mice . ( A ) Representative comet tails of WT , cGAS - / - and Sting - / - BMDMos exposed to γ - irradiation ( IR : 10 Gy ) on ice then incubated at 37 ̊ C for indicated duration . ( B ) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments .

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BMDMos from cGAS-/- mice exhibit enhanced DNA repair efficiency than those from WT and Sting-/- mice. (A) Representative comet tails of WT, cGAS-/- and Sting-/-BMDMos exposed to γ-irradiation (IR: 10 Gy) on ice then incubated at 37 ̊C for indicated duration. (B) Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of 3 independent experiments.


cGAS promotes micronuclei generation in BMDMos independently of STING . Confocal microscopic visualization of micronuclei ( indicated by arrow head ) and cGAS staining in WT , cGAS - / - and Sting - / - BMDMos exposed to γ - irradiation ( 10Gy ) . Scale bar : 10 μm .

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cGAS promotes micronuclei generation in BMDMos independently of STING. Confocal microscopic visualization of micronuclei (indicated by arrow head) and cGAS staining in WT, cGAS-/- and Sting-/- BMDMos exposed to γ-irradiation (10Gy). Scale bar: 10 μm.


cGAS promotes micronuclei generation in BMDMos independently of STING . Average MNs / cell in corresponding representative images . Bar graphs show mean values from eight different microscopic fields with over 200 cells .

cGAS promotes micronuclei generation in BMDMos independently of STING. Average MNs/cell in corresponding representative images. Bar graphs show mean values from eight different microscopic fields with over 200 cells.


γ - irradiation - induced cell death in WT , cGAS - / -

γ-irradiation-induced cell death in WT, cGAS-/- BMDMos


γ - irradiation - induced cell death in Sting - / - and cGAS - / - Sting - / - BMDMos ( F ) .

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γ-irradiation-induced cell death in Sting-/- and cGAS-/- Sting-/- BMDMos (F).


Kinetics of in vivo depletion of indicated bone marrow cells in WT mice ( n = 3 ) after γ - irradiation ( 9 Gy ) .

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Kinetics of in vivo depletion of indicated bone marrow cells in WT mice (n=3) after γ-irradiation (9 Gy).


Bone marrow cells in WT ( n = 3 ) and cGAS - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in WT (n=3) and cGAS-/- (n=3) mice 10 hours post γ-irradiation.


Bone marrow cells in Sting - / - ( n = 3 ) and cGAS - / - Sting - / - ( n = 3 ) mice 10 hours post γ - irradiation .

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Bone marrow cells in Sting-/- (n=3) and cGAS-/- Sting-/- (n=3) mice 10 hours post γ-irradiation.


Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells . Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1 . 2 .

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Obtained results showing enhanced HR efficiency upon knockdown of endogenous cGAS in U2OS cells. Immunoblot inserts in depict knockdown efficiency of cGAS and Histone H1.2.


Results showing the effect of hcGAS , hcGAS △ cGAMP , hcGAS △ DNA or hcGAS △ Oligo on HR in HEK293 cells . Corresponding immunoblot inserts depict cGAS expression .

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Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on HR in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


Results showing the effect of hcGAS , hcGAS △ cGAMP , hcGAS △ DNA or hcGAS △ Oligo on NHEJ in HEK293 cells . Corresponding immunoblot inserts depict cGAS expression .

Results showing the effect of hcGAS, hcGAS△cGAMP, hcGAS△DNA or hcGAS△Oligo on NHEJ in HEK293 cells. Corresponding immunoblot inserts depict cGAS expression.


cGAS is not recruited to DSB sites : Confocal microscopic images of GFP - NLS - or GFP - hcGAS - expressing U2OS - DSB reporter cells incubated ( or not ) with Shield - 1 and 4 - OHT to induce the expression and translocation of mCherry - LacI - FokI ( red ) to specific DSB sites . Scale bar : 10μm . The arrowheads indicate DSB sites .

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cGAS is not recruited to DSB sites: Confocal microscopic images of GFP-NLS- or GFP-hcGAS-expressing U2OS-DSB reporter cells incubated (or not) with Shield-1 and 4-OHT to induce the expression and translocation of mCherry-LacI-FokI (red) to specific DSB sites. Scale bar: 10μm. The arrowheads indicate DSB sites.


cGAS does not colocalize with γ - H2AX at DSB sites : GFP - NLS - or GFP - hcGAS - expressing HEK293 cells exposed ( or not ) to γ - irradiation ( IR : 10 Gy ) then stained for γ - H2AX . Scale bar : 10 μm .

cGAS does not colocalize with γ-H2AX at DSB sites: GFP-NLS- or GFP-hcGAS- expressing HEK293 cells exposed (or not) to γ-irradiation (IR: 10 Gy) then stained for γ-H2AX. Scale bar: 10 μm.


Nuclear cGAS is mainly chromatin - bound and remains unaltered upon γ - irradiation . Cytosolic ( cyto ) and nuclear fractions of γ - irradiated ( 10 Gy , 30 min ) BMDMos analyzed for cGAS and indicated molecules .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic (cyto) and nuclear fractions of γ-irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules.


Nuclear cGAS is mainly chromatin - bound and remains unaltered upon γ - irradiation . Cytosolic , soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins .

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Nuclear cGAS is mainly chromatin-bound and remains unaltered upon γ-irradiation. Cytosolic, soluble nuclear and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins.


cGAS co - isolates with DNA repair proteins because of bound chromatin bridges . Nuclease digestion abrogates the co - isolation of cGAS and DNA repair proteins : Lysates of control ( - IR ) and γ - irradiated ( + IR , 10 Gy , 30 min ) GFP - hcGAS expressing HEK293 cells were treated ( or not ) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins . Agarose gel analysis of DNA in corresponding cell lysates in ( E ) .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Nuclease digestion abrogates the co-isolation of cGAS and DNA repair proteins: Lysates of control (-IR) and γ-irradiated (+IR, 10 Gy, 30 min) GFP-hcGAS expressing HEK293 cells were treated (or not) with Benzonase before cGAS immunoprecipitation and analysis for indicated proteins. Agarose gel analysis of DNA in corresponding cell lysates in (E).


cGAS co - isolates with DNA repair proteins because of bound chromatin bridges . Co - isolation of cGAS and DNA repair proteins depends on its binding to DNA : cGAS pulldowns along with lysate inputs of control and γ - irradiated HEK293 cells expressing GFP - hcGAS or GFP - hcGASΔDNA probed for indicated proteins .

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cGAS co-isolates with DNA repair proteins because of bound chromatin bridges. Co-isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ-irradiated HEK293 cells expressing GFP-hcGAS or GFP-hcGASΔDNA probed for indicated proteins.


Confocal images of γ - irradiated GFP - NLS - , GFP - hcGAS - expressing HEK293 cells stained for RAD51 ( red ) with or without γ - irradiation . Scale bar : 10 μm .

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Confocal images of γ-irradiated GFP-NLS-, GFP-hcGAS-expressing HEK293 cells stained for RAD51 (red) with or without γ-irradiation. Scale bar: 10 μm.


Pre - incubation of dsDNA with mcGAS - cat prevents D - loop formation by human RAD51 , but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA . The percentage of D - loop formed in each reaction ( left ) was graphed as the average of triplicates ± SD .

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Pre-incubation of dsDNA with mcGAS-cat prevents D-loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D-loop formed in each reaction (left) was graphed as the average of triplicates ± SD.


Pre - incubation of template dsDNA with hcGAScat blocks subsequent D - loop formation . The percentage of D - loop formation ( below ) was graphed as the average of triplicates ± SD .

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Pre-incubation of template dsDNA with hcGAScat blocks subsequent D-loop formation. The percentage of D-loop formation (below) was graphed as the average of triplicates±SD.


Negative - stain electron micrographs of cGAS - dsDNA complexes following incubation of dsDNA with indicated cGAS variants . Scale bar : 100 nm .

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Negative-stain electron micrographs of cGAS-dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation when pre - incubated with dsDNA . Percentage of D - loop formed in each reaction ( left ) graphed as the average of triplicates ± SD .

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Effect of indicated hcGAS variants on D-loop formation when pre-incubated with dsDNA. Percentage of D-loop formed in each reaction (left) graphed as the average of triplicates± SD.


DR - GFP assay showing that hcGASΔDNA - Y215E is impaired in HR inhibition .

DR-GFP assay showing that hcGASΔDNA-Y215E is impaired in HR inhibition.


hcGASΔDNA - Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24 .

hcGASΔDNA-Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24.


hcGASΔDNA - Y215E and hcGASΔcGAMP are defective in synthase activity .

hcGASΔDNA-Y215E and hcGASΔcGAMP are defective in synthase activity.


Negative - stain electron micrographs showing that hcGAScat - ΔDNA - Y215E is defective in inducing cGAS - dsDNA complexes . Scale bar : 100 nm .

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Negative-stain electron micrographs showing that hcGAScat-ΔDNA-Y215E is defective in inducing cGAS-dsDNA complexes. Scale bar: 100 nm.


Effect of indicated hcGAS variants on D - loop formation .

Effect of indicated hcGAS variants on D-loop formation.


( E ) Representative photos of iWAT , eWAT and BAT from Ftoflox / flox and FtoAKO mice fed a HFD . Scale bar , 1 cm . ( F ) Representative H & E staining of iWAT , eWAT and BAT from Ftoflox / flox and FtoAKO mice fed a HFD . Scale bar , 200 μm . (

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(E) Representative photos of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 1 cm. (F) Representative H&E staining of iWAT, eWAT and BAT from Ftoflox/flox and FtoAKO mice fed a HFD. Scale bar, 200 μm. (


( I - K ) Serum levels of glucose ( I ) , triglyceride ( J ) and free fatty acid ( FFA ) ( K ) in Ftoflox / flox and FtoAKO mice fed a HFD followed by fasting . Data information : The data were presented as the mean ± SD ( n = 6 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(I-K) Serum levels of glucose (I), triglyceride (J) and free fatty acid (FFA) (K) in Ftoflox/flox and FtoAKO mice fed a HFD followed by fasting. Data information: The data were presented as the mean ± SD (n = 6). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( A ) O2 consumption of 16 - week - old Ftoflox / flox and FtoAKO mice fed a HFD . White and gray areas in the graphs indicate day and night , respectively . ( B ) CO2 generation of 16 - week - old Ftoflox / flox and FtoAKO mice fed a HFD . White and gray areas in the graphs indicate day and night , respectively . ( Data information : The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(A) O2 consumption of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. (B) CO2 generation of 16-week-old Ftoflox/flox and FtoAKO mice fed a HFD. White and gray areas in the graphs indicate day and night, respectively. ( Data information: The data were presented as the mean ± SD of n = 6 mice per group or triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗∗p < 0.01, ∗∗∗p < 0.001.


( F ) qPCR analysis of genes involved in thermogenesis , mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox / flox and FtoAKO mice .

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(F) qPCR analysis of genes involved in thermogenesis, mitochondrial biogenesis and adipogenesis in iWAT of Ftoflox/flox and FtoAKO mice.


( G ) Western blot analysis of the protein levels of UCP1 , PPARGC1A and PPARG in iWAT of Ftoflox / flox and FtoAKO mice .

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(G) Western blot analysis of the protein levels of UCP1, PPARGC1A and PPARG in iWAT of Ftoflox/flox and FtoAKO mice.


( I ) Immunofluorescence detection of UCP1 in iWAT from Ftoflox / flox and FtoAKO mice following 6 days cold exposure ( 4 ° C ) . Scale bar , 20 μm .

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(I) Immunofluorescence detection of UCP1 in iWAT from Ftoflox/flox and FtoAKO mice following 6 days cold exposure (4°C). Scale bar, 20 μm.


( M ) Western blot analysis of UCP1 , PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in ( J ) .

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(M) Western blot analysis of UCP1, PPARGC1A and PPARG protein levels in differentiated beige adipocytes described in (J).


( N ) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in ( J ) . Scale bar , 20 μm .

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(N) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (J). Scale bar, 20 μm.


( G ) Western blot analysis of the protein levels of FTO , HIF1A , UCP1 , PPARGC1A and PPARG in differentiated beige adipocytes described in ( F ) .

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(G) Western blot analysis of the protein levels of FTO, HIF1A, UCP1, PPARGC1A and PPARG in differentiated beige adipocytes described in (F).


( C ) The O2 consumption rate ( OCR ) in differentiated beige adipocytes from 3T3 - L1 cells overexpressing Vec or HIF1A plasmid . The average basal and maximal respiration rates are shown in right panel . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(C) The O2 consumption rate (OCR) in differentiated beige adipocytes from 3T3-L1 cells overexpressing Vec or HIF1A plasmid. The average basal and maximal respiration rates are shown in right panel. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( G ) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in ( C ) . ( H ) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in ( D ) . ( Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(G) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (C). (H) qPCR analysis of genes involved in thermogenesis in differentiated beige adipocytes described in (D). ( Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.


( I ) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in ( D ) . Scale bar , 20 μm .

(I) Immunofluorescent staining of UCP1 in differentiated beige adipocytes described in (D). Scale bar, 20 μm.


( C ) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3 - L1 cells . Blue boxes represent exons , and blue lines represent introns . The m6A peaks were in the dashed red box .

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(C) Distribution of m6A peaks in Hif1a mRNA transcript in siCtrl and siFto 3T3-L1 cells. Blue boxes represent exons, and blue lines represent introns. The m6A peaks were in the dashed red box.


( D ) Methylated RNA immunoprecipitation ( MeRIP ) - qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3 - L1 cells . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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(D) Methylated RNA immunoprecipitation (MeRIP)-qPCR analysis of m6A levels of Hif1a mRNA in siCtrl and siFto 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


( H ) Western blot analysis of the protein levels of HIF1A , UCP1 , PPARGC1A , PPARG and GFP in 3T3 - L1 cells infected with lentiviruses pCDHpuro expressing Vec , WT - or MUT - HIF1A .

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(H) Western blot analysis of the protein levels of HIF1A, UCP1, PPARGC1A, PPARG and GFP in 3T3-L1 cells infected with lentiviruses pCDHpuro expressing Vec, WT- or MUT-HIF1A.


( A ) Western blot analysis of the protein levels of YTHDF1 , YTHDF2 and HIF1A in siCtrl , siYthdf1 and siYthdf2 3T3 - L1 cells .

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(A) Western blot analysis of the protein levels of YTHDF1, YTHDF2 and HIF1A in siCtrl, siYthdf1 and siYthdf2 3T3-L1 cells.


( G ) Polysome profiling of 3T3 - L1 cells overexpressing Vec or YTHDC2 plasmid . ( H ) qPCR analysis of mRNA levels of Hif1a in 40 - 80S or polysome fraction in 3T3 - L1 cells overexpressing Vec or YTHDC2 plasmid . ( I ) Polysome profiling of siCtrl and siYthdc2 3T3 - L1 cells . ( J ) qPCR analysis of mRNA levels of Hif1a in 40 - 80S or polysome fraction in siCtrl and siYthdc2 3T3 - L1 cells . Data information : The data were presented as the mean ± SD of triplicate tests ( n = 3 ) . Statistical analyses were performed using Student t ‐ test . ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ p < 0 . 001 .

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

(G) Polysome profiling of 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (H) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in 3T3-L1 cells overexpressing Vec or YTHDC2 plasmid. (I) Polysome profiling of siCtrl and siYthdc2 3T3-L1 cells. (J) qPCR analysis of mRNA levels of Hif1a in 40-80S or polysome fraction in siCtrl and siYthdc2 3T3-L1 cells. Data information: The data were presented as the mean ± SD of triplicate tests (n = 3). Statistical analyses were performed using Student t‐test.∗∗p < 0.01, ∗∗∗p < 0.001.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) in response to different levels of KCl stress . Points reflect the measured values , and lines correspond to best model fit .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) in response to different levels of KCl stress. Points reflect the measured values, and lines correspond to best model fit.


Hsp12 - GFP accumulation ( Y axis ) vs . time ( X axis ) at 0 . 4M KCl .

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Hsp12-GFP accumulation (Y axis) vs. time (X axis) at 0.4M KCl.


Representation of the experiments shown in ( B , C ) in parameter space . Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter ( Y axis ) vs . KCl concentration ( X axis ) of the KCl gradient experiment The inferred total open time - toff − ton ( Y axis ) vs . the inferred production rate ( X axis ) of the KCl gradient

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Representation of the experiments shown in (B,C) in parameter space. Connected line shows the progression in the response to increasing KCl levels The inferred tonparameter (Y axis) vs. KCl concentration (X axis) of the KCl gradient experiment The inferred total open time - toff− ton(Y axis) vs. the inferred production rate (X axis) of the KCl gradient experiment


Summary of Hsp12 - GFP dynamics single mutant screen . 68 mutants were divided into three groups : increased / decreased / did not change the total GFP produced . Each group was independently hierarchically clustered . The values shown are the Z - Scores of each parameter : ton - response onset time ; production rate ; total open time - length of transcriptional window ( toff − ton ) ; Basal GFP - GFP levels measured before exposure to stress ; Delta GFP - amount of GFP produced throughout the experiment ( Max GFP - Basal GFP ) .

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

Summary of Hsp12-GFP dynamics single mutant screen. 68 mutants were divided into three groups: increased/decreased/did not change the total GFP produced. Each group was independently hierarchically clustered. The values shown are the Z-Scores of each parameter: ton- response onset time; production rate; total open time - length of transcriptional window (toff − ton); Basal GFP - GFP levels measured before exposure to stress; Delta GFP - amount of GFP produced throughout the experiment (Max GFP - Basal GFP).


mRNA levels of HSP12 ( Y axis ) as a function of time ( X axis ) in six representative strains .

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mRNA levels of HSP12 (Y axis) as a function of time (X axis) in six representative strains.


Comparison of RNA levels to protein levels . X axis is the sum over the HSP12 mRNA counts ( see D ) . Y axis is the maximum over the Hsp12 - GFP values . The red line indicates the best linear fit .

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Comparison of RNA levels to protein levels. X axis is the sum over the HSP12 mRNA counts (see D). Y axis is the maximum over the Hsp12-GFP values. The red line indicates the best linear fit.


Comparing the total open time parameter inferred from mRNA data ( X axis ) to the one inferred from protein data ( Y axis ) .

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Comparing the total open time parameter inferred from mRNA data (X axis) to the one inferred from protein data (Y axis).


Time - lapse microscopy of Hsp12 - GFP in response to stress ( 0 . 4M KCl ) . Examples of raw images ( top ) and traces of individual cells ( bottom ) . Traces are colored according to their basal GFP level .

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Time-lapse microscopy of Hsp12-GFP in response to stress (0.4M KCl). Examples of raw images (top) and traces of individual cells (bottom). Traces are colored according to their basal GFP level.


Scatter plot showing the production rate estimated from single traces vs . the production rate estimated from the population average . Each point represents a different yeast strain .

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Scatter plot showing the production rate estimated from single traces vs. the production rate estimated from the population average. Each point represents a different yeast strain.


Estimated parameters ( median over repeats ) of all ~ 1600 double mutant strains . The color represents the total Hsp12 - GFP produced . The same amount of GFP can be produced with multiple parameter combinations . The ellipses illustrate iso - expression areas in the graph . Three examples of individual strains ( marked in B ) with nearly the same total expression but dramatically different dynamics .

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Estimated parameters (median over repeats) of all ~1600 double mutant strains. The color represents the total Hsp12-GFP produced. The same amount of GFP can be produced with multiple parameter combinations. The ellipses illustrate iso-expression areas in the graph. Three examples of individual strains (marked in B) with nearly the same total expression but dramatically different dynamics.


Illustration of the genetic interaction trends of individual mutants . For each single mutant we plot the median of all the double mutants that contain it ( purple dots ) . The gray dots are all individual double mutant strains ( B ) .

Illustration of the genetic interaction trends of individual mutants. For each single mutant we plot the median of all the double mutants that contain it (purple dots). The gray dots are all individual double mutant strains (B).


Illustration of the interactions of Δhog1 in the production rate parameter . Shown the production rate of the single mutant ( X axis ) versus the production rate of the double mutant with Δhog1 ( Y axis ) . The gray line marks the Hog1 single mutant levels . Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 ( pink dots ) .

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

Illustration of the interactions of Δhog1 in the production rate parameter. Shown the production rate of the single mutant (X axis) versus the production rate of the double mutant with Δhog1 (Y axis). The gray line marks the Hog1 single mutant levels. Points close to the gray line are ones where the value of the double mutant is close to the value of Δhog1 and defined as epistasis of Δhog1 (pink dots).


Illustration of the interactions of Δhog1 for the total open time parameter .

Illustration of the interactions of Δhog1 for the total open time parameter.


Epistatic interactions of Δmck1 in the production rate parameter


Epistatic interactions of Δmck1 in the total open time parameter


Upper : HSP12 mRNA in response to osmotic stress ( 0 . 4M KCl ) . Lower : Estimate of Msn2 / 4 dependent production of HSP12 mRNA over time ( difference between WT and Δmsn2Δmsn4 production profiles , Figure EV4A , Methods ) .

Upper: HSP12 mRNA in response to osmotic stress (0.4M KCl). Lower: Estimate of Msn2/4 dependent production of HSP12 mRNA over time (difference between WT and Δmsn2Δmsn4 production profiles, Figure EV4A, Methods).


Δmck1 effect on the response of all stress genes . The integral over mRNA values in WT strain ( X axis ) vs . the integral in the Δmck1 strain ( Y values ) . The inset shows the integral for the HSP12 gene .

Δmck1 effect on the response of all stress genes. The integral over mRNA values in WT strain (X axis) vs. the integral in the Δmck1 strain (Y values). The inset shows the integral for the HSP12 gene.


Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints ( See 2C ) . Each gene is presented relative to its pre - stress level and normalized by the median over WT values to visualize the dynamic range in all genes . The genes are separated into MSN dependent and independent genes ( Figure EV4B ) . The MSN - dependent genes are sorted based on the onset time to three groups ( Early , Average and Late onset ) .

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

Heatmap showing expression of stress responsive genes in WT and two mutants over eight timepoints (See 2C). Each gene is presented relative to its pre-stress level and normalized by the median over WT values to visualize the dynamic range in all genes. The genes are separated into MSN dependent and independent genes (Figure EV4B). The MSN-dependent genes are sorted based on the onset time to three groups (Early, Average and Late onset).


The behaviour of early and late onset genes . Similar to ( C ) but showing the average over the genes in each group .

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The behaviour of early and late onset genes. Similar to (C) but showing the average over the genes in each group.


Msn2 - GFP localization following stress ( 0 . 4M KCl ) in selected strains . Top : The percentage of nuclear cells ( Y axis ) over time ( X axis ) . Bottom : count of the number of nuclear import events in live - cell time - lapse microscopy at the two relevant periods .

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Msn2-GFP localization following stress (0.4M KCl) in selected strains. Top: The percentage of nuclear cells (Y axis) over time (X axis). Bottom: count of the number of nuclear import events in live-cell time-lapse microscopy at the two relevant periods.


( A ) Representative images of a PDGFB tumour : H & E on the right to identify the tumour , an adjacent section stained for Iba1 ( red ) , p - S6 ( green ) and DAPI ( blue ) at the centre and the region selection for quantification on the left ( non - tumour in light blue , tumour edge in orange and tumour core in dark blue ) .

(A) Representative images of a PDGFB tumour: H&E on the right to identify the tumour, an adjacent section stained for Iba1 (red), p-S6 (green) and DAPI (blue) at the centre and the region selection for quantification on the left (non-tumour in light blue, tumour edge in orange and tumour core in dark blue).


( B ) Representative images of core , edge and adjacent non - tumour brain tissue for Iba1 , p - S6 and DAPI staining in Pten - / - p53 - / - ( right ) and GL261 ( left ) tumours . Percentage of Iba1 + cells co - expressing p - S6 in the three defined regions . Five high grade glioma models were analysed - GL261 ( n = 3 ) , PDGFB ( n = 3 ) , Ntv - a ; PDGFB + shp53 ( n = 2 ) , Pten - / - p53 - / - ( n = 3 ) , and Pten - / - p53 - / - Idh1mut ( n = 3 ) ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(B) Representative images of core, edge and adjacent non-tumour brain tissue for Iba1, p-S6 and DAPI staining in Pten-/-p53-/- (right) and GL261 (left) tumours. Percentage of Iba1+ cells co-expressing p-S6 in the three defined regions. Five high grade glioma models were analysed - GL261 (n=3), PDGFB (n=3), Ntv-a;PDGFB+shp53 (n=2), Pten-/-p53-/- (n=3), and Pten-/-p53-/-Idh1mut (n=3) (mean ±SEM; Two-Way ANOVA Tukey test).


( F , G ) Expression levels of ( F ) Rps6 and ( G ) Eif4ebp1 in healthy versus GL261 microglia and macrophage ( n = 3 ; mean ± SEM , likelihood ratio test in edgeR ) .

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(F,G) Expression levels of (F) Rps6 and (G) Eif4ebp1 in healthy versus GL261 microglia and macrophage (n=3; mean ±SEM, likelihood ratio test in edgeR).


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr ( B and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr (B and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( C ) p - S6 S240 / 244 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Flow cytometry analysis was carried out in microglia for (C) p-S6 S240/244 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 4hr and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( E ) p - 4EBP1 T37 / 46 Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (E) p-4EBP1 T37/46 Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( G ) p - AKT S473 ; Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin , was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Flow cytometry analysis was carried out in microglia for (G) p-AKT S473; Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+Torin, was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0 . 5hr ( H ) and normalised against non - phosphorylated protein and vinculin analysed on the same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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

Signalling was analysed in microglia by immunoblotting of whole cell lysates collected at 0.5hr (H) and normalised against non-phosphorylated protein and vinculin analysed on the same blot. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM +LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


Flow cytometry analysis was carried out in microglia for ( I ) p - AKT T308 . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

Flow cytometry analysis was carried out in microglia for (I) p-AKT T308. Each treatment (mNSC-CM, Pten-/-p53-/- mGIC-CM, Pten-/-p53-/- mGIC-CM+ LY) was normalised to unconditioned control (n=3; mean ±SEM, Two-Way ANOVA Tukey test). *p≤0.05, **p≤0.01, ***p≤0.001 comparing mGICPten-/-;p53-/--CM versus mGICPten-/-;p53-/--CM +inhibitor.


( B ) Survival analysis for Cx3cr1 - Rheb1Δ / Δ ( n = 7 ) and Rheb1fl / fl ( n = 7 ) mice . Chi square test .

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(B) Survival analysis for Cx3cr1-Rheb1Δ/Δ (n=7) and Rheb1fl/fl (n=7) mice. Chi square test.


( C ) Representative images and quantification of the tumour volume with MRI of Rheb1fl / fl ( n = 8 ) compared to Cx3cr1 - Rheb1Δ / Δ ( n = 8 ) mice . ( mean ± SEM ; Unpaired parametric t test ) .

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(C) Representative images and quantification of the tumour volume with MRI of Rheb1fl/fl (n=8) compared to Cx3cr1-Rheb1Δ/Δ (n=8) mice. (mean ±SEM; Unpaired parametric t test).


( D ) H & E staining showing representative histological features ( overview and high magnification of microvascular proliferation ) of Rheb1fl / fl and Cx3cr1 - Rheb1Δ / Δ GL261 tumours . Scale bar is 125 μm ( top , H & E ) and 80 μm ( all other images )

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(D) H&E staining showing representative histological features (overview and high magnification of microvascular proliferation) of Rheb1fl/fl and Cx3cr1-Rheb1Δ/Δ GL261 tumours. Scale bar is 125 μm (top, H&E) and 80 μm (all other images)


( E ) Percentage of GFP + CD45 - GL261 tumour cells in Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) and Rheb1fl / fl ( n = 7 ) tumours , with representative FACS plot ( mean ± SEM ; Unpaired parametric t test ) .

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(E) Percentage of GFP+ CD45- GL261 tumour cells in Cx3cr1-Rheb1Δ/Δ (n=5) and Rheb1fl/fl (n=7) tumours, with representative FACS plot (mean ±SEM; Unpaired parametric t test).


( B ) Levels of TAM - MG ( P2RY12 + CD49d - ) and TAM - BMDM ( P2RY12 - CD49d + ) gated from CD45 + population in GL261 tumours from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice by flow cytometry ( representative flow cytometry plots on top and quantification at the bottom ; mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(B) Levels of TAM-MG (P2RY12+ CD49d-) and TAM-BMDM (P2RY12- CD49d+) gated from CD45+ population in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice by flow cytometry (representative flow cytometry plots on top and quantification at the bottom; mean ±SEM; Two-Way ANOVA Tukey test).


( C ) Percentage of CD8 CTL ( top ) and CD4 Th ( CD4 ) cells in GL261 tumours from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 5 ) mice assessed by flow cytometry ( representative flow cytometry plots , left and quantification , right ; mean ± SEM ; Unpaired t test ) .

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(C) Percentage of CD8 CTL (top) and CD4 Th (CD4) cells in GL261 tumours from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=5) mice assessed by flow cytometry (representative flow cytometry plots, left and quantification, right; mean ±SEM; Unpaired t test).


( D ) Tumour tissues from Rheb1fl / fl ( n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( n = 4 ) were stained for CD8 , CD4 and FoxP3 . DAB staining was quantified as % of positive cells using Definiens software . Representative images of the stained tissues are shown . ( mean ± SEM ; unpaired parametric t test ) . Scale bar is 100µm .

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(D) Tumour tissues from Rheb1fl/fl (n=4) and Cx3cr1-Rheb1Δ/Δ (n=4) were stained for CD8, CD4 and FoxP3. DAB staining was quantified as % of positive cells using Definiens software. Representative images of the stained tissues are shown. (mean ±SEM; unpaired parametric t test). Scale bar is 100µm.


( B ) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores ( ssGSEA ) for lymphocytes in the Cx3cr1 - Rheb1Δ / Δ ( n = 3 ) vs Rheb1fl / fl ( n = 3 ) GL261 tumours .

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(B) Heatmap of the differential correlation analysis between CIBERSORT cell fractions and signatures enrichment scores (ssGSEA) for lymphocytes in the Cx3cr1-Rheb1Δ/Δ (n=3) vs Rheb1fl/fl (n=3) GL261 tumours.


( C , D ) CD4 Th cells ( C ) and CD8 CTL cells ( D ) expression of Ki67 , perforin , granzyme b , and IFNγ , in Cx3cr1 - Rheb1fl / fl ( n = 5 ) and Rheb1Δ / Δ ( n = 5 ) GL261 tumours . Representative FACS plots show control samples ( tumour - derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange ) compared to stimulated cells derived from Rheb1fl / fl ( fl / fl , red ) and Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , blue ) GL261 tumours ( cells stimulated in culture with protein inhibitor cocktail ) ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(C,D) CD4 Th cells (C) and CD8 CTL cells (D) expression of Ki67, perforin, granzyme b, and IFNγ, in Cx3cr1-Rheb1fl/fl (n=5) and Rheb1Δ/Δ (n=5) GL261 tumours. Representative FACS plots show control samples (tumour-derived cells stimulated in culture in the absence of protein inhibitor cocktail - in orange) compared to stimulated cells derived from Rheb1fl/fl (fl/fl, red) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, blue) GL261 tumours (cells stimulated in culture with protein inhibitor cocktail) (mean ±SEM; Two-Way ANOVA Tukey test).


( E , F ) Expression of CD44 ( E ) and CD62L ( F ) by CD4 Th and CD8 CTL in Cx3cr1 - Rheb1fl / fl ( n = 5 ; fl / fl , red ) and Rheb1Δ / Δ ( n = 5 ; Δ / Δ , blue ) GL261 tumours with representative FACS plots show control samples ( mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(E,F) Expression of CD44 (E) and CD62L (F) by CD4 Th and CD8 CTL in Cx3cr1-Rheb1fl/fl (n=5; fl/fl, red) and Rheb1Δ/Δ (n=5; Δ/Δ, blue) GL261 tumours with representative FACS plots show control samples (mean ±SEM; Two-Way ANOVA Tukey test).


( B ) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change ( Log ( FC ) ) .

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(B) Frequency of occurrence of differentially regulated genes across significantly deregulated canonical pathways compared to the Log fold change (Log(FC)).


( C ) Upstream regulators as identified by the IPA software from differentially regulated genes .

(C) Upstream regulators as identified by the IPA software from differentially regulated genes.


( E ) Microglia were pre - treated with Torin , or medium as indicated and then stimulated with mNSC - CM , mGICPten - / - ; p53 - / - - CM , or GL261 - CM . p - NF - ĸB ( p - P65 ) and p - STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr .

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(E) Microglia were pre-treated with Torin, or medium as indicated and then stimulated with mNSC-CM, mGICPten-/-;p53-/--CM, or GL261-CM. p-NF-ĸB (p-P65) and p-STAT3 were analysed by immunoblotting of whole cell lysates collected at 4hr.


( F ) Immunofluorescence images of NF - ĸB ( P65 ) in conditioned microglia . Quantification of nuclear translocation of NF - κB from staining . Units represent the % of voxels in the NF - κB channel colocalised with DAPI ( n = 3 ; mean ± SEM ; One - Way ANOVA Tukey test - right ) . Scale bar is 50µm .

(F) Immunofluorescence images of NF-ĸB (P65) in conditioned microglia. Quantification of nuclear translocation of NF-κB from staining. Units represent the % of voxels in the NF-κB channel colocalised with DAPI (n=3; mean ±SEM; One-Way ANOVA Tukey test - right). Scale bar is 50µm.


( G ) Production of Il - 12 ( Il - 12p40 ) , Tnf , Il6 , and Il10 by conditioned microglia was determined by qPCR . Each treatment was normalised to housekeeping gene and the unconditioned control . ( n = 3 ; mean ± SEM ; Two - Way ANOVA Tukey test ) .

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(G) Production of Il-12 (Il-12p40), Tnf, Il6, and Il10 by conditioned microglia was determined by qPCR. Each treatment was normalised to housekeeping gene and the unconditioned control. (n=3; mean ±SEM; Two-Way ANOVA Tukey test).


( H , I ) MFI levels of pSTAT3 in TAM - MG ( CD45 + P2RY12 + CD49d - ) ( H ) and p - NF - κB ( p - P65 ) in TAM - BMDM ( CD45 + P2RY12 - CD49d + ) ( I ) from Rheb1fl / fl ( fl / fl , blue , n = 4 ) and Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , red , n = 5 ) GL261 tumours . Representative FACS plots are displayed for each cell type ( mean ± SEM , Two - Way ANOVA Tukey test ) .

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(H,I) MFI levels of pSTAT3 in TAM-MG (CD45+ P2RY12+ CD49d-) (H) and p-NF-κB (p-P65) in TAM-BMDM (CD45+ P2RY12- CD49d+) (I) from Rheb1fl/fl (fl/fl, blue, n=4) and Cx3cr1-Rheb1Δ/Δ (Δ/Δ, red, n=5) GL261 tumours. Representative FACS plots are displayed for each cell type (mean ±SEM, Two-Way ANOVA Tukey test).


( A ) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM - MG or TAM - BMDM signatures in TCGA GBM transcriptomic data . Comparison carried out on all IDH - wild - type samples , and in a subgroup specific manner according to Wang ' s classifier . Size of circle is indicative of Rsquare value and bold outline represents a p - value ≤ 0 . 05 .

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(A) Correlation between ssGSEA enrichment scores for the mTOR signature versus TAM-MG or TAM-BMDM signatures in TCGA GBM transcriptomic data. Comparison carried out on all IDH-wild-type samples, and in a subgroup specific manner according to Wang's classifier. Size of circle is indicative of Rsquare value and bold outline represents a p-value ≤0.05.


( B ) Separation of IDH - wildtype GBM samples between those displaying high mTOR and microglia enrichment ( + ve correlation , group 1 in orange ) and those without this signature ( group 2 in green ) .

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(B) Separation of IDH-wildtype GBM samples between those displaying high mTOR and microglia enrichment (+ve correlation, group 1 in orange) and those without this signature (group 2 in green).


( C ) CIBERSORT cell fractions calculated from the TPM of TCGA IDH - wild type GBM samples from group 1 compared to those from group 2 .

(C) CIBERSORT cell fractions calculated from the TPM of TCGA IDH-wild type GBM samples from group 1 compared to those from group 2.


( F ) Staining by flow cytometry of EPSC , YS - EBs and matched - iMGL with indicated markers ( n = 3 ; mean ± SEM ) .

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(F) Staining by flow cytometry of EPSC, YS-EBs and matched-iMGL with indicated markers (n=3; mean ±SEM).


( G , H ) Mature iMGL were immunostained for ( G ) PU1 and CD11b and ( H ) Iba1 and TREM2 . Scale bar 20µm .

(G,H) Mature iMGL were immunostained for (G) PU1 and CD11b and (H) Iba1 and TREM2. Scale bar 20µm.


( I - K ) ( I ) P - S6 , ( J ) p - STAT3 , and ( K ) p - NF - ĸB ( p - P65 ) were analysed by flow cytometry in iMGL , pre - treated with Torin , as indicated and then stimulated with matched patient EPSC - or hGIC - CM . ( n = 3 ; mean ± SEM , Two - Way ANOVA Tukey test ) .

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(I-K) (I) P-S6, (J) p-STAT3, and (K) p-NF-ĸB (p-P65) were analysed by flow cytometry in iMGL, pre-treated with Torin, as indicated and then stimulated with matched patient EPSC- or hGIC-CM. (n=3; mean ±SEM, Two-Way ANOVA Tukey test).


( A ) The AH109 strain was co ‐ transformed with pGBKT7 containing the C ‐ terminal ( CT ) domain of mAtg9 ( bait ) and pGADT7 , or pACT2 , containing the CT domain of p38IP ( prey ) . The interaction was then visualized by growth of co ‐ transformed yeast on SD ‐ Trp / Leu / His plates for 3 days at 30 ° C .

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(A) The AH109 strain was co‐transformed with pGBKT7 containing the C‐terminal (CT) domain of mAtg9 (bait) and pGADT7, or pACT2, containing the CT domain of p38IP (prey). The interaction was then visualized by growth of co‐transformed yeast on SD‐Trp/Leu/His plates for 3 days at 30°C.


( C ) HEK293A cells were transfected with RFP - mAtg9 and HA - p38IP . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody and the co ‐ immunoprecipitated RFP - mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP was detected with an anti ‐ HA antibody . Asterisk indicates a non ‐ specific band .

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(C) HEK293A cells were transfected with RFP-mAtg9 and HA-p38IP. HA-p38IP was immunoprecipitated with an anti‐HA antibody and the co‐immunoprecipitated RFP-mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP was detected with an anti‐HA antibody. Asterisk indicates a non‐specific band.


Indirect immunofluorescence analysis of mAtg9 with ( D ) HA - full ‐ length p38IP ( HA - FL ) , ( E ) HA - C ‐ terminal p38IP ( HA - CT ) , and ( F ) HA - N ‐ terminal p38IP ( HA - NT ) . Bars = 5 μm . The co ‐ localization seen in ( D ) and ( E ) was not because of overlap of randomly distributed vesicles . Cross ‐ correlation function ( CCF ) analysis ( van Steensel et al , 1996 ) showed a peak and maximum Rp value around ΔX = 0 , indicating that co ‐ localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1 ) .

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Indirect immunofluorescence analysis of mAtg9 with (D)HA-full‐length p38IP (HA-FL), (E)HA-C‐terminal p38IP (HA-CT), and (F) HA-N‐terminal p38IP (HA-NT). Bars=5 μm. The co‐localization seen in (D) and (E) was not because of overlap of randomly distributed vesicles. Cross‐correlation function (CCF) analysis ( van Steensel et al, 1996) showed a peak and maximum Rp value around ΔX=0, indicating that co‐localization between mAtg9 and p38IP is positively correlated and non random ( Supplementary Figure S1).


( A ) HEK293A cells were transfected with HA - p38IP alone or co ‐ transfected with HA - p38IP and RFP - mAtg9 , homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol . Equal protein amounts were resolved by SDS - PAGE and immunoblotted with anti ‐ mannose ‐ 6 ‐ phosphate receptor ( MPR ) antibody as a control for membrane ‐ associated proteins , anti ‐ superoxide dismutase ( SOD ) antibody as a control for cytosolic proteins , anti ‐ mAtg9 antibody , and anti ‐ HA antibody . HA - p38IP levels were quantified using ImageJ software and plotted relative to HA - p38IP in the PNS ( n = 4 ) ( * * P = 0 . 0051 ) .

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(A) HEK293A cells were transfected with HA-p38IP alone or co‐transfected with HA-p38IP and RFP-mAtg9, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐mannose‐6‐phosphate receptor (MPR) antibody as a control for membrane‐associated proteins, anti‐superoxide dismutase (SOD) antibody as a control for cytosolic proteins, anti‐mAtg9 antibody, and anti‐HA antibody. HA-p38IP levels were quantified using ImageJ software and plotted relative to HA-p38IP in the PNS (n=4) (**P=0.0051).


( B ) HEK293 cells were depleted of mAtg9 using siRNA , then transfected with HA - p38IP and incubated in full medium or EBSS ( data not shown ) . The asterisk indicates mAtg9 ‐ depleted cells . Bar is 5 μM . The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software ( * * * P = 0 . 0001 , Students t ‐ test )

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(B) HEK293 cells were depleted of mAtg9 using siRNA, then transfected with HA-p38IP and incubated in full medium or EBSS (data not shown). The asterisk indicates mAtg9‐depleted cells. Bar is 5 μM. The intensity of the nuclear versus cytosolic fluorescence in control and cells depleted of mAtg9 under fed and starvation conditions was analysed using ImageJ software (***P=0.0001, Students t‐test)


( C ) HEK293A cells were transfected with HA - p38IP and processed for indirect immunofluorescence . HA - p38IP was detected with an anti ‐ HA antibody and endogenous mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS . HA - p38IP co ‐ localizes with mAtg9 in the periphery of the cell ( arrowheads ) . Bars ( upper panels ) 5 μM , ( lower panels ) 1 μM .

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(C) HEK293A cells were transfected with HA-p38IP and processed for indirect immunofluorescence. HA-p38IP was detected with an anti‐HA antibody and endogenous mAtg9 was detected with an anti‐mAtg9 antibody. HA-p38IP localizes to the nucleus and cytoplasmic puncta in full medium or EBSS. HA-p38IP co‐localizes with mAtg9 in the periphery of the cell (arrowheads). Bars (upper panels) 5 μM, (lower panels) 1 μM.


( D ) Anti ‐ mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA - p38IP alone or with RFP - mAtg9 and HA - p38IP . A total of 5 % of lysates or immunoprecipitates were probed with anti ‐ HA or anti ‐ mAtg9 antibodies . The left and right hand bottom panels were obtained from the same immunoblot , but exposed for different times to allow the endogenous mAtg9 to be visualized better .

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(D) Anti‐mAtg9 antibody was used to immunoprecipitate mAtg9 from HEK293 cells transfected with HA-p38IP alone or with RFP-mAtg9 and HA-p38IP. A total of 5% of lysates or immunoprecipitates were probed with anti‐HA or anti‐mAtg9 antibodies. The left and right hand bottom panels were obtained from the same immunoblot, but exposed for different times to allow the endogenous mAtg9 to be visualized better.


( E ) HEK293 cells were transfected with control or p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium or EBSS for 2 h , then fixed and immunostained to detect endogenous mAtg9 localization . mAtg9 localization was quantified by visually scoring cells for either a juxta ‐ nuclear or dispersed phenotype . Bars = 5 μm ( data are represented as mean ± s . e . m . of 200 cells , * P = 0 . 0413 , Student ' s t ‐ test ) .

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(E) HEK293 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. mAtg9 localization was quantified by visually scoring cells for either a juxta‐nuclear or dispersed phenotype. Bars=5 μm (data are represented as mean±s.e.m. of 200 cells, *P=0.0413, Student's t‐test).


( A ) 293 / GFP - LC3 cells were transfected with control or p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium ( FM ) , full medium with leupeptin ( FM Leu ) , EBSS ( ES ) , or EBSS with leupeptin ( ES Leu ) for 2 h . FM Leu ( data not shown ) is identical to FM alone . Quantification of GFP - LC3 ‐ positive autophagosome number was performed by counting in a blinded experiment . Bars = 5 μm ( data are represented as mean ± s . e . m . of 60 cells , * * * P = 0 . 0002 , Students t ‐ test ) .

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(A) 293/GFP-LC3 cells were transfected with control or p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium (FM), full medium with leupeptin (FM Leu), EBSS (ES), or EBSS with leupeptin (ES Leu) for 2 h. FM Leu (data not shown) is identical to FM alone. Quantification of GFP-LC3‐positive autophagosome number was performed by counting in a blinded experiment. Bars=5 μm (data are represented as mean±s.e.m. of 60 cells, ***P=0.0002, Students t‐test).


( B ) HEK293A cells were transfected with siRNA control , siRNA for p38IP , and siRNA for ULK1 . Cell lysates were analysed by SDS - PAGE for endogenous LC3 lipidation using an anti ‐ LC3 antibody , or immunoblotted with anti ‐ ULK1 antibody .

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(B) HEK293A cells were transfected with siRNA control, siRNA for p38IP, and siRNA for ULK1. Cell lysates were analysed by SDS-PAGE for endogenous LC3 lipidation using an anti‐LC3 antibody, or immunoblotted with anti‐ULK1 antibody.


( C ) HeLa cells were transfected with siRNA control , and siRNA for p38IP . After incubation for 2 h as in ( A ) , samples were immunoblotted with anti ‐ LC3 and anti ‐ actin antibodies . Endogenous LC3II / LC3I levels were quantified and the ratio presented as arbitrary units . In ( B ) , n = 4 , * * * P = 0 . 0001 and * P = 0 . 0324 , Students t ‐ test . In ( C ) , data are representative of two experiments .

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(C) HeLa cells were transfected with siRNA control, and siRNA for p38IP. After incubation for 2 h as in (A), samples were immunoblotted with anti‐LC3 and anti‐actin antibodies. Endogenous LC3II/LC3I levels were quantified and the ratio presented as arbitrary units. In (B), n=4, ***P=0.0001 and *P=0.0324, Students t‐test. In (C), data are representative of two experiments.


( D ) 293 / GFP - LC3 cells were transfected with siRNA control or siRNA p38IP , and incubated in full medium , or for 2 and 4 h in EBSS . Samples were immunoblotted with anti ‐ p62 antibodies , and actin as a loading control .

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(D) 293/GFP-LC3 cells were transfected with siRNA control or siRNA p38IP, and incubated in full medium, or for 2 and 4 h in EBSS. Samples were immunoblotted with anti‐p62 antibodies, and actin as a loading control.


( E ) HEK293A cells were transfected with HA - p38IP or myc - ULK1 C ‐ terminal domain ( CTD ) . After 24 h , cells were labelled with [ 14C ] valine as described in Material and Methods section , and incubated in either full medium or EBSS for 2 h . Cells were then collected and analysed for long ‐ lived protein degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS mock versus EBSS HA - p38IP ( * * * P = 0 . 036 ) ; EBSS mock versus ULK1 CTD ( * * * P = 0 . 0001 ) ; Students t ‐ test ) .

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(E) HEK293A cells were transfected with HA-p38IP or myc-ULK1 C‐terminal domain (CTD). After 24 h, cells were labelled with [14C]valine as described in Material and Methods section, and incubated in either full medium or EBSS for 2 h. Cells were then collected and analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS mock versus EBSS HA-p38IP (***P=0.036); EBSS mock versus ULK1 CTD (***P=0.0001); Students t‐test).


( A ) 293 / GFP - LC3 cells were treated with 10 μM anisomycin for 30 min , or exposed to UV irradiation for 3 min followed by a 40 ‐ min recovery and incubation in full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . Cells were then fixed and visualized by confocal microscopy . GFP - LC3 ‐ positive structures per cell were quantified as in Figure 3A . Bars = 5 μm . Data are represented as mean ± s . e . m . n = 60 cells , mock versus anisomycin EBSS ( * * * P = 0 . 0001 ) ; mock versus UV EBSS ( * * * P = 0 . 0001 ) ; mock versus anisomycin EBSS with leupeptin ( * * * P = 0 . 0001 ) ; mock versus EBSS with leupeptin UV ( * * * P = 0 . 0001 ) . All analysed using Student ' s t - test .

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

(A) 293/GFP-LC3 cells were treated with 10 μM anisomycin for 30 min, or exposed to UV irradiation for 3 min followed by a 40‐min recovery and incubation in full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells were then fixed and visualized by confocal microscopy. GFP-LC3‐positive structures per cell were quantified as in Figure 3A. Bars=5 μm. Data are represented as mean±s.e.m. n=60 cells, mock versus anisomycin EBSS (***P=0.0001); mock versus UV EBSS (***P=0.0001); mock versus anisomycin EBSS with leupeptin (***P=0.0001); mock versus EBSS with leupeptin UV (***P=0.0001). All analysed using Student's t-test.


( B ) HEK293A cells were pretreated with 10 μM anisomycin for 30 min , or UV irradiation for 3 min followed by a 40 ‐ min recovery , before incubation in EBSS for 2 h . Images were analysed as in Figure 2E . Bars = 5 μm . Data are represented as mean ± s . e . m . of 60 cells , EBSS mock versus EBSS anisomycin ( * P = 0 . 0142 ) ; EBSS mock versus EBSS UV ( * P = 0 . 0150 ) ; Student ' s t ‐ test .

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

(B) HEK293A cells were pretreated with 10 μM anisomycin for 30 min, or UV irradiation for 3 min followed by a 40‐min recovery, before incubation in EBSS for 2 h. Images were analysed as in Figure 2E. Bars=5 μm. Data are represented as mean±s.e.m. of 60 cells, EBSS mock versus EBSS anisomycin (*P=0.0142); EBSS mock versus EBSS UV (*P=0.0150); Student's t‐test.


( A ) 293 / GFP - LC3 cells were pre ‐ treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS . Cell lysates were analysed by SDS - PAGE for GFP - LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ β tubulin . The GFP - LC3 lipidation was quantified as the amount of GFP - LC3II / GFP - LC3I ( data are represented as mean ± s . e . m . of triplicates , P = 0 . 0091 , Student ' s t ‐ test ) .

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

(A) 293/GFP-LC3 cells were pre‐treated with 10 μM anisomycin for 30 min before incubation in either full medium or EBSS. Cell lysates were analysed by SDS-PAGE for GFP-LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐β tubulin. The GFP-LC3 lipidation was quantified as the amount of GFP-LC3II/GFP-LC3I (data are represented as mean±s.e.m. of triplicates, P=0.0091, Student's t‐test).


( B ) HEK293A cells labelled with [ 14C ] valine , as described in Material and Methods section , were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium , EBSS , or EBSS with leupeptin for 2 h . Cells were then analysed for long ‐ lived protein degradation ( data are represented as mean ± s . e . m . of triplicates , representative of two experiments , EBSS untreated versus anisomycin treated ( P = 0 . 0363 ) ; EBSS untreated versus leupeptin treated ( P = 0 . 0001 ) , Student ' s t ‐ test ) .

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

(B) HEK293A cells labelled with [14C]valine, as described in Material and Methods section, were pretreated with 10 μM anisomycin for 30 min before incubation in either full medium, EBSS, or EBSS with leupeptin for 2 h. Cells were then analysed for long‐lived protein degradation (data are represented as mean±s.e.m. of triplicates, representative of two experiments, EBSS untreated versus anisomycin treated (P=0.0363); EBSS untreated versus leupeptin treated (P=0.0001), Student's t‐test).


( C ) HEK293 cells were transfected with control siRNA or siRNA for p38IP . After 24 ‐ h incubation , control or p38IP ‐ depleted cells were pre ‐ treated with anisomycin , followed by a 2 ‐ h incubation with full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin . Cell lysates were analysed with anti ‐ p38α or anti ‐ LC3 antibodies . LC3II / LC3I levels were quantified and presented normalized to control , untreated cells incubated in EBSS . Data are representative of two experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O 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 O O O O O O O O O O O O O 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) HEK293 cells were transfected with control siRNA or siRNA for p38IP. After 24‐h incubation, control or p38IP‐depleted cells were pre‐treated with anisomycin, followed by a 2‐h incubation with full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin. Cell lysates were analysed with anti‐p38α or anti‐LC3 antibodies. LC3II/LC3I levels were quantified and presented normalized to control, untreated cells incubated in EBSS. Data are representative of two experiments.


( D ) HEK293A cells transiently transfected with HA ‐ p38IP and RFP ‐ mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated . 5 % of the lysates or the immunoprecipitates were probed with anti ‐ HA and anti ‐ mAtg9 antibodies ( data is representative of 3 experiments , * P = 0 . 0332 ) .

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

(D) HEK293A cells transiently transfected with HA‐p38IP and RFP‐mAtg9 were treated with anisomycin for 30 min and mAtg9 was immunoprecipitated. 5% of the lysates or the immunoprecipitates were probed with anti‐HA and anti‐mAtg9 antibodies (data is representative of 3 experiments, *P=0.0332).


( A ) HEK293A cells were transfected with RFP - mAtg9 and Flag - p38 ( lanes 2 and 7 ) , RFP - mAtg9 , Flag - p38 and HA - p38IP ( lanes 3 and 8 ) , or RFP - mAtg9 and HA - p38IP ( lanes 4 and 9 ) . RFP - mAtg9 was immunoprecipitated with an anti ‐ mAtg9 antibody , the co ‐ immunoprecipitated HA - p38IP was detected with an anti ‐ HA antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ Flag antibody . RFP - mAtg9 was detected with an anti ‐ Atg9 antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with RFP-mAtg9 and Flag-p38 (lanes 2 and 7), RFP-mAtg9, Flag-p38 and HA-p38IP (lanes 3 and 8), or RFP-mAtg9 and HA-p38IP (lanes 4 and 9). RFP-mAtg9 was immunoprecipitated with an anti‐mAtg9 antibody, the co‐immunoprecipitated HA-p38IP was detected with an anti‐HA antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐Flag antibody. RFP-mAtg9 was detected with an anti‐Atg9 antibody.


( B ) HEK293A cells were transfected with RFP - Atg9 and HA - p38IP ( lanes 1 and 5 ) , Flag - Flag , Flag - p38 , and HA - p38IP ( lanes 2 and 6 ) , or Flag - p38 and HA - p38IP ( lanes 3 and 7 ) . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody , the co ‐ immunoprecipitated RFP - Atg9 was detected with an anti ‐ Atg9 antibody , and co ‐ immunoprecipitated Flag - p38 was detected with an anti ‐ p38α antibody . HA - p38IP was detected with an anti ‐ HA antibody .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with RFP-Atg9 and HA-p38IP (lanes 1 and 5), Flag-Flag, Flag-p38, and HA-p38IP (lanes 2 and 6), or Flag-p38 and HA-p38IP (lanes 3 and 7). HA-p38IP was immunoprecipitated with an anti‐HA antibody, the co‐immunoprecipitated RFP-Atg9 was detected with an anti‐Atg9 antibody, and co‐immunoprecipitated Flag-p38 was detected with an anti‐p38α antibody. HA-p38IP was detected with an anti‐HA antibody.


( C ) HEK293A cells were transfected with Flag - p38α for 24 h . Cells were incubated in either full medium , EBSS , or EBSS plus leupeptin for 2 h , lysed , and analysed for endogenous LC3 lipidation using an anti ‐ LC3 antibody , and immunoblotted with anti ‐ Flag antibody . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . of triplicates , n = 2 experiments , EBSS control versus EBSS with Flag ‐ p38α , * * * P = 0 . 0026 ) .

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

(C) HEK293A cells were transfected with Flag-p38α for 24 h. Cells were incubated in either full medium, EBSS, or EBSS plus leupeptin for 2 h, lysed, and analysed for endogenous LC3 lipidation using an anti‐LC3 antibody, and immunoblotted with anti‐Flag antibody. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean±s.e.m. of triplicates, n=2 experiments, EBSS control versus EBSS with Flag‐p38α, ***P=0.0026).


( A ) 293 / GFP - LC3 cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS with leupeptin for 2 h , then fixed and visualized by confocal microscopy . Bars = 5 μm ( data are represented as mean ± s . e . m . n = 60 cells , EBSS control versus p38α siRNA ( * * * P = 0 . 0001 ) ; EBSS with leupeptin control versus p38α siRNA ( * * * P = 0 . 0001 ) , Student ' s t ‐ test ) .

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

(A) 293/GFP-LC3 cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS with leupeptin for 2 h, then fixed and visualized by confocal microscopy. Bars=5 μm (data are represented as mean±s.e.m. n=60 cells, EBSS control versus p38α siRNA (***P=0.0001); EBSS with leupeptin control versus p38α siRNA (***P=0.0001), Student's t‐test).


( B ) HEK293A cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium , EBSS , or EBSS containing leupeptin for 2 h . Cells lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ actin and anti ‐ p38α antibodies . LC3 lipidation was quantified as the amount of LC3II / LC3I ( data are represented as mean ± s . e . m . n = 5 , full medium control versus p38α siRNA ( * * P = 0 . 0056 ) ; EBSS control versus p38α siRNA ( * * P = 0 . 048 ) ; EBSS with Leu control versus p38α siRNA ( * * P = 0 . 04 ) , Student ' s t ‐ test ) .

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

(B) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium, EBSS, or EBSS containing leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐actin and anti‐p38α antibodies. LC3 lipidation was quantified as the amount of LC3II/LC3I (data are represented as mean ±s.e.m. n=5, full medium control versus p38α siRNA (**P=0.0056); EBSS control versus p38α siRNA (**P=0.048); EBSS with Leu control versus p38α siRNA (**P=0.04), Student's t‐test).


( C ) HEK293A cells were transfected with control or p38α siRNA . At 72 h after transfection , cells were incubated in either full medium or EBSS for 2 h , then fixed and immunostained to detect endogenous mAtg9 localization . The percentage of cells with dispersed mAtg9 was quantified as in Figure 2E ( data are represented as mean ± s . e . m . n = 200 cells , full medium control versus p38α siRNA ( * * P = 0 . 0027 ) ; EBSS control versus p38α siRNA ( * P = 0 . 0459 ) ; Student ' s t ‐ test ) . Bars = 5

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with control or p38α siRNA. At 72 h after transfection, cells were incubated in either full medium or EBSS for 2 h, then fixed and immunostained to detect endogenous mAtg9 localization. The percentage of cells with dispersed mAtg9 was quantified as in Figure 2E (data are represented as mean±s.e.m. n=200 cells, full medium control versus p38α siRNA (**P=0.0027); EBSS control versus p38α siRNA (*P=0.0459); Student's t‐test). Bars=5 μm


( D ) HEK293A cells were transfected with siRNA for p38α , homogenized and subjected to centrifugation , and the resulting post ‐ nuclear supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol as in Figure 2A . Equal protein amounts were resolved by SDS - PAGE and immunoblotted with anti ‐ MPR , anti ‐ HA , and anti ‐ p38α antibodies . Data are representative of four experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293A cells were transfected with siRNA for p38α, homogenized and subjected to centrifugation, and the resulting post‐nuclear supernatant (PNS) was fractionated by centrifugation at 100 000 g into membrane pellet and cytosol as in Figure 2A. Equal protein amounts were resolved by SDS-PAGE and immunoblotted with anti‐MPR, anti‐HA, and anti‐p38α antibodies. Data are representative of four experiments.


( E ) 72 h cells were transfected with control , p38α siRNA or both p38α siRNA , and p38IP siRNA . At 72 h after transfection , cells were incubated in either full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . Cells lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . The membrane was also probed with anti ‐ p38α . LC3 lipidation was quantified as the amount of LC3II / LC3I . Data are representative of two experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O 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) 72 h cells were transfected with control, p38α siRNA or both p38α siRNA, and p38IP siRNA. At 72 h after transfection, cells were incubated in either full medium, full medium with leupeptin, EBSS, or EBSS with leupeptin for 2 h. Cells lysates were analysed for LC3 lipidation using an anti‐LC3 antibody. The membrane was also probed with anti‐p38α. LC3 lipidation was quantified as the amount of LC3II/LC3I. Data are representative of two experiments.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography .

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

C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heme oxygenase activity is increased by CoPP and decreased by SnPP in the liver as measured by gas chromatography.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Total leukocyte and red blood cell count in PB . CoPP increases the number of all WBC populations . Granulocyte and monocyte percentage are increasing , whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP .

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

C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Total leukocyte and red blood cell count in PB. CoPP increases the number of all WBC populations. Granulocyte and monocyte percentage are increasing, whereas lymphocyte percentage is decreasing among PB leukocytes after CoPP.


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases the concentrations of a group of cytokines ( red box ) .

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

C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).


C3H mice were injected with CoPP , SnPP or solvent controls ( NaCl , DMSO ) each second day for 5 days . Samples were collected 24 hours after the last injection . Selected cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP increases concentrations of G - CSF , MCP - 1 , IP - 10 , IL - 6 and IL - 5 . Results are shown as mean + SEM , one - way Anova with Bonferroni post - test , n = 5 mice per group .

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

C3H mice were injected with CoPP, SnPP or solvent controls (NaCl, DMSO) each second day for 5 days. Samples were collected 24 hours after the last injection. Selected cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases concentrations of G-CSF, MCP-1, IP-10, IL-6 and IL-5. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 5 mice per group.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . ( A ) Cell numbers of main leukocyte populations in PB measured by flow cytometry . Both G - CSF and CoPP increase numbers of CD45 + cells in blood . G - CSF and CoPP similarly increase monocyte numbers , but the increase of granulocytes is higher after G - CSF . Lymphocytes , including T cells , are increased only by G - CSF and not by CoPP .

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

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. (A) Cell numbers of main leukocyte populations in PB measured by flow cytometry. Both G-CSF and CoPP increase numbers of CD45+ cells in blood. G-CSF and CoPP similarly increase monocyte numbers, but the increase of granulocytes is higher after G-CSF. Lymphocytes, including T cells, are increased only by G-CSF and not by CoPP.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Cytokine and growth factor concentrations in plasma measured by Luminex assay . CoPP induces a group of cytokines ( red box ) which are not induced by G - CSF .

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

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Plasma concentrations of selected cytokines and growth factors . CoPP highly increases concentration of endogenous G - CSF and IL - 6 that are not increased by G - CSF .

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

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP decreases percentage of Ly6G + macrophages in the

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 B-CONTROLLED_VAR O O O O O O O O

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP decreases percentage of Ly6G+ macrophages in the BM


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . Treatment with G - CSF and CoPP increases relative spleen weight .

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 B-CONTROLLED_VAR O O O O O

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. Treatment with G-CSF and CoPP increases relative spleen weight.


C57BL6xFVB mice were injected with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) daily for 5 days . Samples were collected 6 h after the last injection . viSNE maps of CD11b + CD11c - blood cells , colored by SSC value . Three independent experiments were performed and the results were pooled . Results are shown as mean + SEM , one - way Anova with Bonferroni post - test , n = 7 mice per group .

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

C57BL6xFVB mice were injected with G-CSF, CoPP or solvent controls (NaCl, DMSO) daily for 5 days. Samples were collected 6 h after the last injection. viSNE maps of CD11b+ CD11c- blood cells, colored by SSC value. Three independent experiments were performed and the results were pooled. Results are shown as mean + SEM, one-way Anova with Bonferroni post-test, n = 7 mice per group.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . ( A ) Representative flow cytometry plots show higher granularity ( SSC ) and Ly6G expression in CoPP - mobilized granulocytes than in G - CSF mobilized granulocytes .

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

C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. (A) Representative flow cytometry plots show higher granularity (SSC) and Ly6G expression in CoPP-mobilized granulocytes than in G-CSF mobilized granulocytes.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after the last injection . Relative abundance of different granulocyte phenotypes in blood of mice treated with G - CSF and CoPP . Mice treated with CoPP have higher proportion of granulocytes with mature phenotype , than mice treated with G - CSF .

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

C57BL/6 mice were treated with G-CSF, CoPP or solvent controls (NaCl, DMSO) for 5 consecutive days. Samples were collected 6 hours after the last injection. Relative abundance of different granulocyte phenotypes in blood of mice treated with G-CSF and CoPP. Mice treated with CoPP have higher proportion of granulocytes with mature phenotype, than mice treated with G-CSF.