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

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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 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 to . X axis is sum over HSP12 mRNA ( see D ) . Y axis is maximum over Hsp12 - GFP values . red indicates 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 total open time parameter inferred from mRNA data ( X axis ) to one inferred from 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 ( top ) traces of ( 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 production rate estimated from single traces vs . production rate estimated from population average . Each point represents a different yeast .

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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 ~ 1600 double mutant strains . color represents total Hsp12 - GFP produced . same amount of GFP can be produced with multiple parameter combinations . ellipses illustrate iso - expression areas in graph . Three examples of strains ( marked in B ) with nearly 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 genetic interaction trends of mutants . For each single mutant we plot median of double mutants that contain it ( purple dots ) . gray dots are double mutant strains ( B ) .

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

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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 interactions of Δhog1 for total open time parameter .

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


Epistatic interactions of Δmck1 in production rate

Epistatic interactions of Δmck1 in the production rate parameter


Epistatic interactions of Δmck1 in total open time

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 Δmsn2Δmsn4 production profiles , Figure EV4A , Methods ) .

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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 response of stress genes . integral over mRNA values in WT ( X axis ) vs . integral in Δmck1 ( Y values ) . inset shows integral for HSP12 gene .

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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.


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

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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).


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

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 : of ( Y axis ) over time ( X axis ) . Bottom : of number of import events in live - time - lapse microscopy at 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 of a PDGFB tumour : H & E on right to identify tumour , an adjacent section stained for Iba1 ( red ) , p - S6 ( green ) DAPI ( blue ) at centre region selection for on left ( non - tumour in blue , tumour edge in orange tumour in blue ) .

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(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 of , edge adjacent non - tumour brain tissue for Iba1 , p - S6 DAPI in Pten - / - p53 - / - ( right ) GL261 ( left ) tumours . of Iba1 + co - expressing p - S6 in 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 ) , Pten - / - p53 - / - Idh1mut ( n = 3 ) ( mean ± ; Two - Way ANOVA Tukey ) .

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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 of ( F ) Rps6 ( G ) Eif4ebp1 in healthy versus GL261 microglia macrophage ( n = 3 ; mean ± , likelihood 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 lysates collected at 4hr ( B normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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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 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 ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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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 lysates collected at 4hr normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + Torin was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * 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 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

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 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 ± , Two - Way ANOVA Tukey ) . * 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 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 ± , Two - Way ANOVA Tukey ) . * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 comparing mGICPten - / - ; p53 - / - - CM versus mGICPten - / - ; p53 - / - - CM + inhibitor .

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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 lysates collected at 0 . 5hr ( H ) normalised against non - phosphorylated vinculin analysed on same blot . Each treatment ( mNSC - CM , Pten - / - p53 - / - mGIC - CM , Pten - / - p53 - / - mGIC - CM + LY ) was normalised to unconditioned control ( n = 3 ; mean ± , Two - Way ANOVA Tukey ) . * 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

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 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 ± , Two - Way ANOVA Tukey ) . * 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 for Cx3cr1 - Rheb1Δ / Δ ( n = 7 ) Rheb1fl / fl ( n = 7 ) mice . Chi square .

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


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

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

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

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

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

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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 ) Cx3cr1 - Rheb1Δ / Δ ( n = 4 ) were stained for CD8 , CD4 FoxP3 . DAB was quantified as % of positive using Definiens software . Representative of stained tissues are shown . ( mean ± ; unpaired parametric t ) . 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 ) of differential correlation between CIBERSORT fractions signatures enrichment scores ( ssGSEA ) for lymphocytes in 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 ( C ) CD8 CTL ( D ) expression of Ki67 , perforin , granzyme b , IFNγ , in Cx3cr1 - Rheb1fl / fl ( n = 5 ) Rheb1Δ / Δ ( n = 5 ) GL261 tumours . Representative FACS plots show control samples ( tumour - derived stimulated in culture in absence of inhibitor cocktail - in orange ) compared to stimulated derived from Rheb1fl / fl ( fl / fl , red ) Cx3cr1 - Rheb1Δ / Δ ( Δ / Δ , blue ) GL261 tumours ( stimulated in culture with inhibitor cocktail ) ( mean ± ; Two - Way ANOVA Tukey ) .

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

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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 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 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 then stimulated with mNSC - CM , mGICPten - / - ; p53 - / - - CM , or GL261 - CM . p - NF - ĸB ( p - P65 ) p - STAT3 were analysed by immunoblotting of whole 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 of NF - ĸB ( P65 ) in conditioned microglia . of translocation of NF - κB from . Units represent % of voxels in NF - κB channel colocalised with DAPI ( n = 3 ; mean ± ; One - Way ANOVA Tukey - 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 , Il10 by conditioned microglia was determined by qPCR . Each treatment was normalised to housekeeping gene unconditioned control . ( n = 3 ; mean ± ; Two - Way ANOVA Tukey ) .

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

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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 mTOR signature versus TAM - MG or TAM - BMDM signatures in TCGA GBM transcriptomic data . Comparison carried out on IDH - wild - type samples , in a subgroup specific manner according to Wang ' s classifier . of circle is indicative of Rsquare value 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 microglia enrichment ( + ve correlation , group 1 in orange ) 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 fractions calculated from TPM of TCGA IDH - wild type GBM samples from group 1 compared to those from group 2 .

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

(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 CD11b ( H ) Iba1 TREM2 . Scale bar 20µm .

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

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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 ) AH109 was co ‐ transformed with pGBKT7 containing C ‐ terminal ( CT ) domain of mAtg9 ( bait ) pGADT7 , or pACT2 , containing CT domain of p38IP ( prey ) . 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 were transfected with RFP - mAtg9 HA - p38IP . HA - p38IP was immunoprecipitated with an anti ‐ HA antibody 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 of mAtg9 with ( D ) HA - full ‐ p38IP ( HA - FL ) , ( E ) HA - C ‐ terminal p38IP ( HA - CT ) , ( F ) HA - N ‐ terminal p38IP ( HA - NT ) . Bars = 5 μm . co ‐ localization seen in ( D ) ( E ) was not because of overlap of randomly distributed vesicles . Cross ‐ correlation function ( CCF ) ( van Steensel et al , 1996 ) showed a peak maximum Rp value around ΔX = 0 , indicating that co ‐ localization between mAtg9 p38IP is positively correlated 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 were transfected with HA - p38IP alone or co ‐ transfected with HA - p38IP RFP - mAtg9 , homogenized subjected to centrifugation , resulting post ‐ supernatant ( PNS ) was fractionated by centrifugation at 100 000 g into pellet cytosol . Equal amounts were resolved by SDS - PAGE immunoblotted with anti ‐ mannose ‐ 6 ‐ phosphate receptor ( MPR ) antibody as a control for ‐ associated , anti ‐ superoxide dismutase ( SOD ) antibody as a control for , anti ‐ mAtg9 antibody , anti ‐ HA antibody . HA - p38IP were quantified using ImageJ software plotted relative to HA - p38IP in 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 were depleted of mAtg9 using siRNA , then transfected with HA - p38IP incubated in full medium or EBSS ( data not shown ) . asterisk indicates mAtg9 ‐ depleted . Bar is 5 μM . of versus in control depleted of mAtg9 under fed starvation conditions was analysed using ImageJ software ( * * * P = 0 . 0001 , Students t ‐ )

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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 were transfected with HA - p38IP processed for indirect immunofluorescence . HA - p38IP was detected with an anti ‐ HA antibody endogenous mAtg9 was detected with an anti ‐ mAtg9 antibody . HA - p38IP localizes to nucleus in full medium or EBSS . HA - p38IP co ‐ localizes with mAtg9 in periphery of ( 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 transfected with HA - p38IP alone or with RFP - mAtg9 HA - p38IP . A total of 5 % of lysates or immunoprecipitates were probed with anti ‐ HA or anti ‐ mAtg9 antibodies . left right bottom panels were obtained from same immunoblot , but exposed for different times to allow 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 were transfected with control or p38IP siRNA . At 72 h after transfection , were incubated in either full medium or EBSS for 2 h , then fixed immunostained to detect endogenous mAtg9 localization . mAtg9 localization was quantified by visually scoring for either a juxta ‐ or dispersed phenotype . Bars = 5 μm ( data are represented as mean ± s . e . m . of 200 , * P = 0 . 0413 , Student ' s t ‐ ) .

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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 were transfected with control or p38IP siRNA . At 72 h after transfection , 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 . 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 , * * * P = 0 . 0002 , Students t ‐ ) .

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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 were transfected with siRNA control , siRNA for p38IP , siRNA for ULK1 . 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 were transfected with siRNA control , siRNA for p38IP . After incubation for 2 h as in ( A ) , samples were immunoblotted with anti ‐ LC3 anti ‐ actin antibodies . Endogenous LC3II / LC3I were quantified presented as arbitrary units . In ( B ) , n = 4 , * * * P = 0 . 0001 * P = 0 . 0324 , Students t ‐ . 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 were transfected with siRNA control or siRNA p38IP , incubated in full medium , or for 2 4 h in EBSS . Samples were immunoblotted with anti ‐ p62 antibodies , 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 were transfected with HA - p38IP or myc - ULK1 C ‐ terminal domain ( CTD ) . After 24 h , were labelled with [ 14C ] valine as described in Material Methods section , incubated in either full medium or EBSS for 2 h . were then collected analysed for long ‐ lived 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 ‐ ) .

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

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(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 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 . were analysed as in Figure 2E . Bars = 5 μm . Data are represented as mean ± s . e . m . of 60 , EBSS mock versus EBSS anisomycin ( * P = 0 . 0142 ) ; EBSS mock versus EBSS UV ( * P = 0 . 0150 ) ; Student ' s t ‐ .

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

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(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 labelled with [ 14C ] valine , as described in Material 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 . were then analysed for long ‐ lived 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 ‐ ) .

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

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

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

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

(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 were transfected with Flag - p38α for 24 h . were incubated in either full medium , EBSS , or EBSS plus leupeptin for 2 h , lysed , analysed for endogenous LC3 lipidation using an anti ‐ LC3 antibody , immunoblotted with anti ‐ Flag antibody . LC3 lipidation was quantified as 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 ) .

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

O O O O O O O O O O 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 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 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 were transfected with control or p38α siRNA . At 72 h after transfection , were incubated in either full medium , EBSS , or EBSS containing leupeptin for 2 h . lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . was also probed with anti ‐ actin anti ‐ p38α antibodies . LC3 lipidation was quantified as 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 ‐ ) .

O O O O O O 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

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

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

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(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 were transfected with control , p38α siRNA or both p38α siRNA , p38IP siRNA . At 72 h after transfection , were incubated in either full medium , full medium with leupeptin , EBSS , or EBSS with leupeptin for 2 h . lysates were analysed for LC3 lipidation using an anti ‐ LC3 antibody . was also probed with anti ‐ p38α . LC3 lipidation was quantified as 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 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

(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 last injection . Heme oxygenase activity is increased by CoPP decreased by SnPP in liver as measured by chromatography .

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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 last injection . Total leukocyte red blood in PB . CoPP increases number of WBC populations . Granulocyte monocyte are increasing , whereas lymphocyte is decreasing among PB leukocytes after CoPP .

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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 last injection . Heatplot of cytokine growth factor concentrations in plasma measured by Luminex . CoPP increases 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 B-CONTROLLED_VAR 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 last injection . Selected cytokine growth factor concentrations in plasma measured by Luminex . CoPP increases concentrations of G - CSF , MCP - 1 , IP - 10 , IL - 6 IL - 5 . Results are shown as mean + , one - way Anova with Bonferroni post - , 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 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

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 last injection . ( A ) numbers of main leukocyte populations in PB measured by flow cytometry . Both G - CSF CoPP increase numbers of CD45 + in blood . G - CSF CoPP similarly increase monocyte numbers , but increase of granulocytes is higher after G - CSF . Lymphocytes , including T , are increased only by G - CSF 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 B-CONTROLLED_VAR 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-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 last injection . Cytokine growth factor concentrations in plasma measured by Luminex . 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 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 last injection . Plasma concentrations of selected cytokines growth factors . CoPP highly increases of endogenous G - CSF 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 B-CONTROLLED_VAR 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 last injection . Treatment with G - CSF CoPP decreases of Ly6G + macrophages in

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 B-CONTROLLED_VAR 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 last injection . Treatment with G - CSF CoPP increases relative spleen .

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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 last injection . viSNE maps of CD11b + CD11c - blood , colored by SSC value . Three independent experiments were performed results were pooled . Results are shown as mean + , one - way Anova with Bonferroni post - , 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

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 last injection . ( A ) Representative flow cytometry plots show higher granularity ( SSC ) Ly6G expression in CoPP - mobilized granulocytes than in G - CSF mobilized granulocytes .

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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 last injection . Relative abundance of different granulocyte in blood of mice treated with G - CSF 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 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.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . Relative abundance of different granulocyte in BM ( C ) of mice treated with G - CSF 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 BM (C) 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.


C57BL / 6 mice were treated with G - CSF , CoPP or solvent controls ( NaCl , DMSO ) for 5 consecutive days . Samples were collected 6 hours after last injection . of isolated from C57BL / 6xFVB mice treated with G - CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli .

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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. The percentage of cells isolated from C57BL/6xFVB mice treated with G-CSF or CoPP that are producing reactive oxygen species after incubation with indicated stimuli.


Gating strategy of KLS ( c - Kit + Lin - Sca - 1 + ) in blood , using CD48 CD150 to distinguish HSC ( hematopoietic ) , MPP ( multipotent progenitors ) HPC ( hematopoietic progenitors ) .

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

Gating strategy of KLS (c-Kit+Lin-Sca-1+) cells in blood, using CD48 and CD150 to distinguish HSC (hematopoietic stem cells), MPP (multipotent progenitors) and HPC (hematopoietic progenitors).


CoPP mobilizes higher numbers of HSC ( KLS CD48 - CD150 + ) , MPP ( KLS CD48 - CD150 - ) , HPC - 1 ( KLS CD48 + CD150 - ) HPC - 2 ( KLS CD48 + CD150 + ) than G - CSF to PB .

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CoPP mobilizes higher numbers of HSC (KLS CD48-CD150+), MPP (KLS CD48-CD150-), HPC-1 (KLS CD48+CD150-) and HPC-2 (KLS CD48+CD150+) cells than G-CSF to the PB.


Treatment with CoPP increases of KLS in BM , what is related to increase in HPC populations , but not in LT - HSC fraction .

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Treatment with CoPP increases percentage of KLS cells in BM, what is related to increase in HPC populations, but not in LT-HSC fraction.


number of KLS - ( c - Kit + Lin - Sca - 1 - ) is higher in mice treated with CoPP than in mice treated with G - CSF . Numbers of lineage committed progenitors : GMP ( granulocyte - macrophage progenitors , KLS - CD48 + CD150 - CD34 + ) MEP ( megakaryocyte - erythroid progenitors , KLS - CD48 + CD150 + CD34 - ) were higher after CoPP administration than after G - CSF . G - CSF CoPP similarly increase EP numbers ( erythrocyte progenitors , KLS - CD48 - CD150 - CD34 - ) .

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The number of KLS- (c-Kit+Lin-Sca-1-) is higher in mice treated with CoPP than in mice treated with G-CSF. Numbers of lineage committed progenitors: GMP (granulocyte-macrophage progenitors, KLS- CD48+CD150-CD34+) and MEP (megakaryocyte-erythroid progenitors, KLS- CD48+CD150+CD34-) were higher after CoPP administration than after G-CSF. G-CSF and CoPP similarly increase EP numbers (erythrocyte progenitors, KLS- CD48-CD150-CD34-).


Both G - CSF CoPP treatments decrease KLS - in BM , however decrease after CoPP is smaller . G - CSF CoPP decrease percentages of MEP EP , but only G - CSF decreases of GMP . Results are shown as mean + values , one - way Anova with Bonferroni post - , n = 7 mice per group .

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Both G-CSF and CoPP treatments decrease the KLS- percentage in the BM, however the decrease after CoPP is smaller. G-CSF and CoPP decrease percentages of MEP and EP, but only G-CSF decreases the percentage of GMP. Results are shown as mean + individual values, one-way Anova with Bonferroni post-test, n = 7 mice per group.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . ( A ) Effect of G - CSF CoPP - induced mobilization in C57BL / 6 - Tg ( UBC - GFP ) 30Sch / J donor mice ( mean + values , one - way Anova with Bonferroni post - , n = 16 mice per group ) : Donor mice treated with CoPP had highest number of mobilized granulocytes ( i ) KLS ( ii ) , compared to control or G - CSF - treated donor

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 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 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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. (A) Effect of G-CSF and CoPP-induced mobilization in C57BL/6-Tg(UBC-GFP)30Sch/J donor mice (mean + individual values, one-way Anova with Bonferroni post-test, n = 16 mice per group): Donor mice treated with CoPP had the highest number of mobilized granulocytes (i) and KLS cells (ii), compared to control or G-CSF-treated donor mice


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Recipient mice which were transplanted with CoPP - mobilized PBMC had highest number of WBC , PLT highest hematocrit values 2 after PBMC transplantation , compared to G - CSF - mobilized control PBMC recipients ( mean + values , one - way Anova with Bonferroni post - , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest number of WBC, PLT and the highest hematocrit values 2 weeks after PBMC transplantation, compared to G-CSF-mobilized and control PBMC recipients (mean + individual values, one-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Recipient mice which were transplanted with CoPP - mobilized PBMC had highest chimerism among CD45 + , granulocytes B 2 , 4 18 after transplantation . Chimerism among T was highest in recipients of G - CSF - mobilized PBMC 2 after transplantation , but 18 after transplantation it was highest in CoPP - mobilized PBMC recipients ( mean + , two - way Anova with Bonferroni post - , NaCl : n = 8 , G - CSF : n = 7 , CoPP : n = 9 mice per group ) .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Recipient mice which were transplanted with CoPP-mobilized PBMC had the highest chimerism among CD45+ cells, granulocytes and B cells 2, 4 and 18 weeks after transplantation. Chimerism among T cells was the highest in recipients of G-CSF-mobilized PBMC 2 weeks after transplantation, but 18 weeks after transplantation it was the highest in CoPP-mobilized PBMC recipients (mean + SEM, two-way Anova with Bonferroni post-test, NaCl: n = 8, G-CSF: n = 7, CoPP: n = 9 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . majority of recipients transplanted with G - CSF CoPP - mobilized PBMC have detectable GFP chimerism among BM KLS .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. The majority of primary recipients transplanted with G-CSF and CoPP-mobilized PBMC have detectable GFP chimerism among BM KLS cells.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . Fraction of secondary recipient mice with chimerism in PB CD45 + exceeding 1 % tends to be higher after CoPP - mobilized PBMC transplant than G - CSF - mobilized PBMC transplant .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Fraction of secondary recipient mice with chimerism in PB CD45+ cells exceeding 1% tends to be higher after CoPP-mobilized PBMC transplant than G-CSF-mobilized PBMC transplant.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . PB chimerism in secondary recipients is higher in CoPP group than in control group ( mean + values , Kruskal - Wallis with Dunn ' s post - , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. PB chimerism in secondary recipients is higher in CoPP group than in the control group (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . of secondary recipient mice with GFP chimerism in BM KLS ( G ) KLS - ( H ) higher that 1 % is similar in G - CSF CoPP groups .

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 B-CONTROLLED_VAR O O

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. Percentage of secondary recipient mice with GFP chimerism in BM KLS (G) and KLS-(H) cells higher that 1% is similar in G-CSF and CoPP groups.


Donor GFP + mice were treated with NaCl , G - CSF or CoPP daily . At 5th day of treatment we transplanted 5x106 of isolated PBMC to lethally irradiated GFP - recipient mice , together with 105 GFP - BM - derived competitor . After 18 or 20 we performed secondary transplantation of recipients ' BM to lethally irradiated secondary recipients followed chimerism for additional 14 . GFP chimerism in KLS ( I ) KLS - ( J ) in BM of secondary recipients ( mean + values , Kruskal - Wallis with Dunn ' s post - , NaCl : n = 7 , G - CSF : n = 6 , CoPP : n = 8 mice per group ) .

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

Donor GFP+ mice were treated with NaCl, G-CSF or CoPP daily. At the 5th day of treatment we transplanted 5x106 of isolated PBMC to the lethally irradiated GFP- recipient mice, together with 105 GFP- BM-derived competitor cells. After 18 or 20 weeks we performed secondary transplantation of primary recipients' BM to lethally irradiated secondary recipients and followed the chimerism for additional 14 weeks. GFP chimerism in KLS (I) and KLS- (J) cells in the BM of secondary recipients (mean + individual values, Kruskal-Wallis test with Dunn's post-test, NaCl: n = 7, G-CSF: n = 6, CoPP: n = 8 mice per group).


Wild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; injections were repeated daily for 5 days mice were sacrificed 6 hours after last CoPP injection . G - CSF IL - 6 in

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

Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. G-CSF and IL-6 concentration in plasma


Wild type C57BL / 6 mice were injected with anti - G - CSF antibody alone or in combination with anti - IL - 6 antibody one hour prior to CoPP treatment ; injections were repeated daily for 5 days mice were sacrificed 6 hours after last CoPP injection . CD45 + , granulocytes HSC numbers in PB measured by flow cytometry .

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

Wild type C57BL/6 mice were injected with anti-G-CSF antibody alone or in combination with anti-IL-6 antibody one hour prior to CoPP treatment; the injections were repeated daily for 5 days and mice were sacrificed 6 hours after the last CoPP injection. CD45+ cells, granulocytes and HSC numbers in PB measured by flow cytometry.


HO - 1 deficient control C57BL / 6xFVB mice were injected with CoPP three times , every second day sacrificed 24 hours after last injection . Total leukocyte in PB ( HO - 1 + / + : n = 5 , HO - 1 - / - DMSO : n = 6 , HO - 1 - / - CoPP : n = 4 mice per group ) . Granulocyte among PB leukocytes .

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

HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Total leukocyte counts in PB (HO-1+/+: n = 5, HO-1-/- DMSO: n = 6, HO-1-/- CoPP: n = 4 mice per group). Granulocyte percentage among PB leukocytes.


HO - 1 deficient control C57BL / 6xFVB mice were injected with CoPP three times , every second day sacrificed 24 hours after last injection . Cytokine growth factor concentrations in plasma ( E - HO - 1 + / + n = 5 , HO - 1 - / - DMSO : n = 5 , HO - 1 - / - CoPP : n = 3 mice per group ) . Results are shown as mean + , two - way Anova with Bonferroni post - .

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 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

HO-1 deficient and control C57BL/6xFVB mice were injected with CoPP three times, every second day and sacrificed 24 hours after last injection. Cytokine and growth factor concentrations in plasma (E - HO-1+/+ n = 5, HO-1-/- DMSO: n = 5, HO-1-/- CoPP: n = 3 mice per group). Results are shown as mean + SEM, two-way Anova with Bonferroni post-test.


Nrf2 - deficient control C57BL / 6 mice were injected with CoPP daily for 5 days sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . CD45 + numbers in PB measured by flow cytometry . Granulocyte among PB leukocytes .

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-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

Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). CD45+ cell numbers in PB measured by flow cytometry. Granulocyte percentage among PB leukocytes.


Nrf2 - deficient control C57BL / 6 mice were injected with CoPP daily for 5 days sacrificed 6 hours after last injection ( DMSO : n = 4 , CoPP : n = 5 mice per group ) . Cytokine growth factor concentrations in

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-CONTROLLED_VAR O O O O O O O O O O O O O O

Nrf2-deficient and control C57BL/6 mice were injected with CoPP daily for 5 days and sacrificed 6 hours after last injection (DMSO: n = 4, CoPP: n = 5 mice per group). Cytokine and growth factor concentrations in plasma


Comparison of constitutively active rhodopsin mutants M257Y6 . 40 ( left [ 21 ] ) , T94I2 . 61 ( middle ) G90D2 . 57 ( right [ 16 ] ) . - trans ( yellow ) cis retinal isomers ( orange ) , side of retinal attachment K2967 . 43 ( blue slate ) , retinal counterion E1133 . 28 ( blue slate ) , GαCT peptides ( cyan ) constitutively activating mutations M257Y6 . 40 , T94I2 . 61 , G90D2 . 57 ( green ) are shown as spheres . Palmitoylation at C323H8 glycosylation at N15NT are shown as sticks . lower panels compare M257Y6 . 40 , T94I2 . 61 G90D2 . 57 retinal - binding pockets . constructs also contain an additional thermostabilizing disulfide bridge , depicted as ( c - c ) , to enhance stability [ 18 ] crystallizability [ 19 ] without changing rhodopsin activation pathway [ 20 ] .

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

Comparison of constitutively active rhodopsin mutants M257Y6.40 (left [21]), T94I2.61 (middle) and G90D2.57 (right [16]). All-trans (yellow) and cis retinal isomers (orange), the side of retinal attachment K2967.43 (blue slate), the retinal counterion E1133.28 (blue slate), GαCT peptides (cyan) and the constitutively activating mutations M257Y6.40, T94I2.61, and G90D2.57 (green) are shown as spheres. Palmitoylation at C323H8 and glycosylation at N15NT are shown as sticks. The lower panels compare the M257Y6.40, T94I2.61 and G90D2.57 retinal-binding pockets. All constructs also contain an additional thermostabilizing disulfide bridge, depicted as (c-c), to enhance the stability [18] and crystallizability [19] without changing the rhodopsin activation pathway [20].


Kinetic characterization of rhodopsin state . Thermal decay of state rhodopsin : A ) WT , B ) T94I C ) G90D rhodopsin . spectra are normalized to OD280 OD500 / OD480 at t = 0 . D ) Absorption maximum plotted as a function of time fitted to a single exponential decay .

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

Kinetic characterization of the rhodopsin dark state. Thermal decay of dark state rhodopsin: A) WT, B) T94I and C) G90D rhodopsin. The spectra are normalized to the OD280 and OD500/OD480 at t=0. D) Absorption maximum plotted as a function of time and fitted to a single exponential decay.


Retinal isomerization in rhodopsin state : E ) WT , F ) T94I G ) G90D rhodopsin , H ) fraction of 11 - cis retinal is plotted as a function of time fitted to a single exponential decay .

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

Retinal isomerization in the rhodopsin dark state: E) WT, F) T94I and G) G90D rhodopsin, H) The fraction of 11-cis retinal is plotted as a function of time and fitted to a single exponential decay.


SB stability in state rhodopsin : I ) WT , J ) T94I K ) G90D rhodopsin . L ) OD440 is plotted as a function of time fitted to a single exponential decay . decay rates were measured at 55 C .

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

SB stability in dark state rhodopsin: I) WT, J) T94I and K) G90D rhodopsin. L) OD440 is plotted as a function of time and fitted to a single exponential decay. All decay rates were measured at 55 C.


Accelerated molecular dynamic simulation of rhodopsin state : WT , G90D2 . 57 , T94I2 . 61 T94I2 . 61 with protonated E1133 . 28 . In absence of G90D2 . 57 T94I2 . 61 state structure , both aspartate isoleucine were placed in WT ( 1GZM [ 48 ] ) using a favorable rotamer without introducing major clashes with of . However , both isoleucine aspartate in state structures introduced clashes with several were therefore optimized using energy minimization before simulation . WT simulation was done with protonated SB while in G90D2 . 57 system , both E1133 . 28 SB were protonated [ 36 ] . protonated state of E1133 . 28 was unknown in T94I2 . 61 , therefore simulation were done for both deprotonated protonated E1133 . 28 . T94I2 . 61 state conformation with deprotonated E1133 . 28 was similar to that of WT was stable , whereas state of G90D2 . 57 T94I2 . 61 with protonated E1133 . 28 was unstable with a concomitant movement of E1133 . 28 residue away from SB , thereby , causing movement of TM3 opening of E1133 . 28 - SB activation switch . In contrast to crystal structures orientation of retinal - ionone ring in simulations was variable with respect to polyene chain . This indicates a degree of positional freedom within hydrophobic binding pocket .

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

Accelerated molecular dynamic simulation of the rhodopsin dark state: WT, G90D2.57, T94I2.61 and T94I2.61 with protonated E1133.28. In the absence of the G90D2.57 and T94I2.61 dark state structure, both aspartate and isoleucine were placed in the WT (1GZM [48]) using a favorable rotamer without introducing major clashes with the rest of the protein. However, both isoleucine and aspartate in the dark state structures introduced clashes with several amino acids and were therefore optimized using energy minimization before simulation. WT simulation was done with the protonated SB while in the G90D2.57 system, both E1133.28 and SB were protonated [36]. The protonated state of E1133.28 was unknown in T94I2.61, therefore simulation were done for both deprotonated and protonated E1133.28. The T94I2.61 dark state conformation with deprotonated E1133.28 was similar to that of the WT and was stable, whereas the dark state of G90D2.57 and T94I2.61 with protonated E1133.28 was unstable with a concomitant movement of E1133.28 residue away from the SB, thereby, causing movement of TM3 and opening of the E1133.28-SB activation switch.In contrast to the crystal structures the orientation of the retinal -ionone ring in the simulations was variable with respect to the polyene chain. This indicates a degree of positional freedom within the hydrophobic binding pocket.


B exclusion chromatography confirms that oligomerisation tendency of FICDR371S is unperturbed relative to wild - type FICD . Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species . elution profile of a monomeric FICDL258D - H363A mutant is also shown for reference , alongside elution times of molecular standards .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Size exclusion chromatography confirms that the oligomerisation tendency of FICDR371S is unperturbed relative to wild-type FICD. Across a range of assessable enzyme concentrations both FICDs elute as predominantly dimeric species. The elution profile of a monomeric FICDL258D-H363A mutant is also shown for reference, alongside the elution times of molecular weight standards.


C Representative immunoblot comparing ability of wild - type Arg371Ser FICD ( at a wide range of enzyme concentrations ) to catalyse accumulation of BiP - AMP in presence of physiological ATP concentrations ( 5 mM ) . At low enzyme concentrations ( early time points ) AMPylation defect imposed by Arg371Ser is manifest . At higher enzyme concentrations where deAMPylation activity of wild - type enzyme begins to dominate , residual AMPylation activity of FICDR371S results in a greater accumulation of BiP - AMP ( relative to same of wild - type enzyme ) . D of AMPylated BiP signals relative to total BiP of experiments as in C ( mean values ± , n = 4 , biological replicates ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O 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 Representative immunoblot comparing the ability of wild-type and Arg371Ser FICD (at a wide range of enzyme concentrations) to catalyse the accumulation of BiP-AMP in the presence of physiological ATP concentrations (5 mM). At low enzyme concentrations (and early time points) the AMPylation defect imposed by Arg371Ser is manifest. At higher enzyme concentrations where the deAMPylation activity of the wild-type enzyme begins to dominate, the residual AMPylation activity of FICDR371S results in a greater accumulation of BiP-AMP (relative to the same concentration of wild-type enzyme). D Quantification of the AMPylated BiP signals relative to total BiP of experiments as in C (mean values ± SEM, n = 4, biological replicates).