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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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


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

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 ) .

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

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

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


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

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

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


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

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

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

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


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

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


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

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

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


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

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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 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 .

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


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

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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 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 .

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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 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 ) .

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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. Heatplot of the cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP increases the concentrations of a group of cytokines (red box).


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

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

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


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

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

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


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

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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. Cytokine and growth factor concentrations in plasma measured by Luminex assay. CoPP induces a group of cytokines (red box) which are not induced by G-CSF.


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

O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-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. Plasma concentrations of selected cytokines and growth factors. CoPP highly increases concentration of endogenous G-CSF and IL-6 that are not increased by G-CSF.


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

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

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


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

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

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


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

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

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


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

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

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


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

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

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

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR 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. 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 + ) cells in blood , using CD48 and CD150 to distinguish HSC ( hematopoietic stem cells ) , MPP ( multipotent progenitors ) and HPC ( hematopoietic progenitors ) .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O 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 - ) and HPC - 2 ( KLS CD48 + CD150 + ) cells than G - CSF to the 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 percentage of KLS cells in BM , what is related to increase in HPC populations , but not in LT - HSC fraction .

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

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


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 - ) .

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

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 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 .

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

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 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

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

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

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

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

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

O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR I-CONTROLLED_VAR 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 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 ) .

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

O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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 ; 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 .

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

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

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

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 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 .

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 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

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 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

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 B-CONTROLLED_VAR 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). Cytokine and growth factor concentrations in plasma


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 ] .

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

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 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 .

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

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 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 .

O O O O B-MEASURED_VAR 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

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 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 .

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

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

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O 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 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 ) .

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 B-MEASURED_VAR 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 B-CONTROLLED_VAR O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O O O O O O O O O O O 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).


F Representative immunoblot comparing the accumulation of BiP - AMP in reactions constituted with the indicated concentrations of wild - type and Arg371Ser FICD , performed as in A ( upper panel ) . The lower panel is a Coomassie - stained gel of the reaction constituents . An N - terminally extended version of the wild - type enzyme ( residues 45 - 458was used in lanes 2 and 5 - 7 , to distinguish its migration on the gel from the mutant ( residues 104 - 445 ) . The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F ( from n = 3 , biological replicates , mean values ± SEM ) .

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

F Representative immunoblot comparing the accumulation of BiP-AMP in reactions constituted with the indicated concentrations of wild-type and Arg371Ser FICD, performed as in A (upper panel). The lower panel is a Coomassie-stained gel of the reaction constituents. An N-terminally extended version of the wild-type enzyme (residues 45-458was used in lanes 2 and 5-7, to distinguish its migration on the gel from the mutant (residues 104-445). The bar diagram provides quantification of the AMPylated BiP signals relative to total BiP of experiments as in F (from n = 3, biological replicates, mean values ± SEM).


A Immunoblots of AMPylated proteins detected with a pan - AMP antibody , BiP , FICD and eIF2α ( a loading control ) in lysates of FICD ∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD . Signals corresponding to AMPylated BiP , auto - AMPylated FICDE234G , total BiP , FICD and eIF2α are indicated . The novel species reactive with the anti - FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S ( * ) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369 .

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

A Immunoblots of AMPylated proteins detected with a pan-AMP antibody, BiP, FICD and eIF2α (a loading control) in lysates of FICD∆ CHO cells transiently transfected with plasmids encoding the indicated derivatives of FICD. Signals corresponding to AMPylated BiP, auto-AMPylated FICDE234G, total BiP, FICD and eIF2α are indicated. The novel species reactive with the anti-FICD antiserum in lysates of cells transfected with plasmids expressing FICDR371S (*) likely reflects a glycosylated isoform arising from the creation of a new glycosylation site at Asn369.


C Quantification of the data from repeats of the experiment as shown in B . The upper two graphs show the percent of cells with high CHOP : : GFP or high XBP : : Turq for each transfection . The lower panel provides quantitation of the median fluorescence intensity for each reporter . Mean ± SD , 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 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

C Quantification of the data from repeats of the experiment as shown in B. The upper two graphs show the percent of cells with high CHOP::GFP or high XBP::Turq for each transfection. The lower panel provides quantitation of the median fluorescence intensity for each reporter. Mean ± SD, n=4, biological replicates.


B On the left are immunoblots of AMPylated proteins detected with pan - AMP , BiP and eIF2α ( a loading control ) reactive antibodies in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) for the indicated time . In the panel on the right are immunoblots of samples immunoprecipitated with a BiP - specific antiserum from the lysates of the samples shown on the left .

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

B On the left are immunoblots of AMPylated proteins detected with pan-AMP, BiP and eIF2α (a loading control) reactive antibodies in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) for the indicated time. In the panel on the right are immunoblots of samples immunoprecipitated with a BiP-specific antiserum from the lysates of the samples shown on the left.


C Immunoblots as in B in lysates of cells of the indicated genotype . The cells were untreated or exposed to the protein synthesis inhibitor , cycloheximide ( Cx , 100 µg / mL ) and / or thapsigargin ( Tg ) for the indicated periods prior to harvest . D Quantification of ratio of the BiP - AMP to total BiP signal ( expressed in arbitrary units ) of repeats of the experiment shown in C . Mean ± SD of the BiP - AMP signal normalized to total BiP signal from the same samples are depicted , ( P values by two tailed t - test , n = 6 , 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-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O 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 B-MEASURED_VAR O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR 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

C Immunoblots as in B in lysates of cells of the indicated genotype. The cells were untreated or exposed to the protein synthesis inhibitor, cycloheximide (Cx, 100 µg/mL) and/or thapsigargin (Tg) for the indicated periods prior to harvest. D Quantification of ratio of the BiP-AMP to total BiP signal (expressed in arbitrary units) of repeats of the experiment shown in C. Mean ± SD of the BiP-AMP signal normalized to total BiP signal from the same samples are depicted, (P values by two tailed t-test, n = 6, biological replicates).


A Representative immunoblot of AMPylated proteins in FICD ∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes ( replicated three times ) . Note that the hyperactive E234G mutant bearing the K124E - H131A mutations that compromise its ability to interact with BiP , is nonetheless consistently auto - AMPylated .

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 B-MEASURED_VAR O O O O O O O O

A Representative immunoblot of AMPylated proteins in FICD∆ CHO cells transiently transfected with expression plasmids encoding the indicated FICD enzymes (replicated three times). Note that the hyperactive E234G mutant bearing the K124E-H131A mutations that compromise its ability to interact with BiP, is nonetheless consistently auto-AMPylated.