c , d . Incidence of papilloma formation ( ns : non - significant ; Gehan - Breslow - Wilcoxon test ) ( c ) and number of tumours per mouse ( d ) in wild - type ( WT , n = 17 ) and PRSS35 - / - ( n = 22 ) mice treated with DMBA - TPA ( * p = 0 . 0171 ; Wilcoxon matched - paired rank test ) . Data represent means ± S . E . M .

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

c, d. Incidence of papilloma formation (ns: non-significant; Gehan-Breslow-Wilcoxon test) (c) and number of tumours per mouse (d) in wild-type (WT, n=17) and PRSS35-/- (n=22) mice treated with DMBA-TPA (*p=0.0171; Wilcoxon matched-paired rank test). Data represent means ± S.E.M.


b . Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures ( col1a1 : collagen type 1 alpha 1 ; αSMA , alpha smooth muscle actin ; Cyr61 : Cysteine - rich angiogenic inducer 61 ( = CCN1 ) ) in primary fibroblast cultures isolated from WT and PRSS35 - / - mice , untreated or after 24 hours stimulation with 10 ng / mL TGFβ1 ( n ≥ 7 biological replicates per condition ) ( * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M . c Relative mRNA expression levels of the indicated cytokines / growth factors in primary fibroblast cultures ( n ≥ 8 per condition ) ( VEGF - α , vascular endothelial growth factor - alpha ; FGF2 : basic fibroblast growth factor ; FGF7 : keratinocyte growth factor ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M .

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

b. Relative mRNA expression levels of the indicated ECM genes in primary fibroblast cultures (col1a1: collagen type 1 alpha 1; αSMA, alpha smooth muscle actin; Cyr61: Cysteine-rich angiogenic inducer 61 (=CCN1)) in primary fibroblast cultures isolated from WT and PRSS35-/- mice, untreated or after 24 hours stimulation with 10 ng/mL TGFβ1 (n≥7 biological replicates per condition) (** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M. c Relative mRNA expression levels of the indicated cytokines/growth factors in primary fibroblast cultures (n≥8 per condition) (VEGF-α, vascular endothelial growth factor-alpha; FGF2: basic fibroblast growth factor; FGF7: keratinocyte growth factor) (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.


d . Picrosirius red staining of normal skin , day 14 post - wounding ( d14pw ) skin or wound - induced papillomas from WT and PRSS35 KO mice . Red colouring indicates thick fibres , green colouring indicates thin fibres . Scale bars : 200 µm . e . Quantification of picrosirius red staining as total collagen density ( upper panel ) and ratio of red versus green picrosirius staining ( lower panel ) . Using QuPath Bioimage analysis software pixels colouring red or green were determined per area . ( n ≥ 4 technical replicates per condition ) ( * p < 0 . 05 , * * p < 0 . 01 , * * * * p < 0 . 0001 . ; Two - way ANOVA with multiple comparisons ) . Data represent means ± S . E . M .

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

d. Picrosirius red staining of normal skin, day 14 post-wounding (d14pw) skin or wound-induced papillomas from WT and PRSS35 KO mice. Red colouring indicates thick fibres, green colouring indicates thin fibres. Scale bars: 200 µm. e. Quantification of picrosirius red staining as total collagen density (upper panel) and ratio of red versus green picrosirius staining (lower panel). Using QuPath Bioimage analysis software pixels colouring red or green were determined per area. (n≥4 technical replicates per condition) (* p<0.05, ** p<0.01, **** p<0.0001.; Two-way ANOVA with multiple comparisons). Data represent means ± S.E.M.


a . Human skin sections were stained with a PRSS35 - specific antibody ( red ) and counterstained with Dapi ( n = 5 per condition ) . Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal - dermal boundary . Scale bars : 100 μm . b . Quantification of PRSS35 positive signal by Volocity analysis ( n ≥ 4 per condition ; * * p < 0 . 01 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M .

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 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

a. Human skin sections were stained with a PRSS35-specific antibody (red) and counterstained with Dapi (n=5 per condition). Boxed areas are magnifications as shown in the lower panel and dotted line represents the epidermal-dermal boundary. Scale bars: 100 μm. b. Quantification of PRSS35 positive signal by Volocity analysis (n≥4 per condition; ** p<0.01; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.


Balb / C mice were aerosolically challenged with Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR strains . Each data point represents log10 CFU obtained from the infected lung of mice and e rror bar depicts the median with interquartile range for each group . Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group .

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

Balb/C mice were aerosolically challenged with Rv, Rv∆aosR, and Rv∆aosR::aosR strains. Each data point represents log10 CFU obtained from the infected lung of mice and error bar depicts the median with interquartile range for each group. Each data point represents log10 CFU obtained from the infected spleen of a mice and the error bar depicts the mean with SD for each group.


Each data point represents Log10 CFU / lung of an infected animal and the error bar depicts the median with interquartile range for each group .

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

Each data point represents Log10 CFU/lung of an infected animal and the error bar depicts the median with interquartile range for each group.


Relative gene expression of selected DEGs measured by qRT - PCR in Rv compared with Rv treated with CHP ( g ) and Rv as compared with Rv ∆ aosR under oxidative conditions ( h ) . Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two 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 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

Relative gene expression of selected DEGs measured by qRT-PCR in Rv compared with Rv treated with CHP (g) and Rv as compared with Rv∆aosR under oxidative conditions (h). Data were normalized with respect to 16s rRNA and results are expressed as mean log2 fold change + SD of three independent replicates and is representative of one of two biological replicates.


WCLs of Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15 % SDS - PAGE , transferred to nitrocellulose membrane , and probed with indicated antibodies .

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

WCLs of Rv, Rv∆aosR, and Rv∆aosR::aosR subjected to 0 or 50 µM of CHP for 6 h were resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane, and probed with indicated antibodies.


pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p . i ( mean ± SEM ; n = 3 ) .

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

pΦ from B6 were infected with indicated strains and intracellular bacterial viability was monitored 96 h p.i (mean ± SEM; n=3).


The promoter of sigA and mec was fused to luciferase , and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured ( mean ± SD ; n = 6 )

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

The promoter of sigA and mec was fused to luciferase, and transformed cells were either left untreated or exposed to CHP stress for 6 h and luciferase activity was measured (mean ±SD; n=6)


Rv : : pN and Rv : : pN - F - sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - AosR . 50 µg WCLs were probed with α - FLAG , α - AosR , and α - SigA as controls .

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

Rv::pN and Rv::pN-F-sigH were either subjected to none or 50 µM of CHP for 6 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-AosR. 50 µg WCLs were probed with α-FLAG,α-AosR, and α-SigA as controls.


Expression of CysO , CysM , AosR and GroEL1 ( control ) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h .

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

Expression of CysO, CysM, AosR and GroEL1 (control) was analyzed in WCLs of indicated bacterial strains treated with 0 or 50 µM of CHP for 6 h.


5 - 7 . 5 x 104 end labelled 100 bp double - stranded oligonucleotide was incubated with no protein ( lanes 1 and 6 ) or 4 μΜ BSA ( lanes 2 and 7 ) or 2 μΜ His - AosR + 2 μΜ BSA ( lanes 3 and 8 ) or , 2 μΜ His - SigH + 2 μΜ BSA ( lanes 4 and 9 ) or 2 μΜ His - AosR + 2 μΜ His - SigH ( lanes 5 and 10 ) . The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols , respectively , and resolved on 8 % PAGE gel . Protein - DNA complexes ( bound ) and free probe are indicated by arrows . To confirm the specificity of DNA - protein interaction , EMSA reaction was performed in the presence of 5 , 10 , 25 , 50 , and 100 molar excess of unlabelled 100 bp double - stranded oligonucleotide .

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

5-7.5 x 104 end labelled 100 bp double-stranded oligonucleotide was incubated with no protein (lanes 1 and 6) or 4 μΜ BSA (lanes 2 and 7) or 2 μΜ His-AosR + 2 μΜ BSA (lanes 3 and 8) or , 2 μΜ His-SigH + 2 μΜ BSA (lanes 4 and 9) or 2 μΜ His-AosR + 2 μΜ His-SigH (lanes 5 and 10). The reactions were either incubated in the presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols, respectively, and resolved on 8% PAGE gel. Protein-DNA complexes (bound) and free probe are indicated by arrows. To confirm the specificity of DNA-protein interaction, EMSA reaction was performed in the presence of 5, 10, 25, 50, and 100 molar excess of unlabelled 100 bp double-stranded oligonucleotide.


Msm : : FLAG - SigH , Msm : : FLAG - SigH - AosR - HA and Msm : : FLAG - SigH - AosRAxxxA - HA were either subjected to none or 50 µM of CHP for 3 h followed by cross - linking . 1 mg WCL was used to immunoprecipitate FLAG - SigH and 1 / 10th IP was probed with α - FLAG and 9 / 10th IP with α - HA . 50 µg WCLs were probed with α - FLAG , α - HA and α - SigA as controls .

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

Msm::FLAG-SigH, Msm::FLAG-SigH-AosR-HA and Msm::FLAG-SigH-AosRAxxxA-HA were either subjected to none or 50 µM of CHP for 3 h followed by cross-linking. 1 mg WCL was used to immunoprecipitate FLAG-SigH and 1/10th IP was probed with α-FLAG and 9/10th IP with α-HA. 50 µg WCLs were probed with α-FLAG, α-HA and α-SigA as controls.


Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified . Data presented is representative of one of two biological replicates , each performed in triplicates ( n = 3 ) . Y - axis represents µM thiol / µg of WCL ( mean ± SD ) . Metabolite levels of reduced mycothiol , MSH ergothioneine , ERG were measured in Rv , Rv ∆ aosR , and Rv ∆ aosR : : aosR in normal and CHP stressed cells using LC - MRM MS / MS analysis . The absolute values obtained under normal conditions were normalized to 100 % for each strain and the relative values were calculated for samples processed from CHP treated cells . The data indicates relative levels of metabolites ( mean percent ± SD ) of four replicates ( n = 4 ) .

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

Indicated bacterial strains were subjected to oxidative stress with 50 µM CHP for 6 h and free thiol content was quantified. Data presented is representative of one of two biological replicates, each performed in triplicates (n=3). Y-axis represents µM thiol /µg of WCL (mean ± SD). Metabolite levels of reduced mycothiol, MSH ergothioneine, ERG were measured in Rv, Rv∆aosR, and Rv∆aosR::aosR in normal and CHP stressed cells using LC-MRM MS/MS analysis. The absolute values obtained under normal conditions were normalized to 100% for each strain and the relative values were calculated for samples processed from CHP treated cells. The data indicates relative levels of metabolites (mean percent ± SD) of four replicates (n=4).


qRT - PCR measurements for the log2 fold induction ( mean ± SD , n = 3 ) for indicated genes in Rv ∆ aosR and Rv ∆ aosR treated with 1mM L - cysteine as compared with Rv . All strains were treated with 50 μM CHP . Data were normalized with respect to 16s rRNA and is representative of one of two 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 B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

qRT-PCR measurements for the log2 fold induction (mean ±SD, n=3) for indicated genes in Rv∆aosR and Rv∆aosR treated with 1mM L-cysteine as compared with Rv. All strains were treated with 50 μM CHP. Data were normalized with respect to 16s rRNA and is representative of one of two biological replicates.


( a , b ) Survival of wild - type ( Oregon R ) , yw , PGRP - LE112 , PGRP - LC7454 and PGRP - LE112 , PGRP - LC7454 flies after injection of wild - type L . monocytogenes ( a ) or Δhly L . monocytogenes ( b ) at 28 ° C .

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

(a,b) Survival of wild-type (Oregon R), yw, PGRP-LE112, PGRP-LC7454 and PGRP-LE112,PGRP-LC7454 flies after injection of wild-type L. monocytogenes (a) or Δhly L. monocytogenes (b) at 28 °C.


( c ) Survival of wild - type , yw and PGRP - LE112 flies , as well as flies treated with RNAi targeting PGRP - LE with the hml - Gal4 driver ( hml - Gal4 > PGRP - LE RNAi ) and PGRP - LE112 flies with hml - Gal4 - driven PGRP - LE expression ( PGRP - LE112 , hml - Gal4 > PGRP - LE ) , after injection of wild - type L . monocytogenes at 28 ° C . NS , not significant ; * , P 0 . 01 ( Wilcoxon - Mann - Whitney test ) : P = 0 . 0006 , 0 . 0043 or 0 . 0043 , for yw versus PGRP - LE112 , PGRP - LC7454 or PGRP - LE112 , PGRP - LC7454 , respectively ( a ) ; P = 0 . 0095 , for yw versus RNAi targeting PGRP - LE ( c ) . Data represent the average of four independent experiments with over 30 flies of each genotype examined at the same time .

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

(c) Survival of wild-type, yw and PGRP-LE112 flies, as well as flies treated with RNAi targeting PGRP-LE with the hml-Gal4 driver (hml-Gal4>PGRP-LE RNAi) and PGRP-LE112 flies with hml-Gal4-driven PGRP-LE expression (PGRP-LE112,hml-Gal4>PGRP-LE), after injection of wild-type L. monocytogenes at 28 °C. NS, not significant; *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0006, 0.0043 or 0.0043, for yw versus PGRP-LE112, PGRP-LC7454 or PGRP-LE112,PGRP-LC7454, respectively (a); P = 0.0095, for yw versus RNAi targeting PGRP-LE (c). Data represent the average of four independent experiments with over 30 flies of each genotype examined at the same time.


( a ) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild - type L . monocytogenes . Hemocyte nuclei ( filled arrowhead ) and DNA of L . monocytogenes ( open arrowhead ) are visualized by DAPI staining ( blue or white ) ; the actin cytoskeleton is stained with rhodamine - labeled phalloidin ( red ) . hml - Gal4 > Atg5IR , expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5 . Scale bars , 10 μm .

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

(a) Microscopy of hemocytes from third instar larvae cultured ex vivo and infected with wild-type L. monocytogenes. Hemocyte nuclei (filled arrowhead) and DNA of L. monocytogenes (open arrowhead) are visualized by DAPI staining (blue or white); the actin cytoskeleton is stained with rhodamine-labeled phalloidin (red). hml-Gal4>Atg5IR, expression of an inverted repeat sequence of Atg5 to induce RNAi against Atg5. Scale bars, 10 μm.


( b ) Intracellular wild - type L . monocytogenes ( WT Lm ) or Δhly L . monocytogenes ( Δhly Lm ) counted manually in DAPI - stained infected hemocytes . LE112 , hml > LE , hml - Gal4 - driven expression of PGRP - LE in PGRP - LE112 hemocytes ; imd1 , imd ; RelE20 , RelishE20 ; hml > Atg5IR , inverted repeat sequence described in a ; w , hml > Atg1 , hml - Gal4 - driven expression of Atg1 in w hemocytes ; LE112 , hml > Atg1 , hml - Gal4 - driven expression of Atg1 in PGRP - LE112 hemocytes ; PGRP - LE112 + rap , PGRP - LE112 hemocytes plus rapamycin . * , P 0 . 001 , compared with wild type ( t - test ) . Data are representative of four experiments ( a ) or at least three independent experiments ( b ; error bars , s . d . of triplicate measurements ) .

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

(b) Intracellular wild-type L. monocytogenes (WT Lm) or Δhly L. monocytogenes (Δhly Lm) counted manually in DAPI-stained infected hemocytes. LE112,hml>LE, hml-Gal4-driven expression of PGRP-LE in PGRP-LE112 hemocytes; imd1, imd; RelE20, RelishE20; hml>Atg5IR, inverted repeat sequence described in a; w, hml>Atg1, hml-Gal4-driven expression of Atg1 in w hemocytes; LE112,hml>Atg1, hml-Gal4-driven expression of Atg1 in PGRP-LE112 hemocytes; PGRP-LE112+rap, PGRP-LE112 hemocytes plus rapamycin. *, P 0.001, compared with wild type (t-test). Data are representative of four experiments (a) or at least three independent experiments (b; error bars, s.d. of triplicate measurements).


Survival of yw flies , PGRP - LE112 flies , flies treated with Atg5 - directed RNAi ( hml - Gal4 > Atg5IR ) and control flies carrying hml - Gal4 ( hml - Gal4 > GFP ) , after injection of wild - type L . monocytogenes ( a ) , Δhly L . monocytogenes ( b ) or E . carotovora ( c ) into adult flies . * , P 0 . 01 ( Wilcoxon - Mann - Whitney test ) : P = 0 . 0024 , Atg5 - directed RNAi versus control ( a ) , P = 0 . 0007 , PGRP - LE112 versus control ( a ) ; P = 0 . 0055 , PGRP - LC7454 versus yw ( c ) . Data represent the average of four independent experiments .

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 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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

Survival of yw flies, PGRP-LE112 flies, flies treated with Atg5-directed RNAi (hml-Gal4>Atg5IR) and control flies carrying hml-Gal4 (hml-Gal4>GFP), after injection of wild-type L. monocytogenes (a), Δhly L. monocytogenes (b) or E. carotovora (c) into adult flies. *, P 0.01 (Wilcoxon-Mann-Whitney test): P = 0.0024, Atg5-directed RNAi versus control (a), P = 0.0007, PGRP-LE112 versus control (a); P = 0.0055, PGRP-LC7454 versus yw (c). Data represent the average of four independent experiments.


( a ) Growth of L . monocytogenes in S2 cells ( S2 ) or S2 cells expressing PGRP - LE ( S2 - LE ) transfected with double - stranded RNA ( RNAi ) specific for Atg5 , Rel or Dif and dl ( Dif - dl ) and infected for 1 . 5 h with wild - type or Δhly L . monocytogenes , followed by 6 h of incubation in medium containing CuSO4 and gentamicin , quantified by plate assay of colony - forming units . * , P 0 . 001 ( t - test ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Growth of L. monocytogenes in S2 cells (S2) or S2 cells expressing PGRP-LE (S2-LE) transfected with double-stranded RNA (RNAi) specific for Atg5, Rel or Dif and dl (Dif-dl) and infected for 1.5 h with wild-type or Δhly L. monocytogenes, followed by 6 h of incubation in medium containing CuSO4 and gentamicin, quantified by plate assay of colony-forming units. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


( b , c ) Fluorescence confocal microscopy of the localization of PGRP - LE together with wild - type L . monocytogenes in S2 cells engineered to express YFP - PGRP - LE ( YFP - LE ) and infected for 0 . 5 h with wild - type L . monocytogenes ( b ) or Δhly L . monocytogenes ( c ) , followed by 1 h of incubation in gentamicin - containing medium , then DAPI staining . Merge , YFP - PGRP - LE ( green ) and DAPI ( magenta ) . Filled arrowheads indicate L . monocytogenes ; open arrowhead indicates accumulation of YFP - PGRP - LE around the bacteria . Scale bars , 5 μm . Data are representative of three experiments .

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

(b,c) Fluorescence confocal microscopy of the localization of PGRP-LE together with wild-type L. monocytogenes in S2 cells engineered to express YFP-PGRP-LE (YFP-LE) and infected for 0.5 h with wild-type L. monocytogenes (b) or Δhly L. monocytogenes (c), followed by 1 h of incubation in gentamicin-containing medium, then DAPI staining. Merge, YFP-PGRP-LE (green) and DAPI (magenta). Filled arrowheads indicate L. monocytogenes; open arrowhead indicates accumulation of YFP-PGRP-LE around the bacteria. Scale bars, 5 μm. Data are representative of three experiments.


( a ) Dot - or ring - shaped GFP - LC3 ( LC3 dot ) signals in S2 cells expressing both PGRP - LE and GFP - LC3 under the control of an actin promoter ( PGRP - LE GFP - LC3 ) or GFP - LC3 alone ( GFP - LC3 ) after infection with wild - type or Δhly L . monocytogenes or after 1 . 5 h of incubation with 5 μM rapamycin ( rap ) . - , no infection .

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

(a) Dot- or ring-shaped GFP-LC3 (LC3 dot) signals in S2 cells expressing both PGRP-LE and GFP-LC3 under the control of an actin promoter (PGRP-LE GFP-LC3) or GFP-LC3 alone (GFP-LC3) after infection with wild-type or Δhly L. monocytogenes or after 1.5 h of incubation with 5 μM rapamycin (rap). -, no infection.


( b ) Colocalization of GFP - LC3 dots and wild - type L . monocytogenes in S2 cells , quantified by confocal microscopy .

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

(b) Colocalization of GFP-LC3 dots and wild-type L. monocytogenes in S2 cells, quantified by confocal microscopy.


( c ) Confocal microscopy of S2 cells expressing PGRP - LE and GFP - LC3 ( green ) infected with wild - type L . monocytogenes and stained with DAPI ( magenta ) . Arrow indicates colocalization of GFP - LC3 and L . monocytogenes .

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

(c) Confocal microscopy of S2 cells expressing PGRP-LE and GFP-LC3 (green) infected with wild-type L. monocytogenes and stained with DAPI (magenta). Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( d ) Immunoblot analysis of lysates of S2 cells left untreated ( - ) or infected with wild - type L . monocytogenes or treated with 5 μM rapamycin .

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

(d) Immunoblot analysis of lysates of S2 cells left untreated (-) or infected with wild-type L. monocytogenes or treated with 5 μM rapamycin.


( e - i ) Ultrastructural analysis of S2 cells expressing PGRP - LE and GFP - LC3 and infected with wild - type L . monocytogenes . ( e , f ) Fluorescence microscopy ( e ) and electron microscopy ( f ) of cells expressing GFP - LC3 ( green ) and stained with DAPI ( magenta ) . ( g ) Enlargement of a bacteria - containing vacuole from f . ( h , i ) Enlargement of the fields outlined in g . Arrows indicate double - membrane structure that surrounds the bacteria ; arrowheads indicate an endoplasmic reticulum - like membrane .

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 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

(e-i) Ultrastructural analysis of S2 cells expressing PGRP-LE and GFP-LC3 and infected with wild-type L. monocytogenes. (e,f) Fluorescence microscopy (e) and electron microscopy (f) of cells expressing GFP-LC3 (green) and stained with DAPI (magenta). (g) Enlargement of a bacteria-containing vacuole from f. (h,i) Enlargement of the fields outlined in g. Arrows indicate double-membrane structure that surrounds the bacteria; arrowheads indicate an endoplasmic reticulum-like membrane.


( j ) Confocal microscopy of hemocytes infected with wild - type L . monocytogenes . Green , GFP - LC3 ; magenta , DAPI . Arrow indicates colocalization of GFP - LC3 and L . monocytogenes .

O O O O O O O O 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

(j) Confocal microscopy of hemocytes infected with wild-type L. monocytogenes. Green, GFP-LC3; magenta, DAPI. Arrow indicates colocalization of GFP-LC3 and L. monocytogenes.


( k ) Dot - or ring - shaped GFP - LC3 signals in ex vivo - cultured hemocytes expressing GFP - LC3 ( infection and treatment , horizontal axis ) .

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

(k) Dot- or ring-shaped GFP-LC3 signals in ex vivo-cultured hemocytes expressing GFP-LC3 (infection and treatment, horizontal axis).


( l ) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L . monocytogenes or treated with rapamycin . Arrowhead indicates processed form of GFP - LC3 .

O O O O O O O O O O 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

(l) Immunoblot analysis of lysates of hemocytes from third instar larvae cultured ex vivo and infected with L. monocytogenes or treated with rapamycin. Arrowhead indicates processed form of GFP-LC3.


( m ) GFP - LC3 dots in S2 cells expressing PGRP - LE and GFP - LC3 and treated with RNAi ( below graph ) and infected for 0 . 5 h with L . monocytogenes then incubated for 1 h in gentamicin - containing medium ; dots quantified by confocal microscopy . Scale bars , 5 μm ( c , j ) , 1 μm ( e , f ) or 500 nm ( g ) . * , P 0 . 001 ( t - test ) . Data are representative of three ( a , k , m ) , two ( b , e - i ) , six ( c ) , four ( d ) or five ( j , l ) experiments ( error bars , s . d . of triplicate measurements ( a , b , l ) or at least triplicate measurements ( m ) ) .

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

(m) GFP-LC3 dots in S2 cells expressing PGRP-LE and GFP-LC3 and treated with RNAi (below graph) and infected for 0.5 h with L. monocytogenes then incubated for 1 h in gentamicin-containing medium; dots quantified by confocal microscopy. Scale bars, 5 μm (c,j), 1 μm (e,f) or 500 nm (g). *, P 0.001 (t-test). Data are representative of three (a,k,m), two (b,e-i), six (c), four (d) or five (j,l) experiments (error bars, s.d. of triplicate measurements (a,b,l) or at least triplicate measurements (m)).


( a ) GFP - LC3 dots in S2 cells expressing GFP - LC3 alone or PGRP - LE and GFP - LC3 , treated for 2 h with TCT ( 100 nM ) , highly purified DAP - type PGN from L . plantarum ( DAP ; 100 μg / ml ) or lysine - type PGN from S . epidermidis ( Lys ) .

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

(a) GFP-LC3 dots in S2 cells expressing GFP-LC3 alone or PGRP-LE and GFP-LC3, treated for 2 h with TCT (100 nM), highly purified DAP-type PGN from L. plantarum (DAP; 100 μg/ml) or lysine-type PGN from S. epidermidis (Lys).


( b ) Dot - or ring - shaped GFP - LC3 signals in hemocytes after infection with wild - type or Δhly L . monocytogenes or treatment with rapamycin ( 5 μM ) or with TCT , DAP - type PGN or lysine - type PGN . * , P 0 . 001 ( t - test ) . Data are representative of two independent experiments ( error bars , s . d . ) .

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

(b) Dot- or ring-shaped GFP-LC3 signals in hemocytes after infection with wild-type or Δhly L. monocytogenes or treatment with rapamycin (5 μM) or with TCT, DAP-type PGN or lysine-type PGN. *, P 0.001 (t-test). Data are representative of two independent experiments (error bars, s.d.).


Left : Fluorescence changes of IRDye700 labelled dsDNA ( at 125 nM ) when incubated at increasing ID2 ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FANCD2 ( D2Ub ) concentrations ( ranging from 1 . 3 nM to 5 . 9 µM ) . Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from model fitting . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 23 - 24 data points each ) .

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

Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).


Assessment of protein - DNA interactions using Electro - mobility shift assays ( EMSA ) . IRDye700 labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of non / single / double - ubiquitinated ID2 ( His6 - TEV - V5 - FANCI and FLAG - FANCD2 ) protein complexes ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FLAG - FANCD2 ( D2Ub ) . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . EMSA gels of ID2 complexes are representative of 3 replicate experiments .

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

Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.


Left : CryoEM 2D classes of ID2Ub and IUbD2Ub - dsDNA . Right : 3D reconstructions of ID2Ub ( EMD - 10843 ) and IUbD2Ub - DNA ( EMD - 10844 ) . Both structures exhibit a torus - like shape .

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

Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI can be efficiently ubiquitinated in the presence of DNA . Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA .

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-MEASURED_VAR O O O O O O O O O O O O O O

Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI efficiently associate with FANCD2 . Fluorescence changes occurring when RED - tris - NTA - labelled FANCI ( IWT or IR1285Q ; both at 60 nM ) is incubated at increasing concentrations of FANCD2 ( ranging from 2 . 48 nM to 5 . 08 µM ) . Titrations were conducted twice ( two technical replicates ) and all data points for each protein - combination were used in fitting of a one - site binding model . Apparent Kd values ( mean ± SEM ) derived from fitting of a one - site binding model to the 24 data points of the two technical replicates are shown .

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-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

Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.


FANCD2 within an IR1285QD2 complex is resistant to ubiquitination . Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA . Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS - PAGE .

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

FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by PIFE . Left : Fluorescence changes when IRD700 - labelled dsDNA ( at 125 nM ) is incubated at increasing concentrations of IR1285Q + D2 or IR1285Q + D2Ub ( complex concentrations ranging from 1 . 54 nM to 3 . 8 µM ) . Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from the one - site binding model . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 22 data points each ) . IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values ( from data points shown in Figure 1A ) are also shown for comparison .

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

FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by EMSAs . IRDye700 - labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of ID2 ( His6 - FANCI or His6 - FANCIR1285Q mixed with non - ubiquitinated or ubiquitinated FLAG - FANCD2 . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . Gels shown are representative of two replicate experiments .

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

FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.


USP1 - UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI ( I ) , but not in the presence of ubiquitinated FANCI ( IUb ) . Ubiquitinated FANCD2 ( D2Ub ) was mixed with a 50 base pair dsDNA and either His6 - TEV - V5 - FANCI ( I ) , ubiquitinated His6 - TEV - V5 - FANCI ( IUb ) or no protein ; protein - DNA mixes were subsequently incubated with USP1 - UAF1 ( at 25 nM or 100 nM ) for indicated time periods . Deubiquitination of D2Ub and IUb was assessed , following SDS - PAGE , by Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody .

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

USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.


USP7 and USP2 can deubiquitinate D2Ub , but their activity towards D2Ub is greatly reduced in the presence of FANCI ( I ) or ubiquitinated FANCI ( IUb ) . Reactions were set - up as in A , but with 100 nM USP7 or USP2 . Deubiquitination was assessed , following SDS - PAGE , by Coomassie staining .

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

USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.


Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity / resistance to USP1 - UAF1 mediated deubiquitination of resulting products . Deubiquitination reactions in the presence or absence of USP1 - UAF1 ( 100 nM ) incubated for 30 minutes .

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

Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.


Time course of USP1 - UAF1 mediated deubiquitination of IUbD2Ub - DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild - type ( WT ) ubiquitin . Progress of deubiquitination reaction was assessed following SDS - PAGE , by both Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . FANCI ubiquitination with indicated ubiquitin - mutants ( or WT ) and subsequent deubiquitination of resulting IUbD2Ub - DNA complexes were conducted twice ; the residual FANCD2 ubiquitination , calculated from the FANCD2 blots for each time - point , was plotted for each type of ubiquitin in the protein complex , and corresponding deubiquitination progress curves were fitted using a one - phase decay model .

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

Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.


A . Viability analysis of GICs treated with the α - AR antagonists prazosin , ARC 239 , doxazosin , BMY 7378 , and terazosin . GICs were treated with the antagonists or corresponding vehicles for 72 h , and viability was assayed using WST - 1 . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability analysis of GICs treated with the α-AR antagonists prazosin, ARC 239, doxazosin, BMY 7378, and terazosin. GICs were treated with the antagonists or corresponding vehicles for 72 h, and viability was assayed using WST-1. *P<0.05, n=4, two-sided Mann-Whitney U-test.


B . Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

B. Quantification of GIC survival using trypan blue exclusion after 24 h and 72 h of treatment with prazosin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Viability analysis of patient - derived GICs ( TG1 , TG16 , GBM5 , GBM44 ) and NSCs ( NSC24 , NSC25 , NSC5031 , NSC8853 ) treated with prazosin for 72 h . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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

C. Viability analysis of patient-derived GICs (TG1, TG16, GBM5, GBM44) and NSCs (NSC24, NSC25, NSC5031, NSC8853) treated with prazosin for 72 h. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Analysis of the sphere - forming capabilities of GICs using the extreme limiting dilution assay . Cells were seeded in presence of vehicle or 30 µM prazosin ( PRZ ) . Sphere formation was scored 10 days post - seeding . Frequency of sphere - forming cells : Control = 1 / 3 . 88 ( lower 8 . 61 , upper 1 . 95 ) ; prazosin 1 / 248 ( lower 1003 , upper 62 ) , n = 12 , p = 1 . 13 10 - 10 . Overall test for difference in stem cell number between groups .

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

D. Analysis of the sphere-forming capabilities of GICs using the extreme limiting dilution assay. Cells were seeded in presence of vehicle or 30 µM prazosin (PRZ). Sphere formation was scored 10 days post-seeding. Frequency of sphere-forming cells: Control=1/3.88 (lower 8.61, upper 1.95); prazosin 1/248 (lower 1003, upper 62), n=12, p= 1.13 10-10. Overall test for difference in stem cell number between groups.


E . Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR , and the neural stem cell markers CD15 and CD133 . Prazosin inhibits cell viability regardless of CD133 or CD15 expression . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability analysis of GICs treated with prazosin after sorting cells according to their expression of EGFR, and the neural stem cell markers CD15 and CD133. Prazosin inhibits cell viability regardless of CD133 or CD15 expression. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Imunocytochemical staining of NSCs and GICs cultured in media favoring neuronal ( ß3 - tubulin ) , astroglial ( GFAP ) or oligodendroglial ( O4 ) differentiation ( Diff media ) . ß3 - tub : ß3 - tubulin .

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

F. Imunocytochemical staining of NSCs and GICs cultured in media favoring neuronal (ß3-tubulin), astroglial (GFAP) or oligodendroglial (O4) differentiation (Diff media). ß3-tub: ß3-tubulin.


G . Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h . Diff : differentiation . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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

G. Viability analysis of differentiated NSCs and GICs treated with prazosin for 72 h. Diff: differentiation. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


B , C . In vivo effect of prazosin treatment ( 1 . 5mg / Kg ) on glioblastoma growth . Tumors were initiated with GBM44 GICs ( B ) or GMB5 GICs ( C ) . Left panels : Bioluminescent in vivo images of tumors in mice treated with prazosin ( PRZ ) or vehicle for 45 days . Middle panels : Quantification of the bioluminescent signals . Fold change in total flux represents the ratio : total flux after treatment / total flux before treatment . * P = 0 . 0002 and * P = 0 . 003 for GBM44 and GBM5 respectively , n = 8 , two - sided Mann - Whitney U - test . Right panels : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle , log - rank Mantel - Cox test . The treatment period is shaded in gray .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, C. In vivo effect of prazosin treatment (1.5mg/Kg) on glioblastoma growth. Tumors were initiated with GBM44 GICs (B) or GMB5 GICs (C). Left panels: Bioluminescent in vivo images of tumors in mice treated with prazosin (PRZ) or vehicle for 45 days. Middle panels: Quantification of the bioluminescent signals. Fold change in total flux represents the ratio: total flux after treatment/total flux before treatment. *P=0.0002 and *P=0.003 for GBM44 and GBM5 respectively, n=8, two-sided Mann-Whitney U-test. Right panels: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle demonstrating a significant survival benefit of prazosin as compared to vehicle, log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Example of hematoxilin / eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin ( PRZ ) or vehicle . i : tumor infiltration . n : tumor necrosis . Scale bar = 2 mm . GBM44 GICs .

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

D. Example of hematoxilin/eosin staining of brain coronal sections from mice sacrificed after 45 days of treatment with prazosin (PRZ) or vehicle. i: tumor infiltration. n: tumor necrosis. Scale bar = 2 mm. GBM44 GICs.


E . Left panel : Representative flow cytometry plots depicting the percentage of CD133 + glioblastoma cells ( GFP + ) isolated from mice treated with prazosin ( PRZ ) or vehicle . Tumors were initiated with GBM44 GICs . Right panel : Quantification of flow cytometry analyses of CD133 expression by GFP + - tumor cells isolated from xenografts of three prazosin - treated ( PRZ ) mice and three vehicle - treated mice . * P = 0 . 0003 , n = 6 , two - sided Mann - Whitney U - test .

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 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

E. Left panel: Representative flow cytometry plots depicting the percentage of CD133+ glioblastoma cells (GFP+) isolated from mice treated with prazosin (PRZ) or vehicle. Tumors were initiated with GBM44 GICs. Right panel: Quantification of flow cytometry analyses of CD133 expression by GFP+-tumor cells isolated from xenografts of three prazosin-treated (PRZ) mice and three vehicle-treated mice. *P=0.0003, n=6, two-sided Mann-Whitney U-test.


F . Secondary grafting of tumor cells ( GBM44 - GFP + ) isolated from vehicle or prazosin - treated mice bearing primary tumors . Mice injected with glioblastoma cells isolated from prazosin - treated mice developed tumors at a lower frequency ( 50 % versus 100 % ) . Moreover , mice injected with glioblastoma cells isolated from prazosin - treated mice presented a statistically significant survival benefit . Left Panel : Bioluminescent in vivo images of secondary grafts . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0004 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle - and prazosin - treated mice .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Secondary grafting of tumor cells (GBM44-GFP+) isolated from vehicle or prazosin-treated mice bearing primary tumors. Mice injected with glioblastoma cells isolated from prazosin-treated mice developed tumors at a lower frequency (50% versus 100%). Moreover, mice injected with glioblastoma cells isolated from prazosin-treated mice presented a statistically significant survival benefit. Left Panel: Bioluminescent in vivo images of secondary grafts. Middle panel: Quantification of the bioluminescent signals. *P=0.0004, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice bearing secondary graft from glioblastoma cells isolated from vehicle- and prazosin-treated mice.


G . Inhibitory effect of low doses of prazosin ( 0 . 15 mg / Kg ) on glioblastoma growth in vivo . Left panel : Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin ( PRZ ) for 45 days . Tumors were initiated with GBM44 GICs ( compare with panel B ) . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin , log - rank Mantel - Cox test . The treatment period is shaded in gray .

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

G. Inhibitory effect of low doses of prazosin (0.15 mg/Kg) on glioblastoma growth in vivo. Left panel: Bioluminescent in vivo images of tumors in mice treated with vehicle or prazosin (PRZ) for 45 days. Tumors were initiated with GBM44 GICs (compare with panel B). Middle panel: Quantification of the bioluminescent signals. *P=0.0007, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice demonstrating a significant survival benefit of a treatment with low doses of prazosin, log-rank Mantel-Cox test. The treatment period is shaded in gray.


A . Viability analysis of GL261 treated with prazosin in vitro . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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

A. Viability analysis of GL261 treated with prazosin in vitro. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


C . Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma . Left panel : Bioluminescent in vivo images of tumors in C57 / Bl6mice treated with vehicle or prazosin ( PRZ ) for 14 days . Middle panel : Quantification of the bioluminescent signals . * P = 0 . 0002 , n = 8 , two - sided Mann - Whitney U - test . Right panel : Kaplan - Meyer survival curves of mice treated with prazosin ( PRZ ) or vehicle . log - rank Mantel - Cox test . The treatment period is shaded in gray .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Prazosin inhibits in vivo tumorgrowth in an immunocompetent model of glioblastoma. Left panel: Bioluminescent in vivo images of tumors in C57/Bl6mice treated with vehicle or prazosin (PRZ) for 14 days.Middle panel: Quantification of the bioluminescent signals. *P=0.0002, n=8, two-sided Mann-Whitney U-test. Right panel: Kaplan-Meyer survival curves of mice treated with prazosin (PRZ) or vehicle. log-rank Mantel-Cox test. The treatment period is shaded in gray.


D . Hematoxilin / eosin staining of C57 / Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin . Scale bar = 2 mm .

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

D. Hematoxilin/eosin staining of C57/Bl6 mice brain coronal sections sacrificed after 14 days of treatment with prazosin. Scale bar = 2 mm.


E . Fluorescence imaging of GL261 intra - striatal grafts at 1 and 2 h after an intra - peritoneal injection of BODIPY FL prazosin , the green - fluorescent derivative of prazosin . Scale bar = 2 mm .

O O O O O O O O O O O O O O O O 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. Fluorescence imaging of GL261 intra-striatal grafts at 1 and 2 h after an intra-peritoneal injection of BODIPY FL prazosin, the green-fluorescent derivative of prazosin. Scale bar = 2 mm.


A . Immunoblotting for pro - caspase - 3 ( pro - CASP3 ) and active caspase - 3 ( CASP3 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin activates caspase - 3 . ZVAD , a caspase inhibitor , prevents prazosin - induced caspase - 3 activation . kDa : kilodaltons .

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

A. Immunoblotting for pro-caspase-3 (pro-CASP3) and active caspase-3 (CASP3) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin activates caspase-3. ZVAD, a caspase inhibitor, prevents prazosin-induced caspase-3 activation. kDa: kilodaltons.


B . Immunoblotting for pro - caspase - 9 ( pro - CASP9 ) and active caspase - 9 ( CASP9 ) in GICs treated with prazosin ( PRZ ) or vehicle ( V ) demonstrating that prazosin does not activate caspase - 9 .

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

B. Immunoblotting for pro-caspase-9 (pro-CASP9) and active caspase-9 (CASP9) in GICs treated with prazosin (PRZ) or vehicle (V) demonstrating that prazosin does not activate caspase-9.


C . Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD , a caspase inhibitor . ZVAD counteracts prazosin - induced GIC death . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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

C. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of ZVAD, a caspase inhibitor. ZVAD counteracts prazosin-induced GIC death. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


D . Prazosin induces glioblastoma cell apoptosis in vivo . GFP + and GFP - cells were analyzed from tumors in vehicle or prazosin - treated mice . Annexin V / DAPI staining was used to identify apoptotic cells by FACS . Tumors were initiated by GBM44 GICs implantation .

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

D. Prazosin induces glioblastoma cell apoptosis in vivo. GFP+ and GFP- cells were analyzed from tumors in vehicle or prazosin-treated mice. Annexin V / DAPI staining was used to identify apoptotic cells by FACS. Tumors were initiated by GBM44 GICs implantation.


E . Dose - response curve of prazosin on GIC survival ( 24h treatment ) . EC50 was determined with curve fit using nonlinear regression .

O O 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. Dose-response curve of prazosin on GIC survival (24h treatment). EC50 was determined with curve fit using nonlinear regression.


F . Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline , a subtype agonist of the α - ARs . Cirazoline did not alter GIC survival and counteracted only poorly prazosin - induced GIC death . * P < 0 . 05 , n = 4 , two - sided Mann - Whitney U - test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Viability analysis of GICs treated with prazosin for 24h in the presence or absence of cirazoline, a subtype agonist of the α-ARs. Cirazoline did not alter GIC survival and counteracted only poorly prazosin-induced GIC death. *P<0.05, n=4, two-sided Mann-Whitney U-test.


G . Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations . Positive control shows that prazosin binds to membrane preparations of yeast expressing 1 - AR . PRZ : prazosin . CRZ : cirazoline .

G. Membrane binding assays demonstrating the absence of prazosin binding sites in GIC membrane preparations. Positive control shows that prazosin binds to membrane preparations of yeast expressing 1-AR. PRZ: prazosin. CRZ: cirazoline.


H . Immunoblotting for phosphorylated ERK1 / 2 ( P - ERK1 / 2 ) and total ERK1 / 2 following prazosin treatment for 30 min . Prazosin induces ERK1 / 2 phosphorylation in GICs .

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

H. Immunoblotting for phosphorylated ERK1/2 (P-ERK1/2) and total ERK1/2 following prazosin treatment for 30 min. Prazosin induces ERK1/2 phosphorylation in GICs.


I . SRE - luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 ( 10 μM ) , an inhibitor of the ERK - activating kinase MEK . U0126 prevented prazosin - induced SRE - luciferase activation in GICs .

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

I. SRE-luciferase reporter activity analysis of GICs treated with prazosin in absence or presence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 prevented prazosin-induced SRE-luciferase activation in GICs.


J . Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 ( 10 μM ) , an inhibitor of the ERK - activating kinase MEK . U0126 did not counteract prazosin - induced GIC death . Blockade of prazosin - induced ERK1 / 2 phosphorylation by U0126 was confirmed by immunoblotting ( insert ) . PRZ : prazosin .

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

J. Viability analysis of GICs treated with prazosin for 24 h in the presence or absence of U0126 (10 μM), an inhibitor of the ERK-activating kinase MEK. U0126 did not counteract prazosin-induced GIC death. Blockade of prazosin-induced ERK1/2 phosphorylation by U0126 was confirmed by immunoblotting (insert). PRZ: prazosin.


A . PKCδ expression analysis in two patient - derived GICs and NSCs by immunoblotting . GICs express higher levels of PKCδ than NSCs . kDa : kilodaltons .

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 O O O O O

A. PKCδ expression analysis in two patient-derived GICs and NSCs by immunoblotting. GICs express higher levels of PKCδ than NSCs. kDa: kilodaltons.


B . PKCδ by immunostaining ( green ) . Nuclei were stained with DAPI ( blue ) . Enhanced PKCδ punctate nuclear expression in prazosin - treated ( PRZ , 5µM ) GICs , as compared to vehicle - treated GICs . PRZ : prazosin . Scale bar : 10 µm .

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

B. PKCδ by immunostaining (green). Nuclei were stained with DAPI (blue). Enhanced PKCδ punctate nuclear expression in prazosin-treated (PRZ, 5µM) GICs, as compared to vehicle-treated GICs. PRZ: prazosin. Scale bar: 10 µm.


C . Total PKCδ ( PKCδ 78kDa ) and cleaved PKCδ ( PKCδ 41kDa ) expression analysis in GICs by immunoblotting . Prazosin ( PRZ ) induces PKCδ cleavage into a 41kDa active fragment . 2 µM rottlerin ( Rott ) , a PKCδ inhibitor , inhibits prazosin - induced PKCδ cleavage . V : vehicle . kDa : kilodaltons .

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

C. Total PKCδ (PKCδ 78kDa) and cleaved PKCδ (PKCδ 41kDa) expression analysis in GICs by immunoblotting. Prazosin (PRZ) induces PKCδ cleavage into a 41kDa active fragment. 2 µM rottlerin (Rott), a PKCδ inhibitor, inhibits prazosin-induced PKCδ cleavage. V: vehicle. kDa: kilodaltons.


D . Pro - caspase - 3 ( Pro - CASP3 ) and activated caspase - 3 ( CASP3 ) expression analysis in GICs by immunoblotting . 2 µM rottlerin ( Rott ) inhibits prazosin ( PRZ ) - induced caspase - 3 activation . V : vehicle . kDa : kilodaltons .

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

D. Pro-caspase-3 (Pro-CASP3) and activated caspase-3 (CASP3) expression analysis in GICs by immunoblotting. 2 µM rottlerin (Rott) inhibits prazosin (PRZ)-induced caspase-3 activation. V: vehicle. kDa: kilodaltons.


E . Inhibition of PKCδ by rottlerin prevents , in a concentration - dependent manner , prazosin - induced GIC death . Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin . * P = 0 . 0286 , n = 4 , two - sided Mann - Whitney U - test .

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

E. Inhibition of PKCδ by rottlerin prevents, in a concentration-dependent manner, prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of rottlerin. *P=0.0286, n=4, two-sided Mann-Whitney U-test.


F . Inhibition of PKCδ by the specific anti - PKCδ RACK peptide δV1 . 1 ( 10 μM ) counteracts prazosin - induced GIC death . Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1 . 1 . * P < 0 . 005 for Prazosin 1 and 5 µM and P < 0 . 001 for Prazosin 10 µM by two - tailed unpaired Student ' s t - test , n = 4 .

O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Inhibition of PKCδ by the specific anti-PKCδ RACK peptide δV1.1 (10 μM) counteracts prazosin-induced GIC death. Viability analysis of GICs treated with prazosin for 72 h in the presence or absence of δV1.1. *P < 0.005 for Prazosin 1 and 5 µM and P<0.001 for Prazosin 10 µM by two-tailed unpaired Student's t-test, n=4.


G . Decreased PKCδ expression using shRNA counteracts prazosin - induced GIC death . Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h . * P < 0 . 005 by two - tailed unpaired Student ' s t - test , n = 4 .

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 O O O O O

G. Decreased PKCδ expression using shRNA counteracts prazosin-induced GIC death. Viability analysis of GICs transduced with scrambled or PKCδ shRNA and treated with prazosin for 72 h. *P < 0.005 by two-tailed unpaired Student's t-test, n=4.


H . Prazosin inhibits AKT phosphorylation in GICs . LY294002 ( LY , 30 µM ) , an inhibitor of the PI3K / AKT pathway , was used as positive control . Terazosin , which does not affect GIC viability , does not alter AKT phosphorylation ( see the corresponding cell viability counting in Fig EV4C ) . Phosphorylated AKT ( P - AKT ) and total AKT ( AKT ) expression analysis in GICs by immunoblotting . V : vehicle . kDa : kilodaltons .

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 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-MEASURED_VAR O O O B-MEASURED_VAR 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

H. Prazosin inhibits AKT phosphorylation in GICs. LY294002 (LY, 30 µM), an inhibitor of the PI3K/AKT pathway, was used as positive control. Terazosin, which does not affect GIC viability, does not alter AKT phosphorylation (see the corresponding cell viability counting in Fig EV4C). Phosphorylated AKT (P-AKT) and total AKT (AKT) expression analysis in GICs by immunoblotting. V: vehicle. kDa: kilodaltons.


I . Prazosin ( 10 µM ) does not inhibit AKT phosphorylation in NSCs . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

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

I. Prazosin (10 µM) does not inhibit AKT phosphorylation in NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


J . β - catenin expression , a downstream target of AKT , is decreased in prazosin - treated GICs , as opposed to NSCs . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

O 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 O O O O O O O

J. β-catenin expression, a downstream target of AKT, is decreased in prazosin-treated GICs, as opposed to NSCs. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


K . L . Inhibition of PKCδ using rottlerin ( Rott , 2 µM ) or δV1 . 1 ( 10 µM ) counteracts prazosin inhibition of AKT phosphorylation . Analysis by immunoblotting . V : vehicle . kDa : kilodaltons .

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-MEASURED_VAR O O O O O O O O O O O O O O

K. L. Inhibition of PKCδ using rottlerin (Rott, 2 µM) or δV1.1 (10 µM) counteracts prazosin inhibition of AKT phosphorylation. Analysis by immunoblotting. V: vehicle. kDa: kilodaltons.


B . Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA . All mice were treated with prazosin ( PRZ ) for 45 days . C . Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth . Fold change in total flux represents the ratio : total flux after treatment / total flux prior treatment . * P = 0 . 0007 , n = 8 , two - sided Mann - Whitney U - test .

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

B. Bioluminescent in vivo images of tumors in mice grafted with GICs expressing either scrambled or PKCδ shRNA. All mice were treated with prazosin (PRZ) for 45 days.C. Quantification of the bioluminescent signals showing that decreased expression of PKC counteracts prazosin inhibition of tumor growth. Fold change in total flux represents the ratio: total flux after treatment/total flux prior treatment. *P=0.0007, n=8, two-sided Mann-Whitney U-test.


Morphology of four - week - old soil - grown plants of Col - 0 , snc1 , and two independent transgenic lines of SNIPER1 OE into snc1 background . OE stands for overexpression of SNIPER1 . Scale bar = 1 cm . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, snc1, and two independent transgenic lines of SNIPER1 OE into snc1 background. OE stands for overexpression of SNIPER1. Scale bar = 1 cm. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of two independent transgenic lines of SNIPER1 OE in Col - 0 background . Scale bar = 1 cm . No difference in growth and development was observed . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . t . DC3000 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . Error bars represent mean ± SD ( n = 4 for day 0 ; n = 5 for day 3 ) . Three independent experiments were carried out with similar results .

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 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

Morphology of four-week-old soil-grown plants of two independent transgenic lines of SNIPER1 OE in Col-0 background. Scale bar = 1 cm. No difference in growth and development was observed. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.t. DC3000 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. Error bars represent mean ± SD (n=4 for day 0; n=5 for day 3). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of Col - 0 , chs1 - 2 ( A ) , chs2 - 1 ( B ) and two independent transgenic lines of SNIPER1 OE into chs1 - 2 ( A ) and chs2 - 1 ( B ) backgrounds . Plants were grown at 16 ° C under long day conditions . Scale bar = 1 cm .

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 B-MEASURED_VAR 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

Morphology of four-week-old soil-grown plants of Col-0, chs1-2 (A), chs2-1 (B) and two independent transgenic lines of SNIPER1 OE into chs1-2 (A) and chs2-1 (B) backgrounds. Plants were grown at 16 °C under long day conditions. Scale bar = 1 cm.


Morphology of four - week - old soil - grown plants of Col - 0 , chs3 - 2D ( C ) , snc2 - 1D eds5 npr1 ( D ) and two independent transgenic lines of SNIPER1 OE into chs3 - 2D ( C ) and snc2 - 1D eds5 npr1 ( D ) backgrounds . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0, chs3-2D (C), snc2-1D eds5 npr1 (D) and two independent transgenic lines of SNIPER1 OE into chs3-2D (C) and snc2-1D eds5 npr1 (D) backgrounds. Scale bar = 1 cm.


Morphology of four - week - old soil - grown plants of Col - 0 , mekk1 - 5 ndr1 , and two independent transgenic lines of SNIPER1 OE into mekk1 - 5 ndr1 background . Scale bar = 1 cm . The mekk1 ndr1 double mutant was used instead of the mekk1 - 5 single mutant is to overcome the sterility . SNIPER1 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results . Quantification of H . a . Noco2 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results . Growth of P . s . m . ES4326 on four - week - old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0 . 001 . mn stands for mekk1 - 5 ndr1 double mutant . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Morphology of four-week-old soil-grown plants of Col-0, mekk1-5 ndr1, and two independent transgenic lines of SNIPER1 OE into mekk1-5 ndr1 background. Scale bar = 1 cm. The mekk1 ndr1 double mutant was used instead of the mekk1-5 single mutant is to overcome the sterility. SNIPER1 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results. Quantification of H.a. Noco2 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results. Growth of P.s.m. ES4326 on four-week-old leaves of the indicated genotypes at 0 and 3 dpi with bacterial inoculum of OD600 = 0.001. mn stands for mekk1-5 ndr1 double mutant. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


In vitro ubiquitination assay using E . coli - expressed SNIPER1 - myc ( E3 ) , AtUBC8 ( E2 ) , AtUBA1 ( E1 ) and / or HIS - FLAG - Ubiquitin . The molecular mass markers are indicated on the left ( kDa ) . Asterisk indicates SNIPER1 - myc . Arrow points to His - FLAG - Ub and straight lines on the right indicate self - ubiquitinated SNIPER1 .

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

In vitro ubiquitination assay using E. coli-expressed SNIPER1-myc (E3), AtUBC8 (E2), AtUBA1 (E1) and/or HIS-FLAG-Ubiquitin. The molecular mass markers are indicated on the left (kDa). Asterisk indicates SNIPER1-myc. Arrow points to His-FLAG-Ub and straight lines on the right indicate self-ubiquitinated SNIPER1.


Immunoblot analysis of SNC1 ( A - B ) , SUMM2 - 3HA ( C ) , RPS4HA ( D ) , RPP4myc ( E ) , RPS2HA ( F ) and RPM1myc ( G ) protein levels in the indicated genotypes . For all panels , equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

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

Immunoblot analysis of SNC1 (A-B), SUMM2-3HA (C), RPS4HA (D), RPP4myc (E), RPS2HA (F) and RPM1myc (G) protein levels in the indicated genotypes. For all panels, equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Growth of P . s . t . avrRps4 ( A ) , avrRpt2 ( B ) and avrRpm1 ( C ) on four - week - old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0 . 0001 . Error bars represent mean ± SD ( n = 5 ) . Three independent experiments were carried out with similar results . Appendix Figure S4 shows the growth of P . s . t . avrRps4 , avrRpt2 and avrRpm1 at 0 dpi . Quantification of H . a . Emwa1 sporulation in the indicated genotypes 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Three independent experiments were carried out with similar results .

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

Growth of P.s.t. avrRps4 (A), avrRpt2 (B) and avrRpm1 (C) on four-week-old leaves of the indicated genotypes at 3 dpi with bacterial inoculum of OD600 = 0.0001. Error bars represent mean ± SD (n=5). Three independent experiments were carried out with similar results. Appendix Figure S4 shows the growth of P.s.t. avrRps4, avrRpt2 and avrRpm1 at 0 dpi. Quantification of H.a. Emwa1 sporulation in the indicated genotypes 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Three independent experiments were carried out with similar results.


Morphology of four - week - old soil - grown plants of Col - 0 and two independent transgenic lines of 35S - SNIPER1H129Y . Scale bar = 1 cm .

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

Morphology of four-week-old soil-grown plants of Col-0 and two independent transgenic lines of 35S-SNIPER1H129Y. Scale bar = 1 cm.


PR1 and PR2 gene expression in the indicated plants as determined by RT - PCR . Error bars represent mean ± SD ( n = 3 ) . Two independent experiments were carried out with similar results .

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

PR1 and PR2 gene expression in the indicated plants as determined by RT-PCR. Error bars represent mean ± SD (n = 3). Two independent experiments were carried out with similar results.


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

Morphology of four-week-old soil-grown plants of Col-0, sniper1-1, sniper2-1, eds1-2, SNIPER1H129Y, SNIPER1H129Y eds1-2, sniper1-1 sniper2-1 eds1-2 and sniper1-c1 sniper2-1 eds1-2. sniper1-1 is an exonic T-DNA insertional mutant. sniper1-c1 is a SNIPER1 knockout mutant containing 807bp deletion that was generated by CRISPR-Cas9 system. Scale bar = 1 cm.


Quantification of H . a . Noco2 sporulation in the indicated plants 7 days post inoculation ( dpi ) with 105 spores per ml water . Error bars represent mean ± SD ( n = 4 ) . Two independent experiments were carried out with similar results .

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

Quantification of H.a. Noco2 sporulation in the indicated plants 7 days post inoculation (dpi) with 105 spores per ml water. Error bars represent mean ± SD (n=4). Two independent experiments were carried out with similar results.


Immunoblot of RPS4HA ( E ) , SNC1 ( F ) , RPS2HA ( G ) , and RPM1myc ( H ) in SNIPER1H129Y background . Equal loading is shown by Ponceau S staining of a non - specific band . The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD ( n = 3 ) . Molecular mass marker in kilo Daltons is indicated on the left . Three independent experiments were carried out with similar results .

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

Immunoblot of RPS4HA (E), SNC1 (F), RPS2HA (G), and RPM1myc (H) in SNIPER1H129Y background. Equal loading is shown by Ponceau S staining of a non-specific band. The numbers below represent the normalized ratio between the intensity of the protein band and the Ponceau S band ± SD (n=3). Molecular mass marker in kilo Daltons is indicated on the left. Three independent experiments were carried out with similar results.


Immunoprecipitation and biotinylation of SUMM2NB - FLAG ( A ) , RPP4NB - FLAG ( B ) , or CHS1NB - FLAG ( C ) by SNIPER1 H129Y - HATurboID in N . benthamiana . Two biological repeats were carried out with similar results .

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

Immunoprecipitation and biotinylation of SUMM2NB-FLAG (A), RPP4NB-FLAG (B), or CHS1NB-FLAG (C) by SNIPER1 H129Y-HATurboID in N. benthamiana. Two biological repeats were carried out with similar results.


Interaction of SNIPER1 H129Y - CLuc and SUMM2NB - NLuc ( D ) , RPP4NB - NLuc ( E ) or CHS1NB - NLuc ( F ) as tested by split - luciferase complementary assay in N . benthamiana . Three biological repeats with four technical repeats each were carried out with similar results .

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

Interaction of SNIPER1 H129Y-CLuc and SUMM2NB-NLuc (D), RPP4NB-NLuc (E) or CHS1NB-NLuc (F) as tested by split-luciferase complementary assay in N. benthamiana. Three biological repeats with four technical repeats each were carried out with similar results.


Ubiquitination of the NB domains from SUMM2 ( A ) , RPP4 ( B ) and CHS1 ( C ) . Immunoblot of bacterial lysates form E . coil strains expressing AtUBA1 - S , MBP - NB - HA , AtUBC8 - S , HIS - FLAG - UBQ10 and SNIPER1 - myc , or strains without one of these components . Two biological repeats were carried out with similar results .

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

Ubiquitination of the NB domains from SUMM2 (A), RPP4 (B) and CHS1 (C). Immunoblot of bacterial lysates form E. coil strains expressing AtUBA1-S, MBP-NB-HA, AtUBC8-S, HIS-FLAG-UBQ10 and SNIPER1-myc, or strains without one of these components. Two biological repeats were carried out with similar results.


( G ) Reduced NMDA / AMPA ratio at SC - CA1 synapses of PtenΔC / ΔC mice ( P17 - 21 ) . The NMDAR component was obtained 60 ms after stimulation . ( ratio , n = 11 cells from 8 mice for WT and 8 ( 8 ) for ΔC ; decay , n = 12 cells from 8 mice for WT and 11 ( 8 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U test ) . The error bars represent SEM . ( H ) Reduced amplitude but not frequency of NMDA - mEPSCs at PtenΔC / ΔC CA1 pyramidal neurons . ( n = 17 cells from 4 mice for WT and 18 ( 4 ) for ΔC , * * P < 0 . 01 , ns , not significant , Mann - Whitney U test , Student ' s t - test ) . The error bars represent SEM . ( I ) Reduced NMDA / AMPA ratio at synapses in layer II / III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC / ΔC mice ( P20 - 21 ) . The NMDAR component was obtained 60 ms after stimulation . ( n = 9 cells from 3 mice for WT and 10 ( 3 ) for ΔC , * P < 0 . 05 , * * P < 0 . 01 , Student ' s t - test , Mann - Whitney U test ) . The error bars represent SEM . Data information : ( C - F ) Results from the last 10 - minute recordings are shown in scatter plots .

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

(G) Reduced NMDA/AMPA ratio at SC-CA1 synapses of PtenΔC/ΔC mice (P17-21). The NMDAR component was obtained 60 ms after stimulation. (ratio, n = 11 cells from 8 mice for WT and 8 (8) for ΔC; decay, n = 12 cells from 8 mice for WT and 11 (8) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test). The error bars represent SEM. (H) Reduced amplitude but not frequency of NMDA -mEPSCs at PtenΔC/ΔC CA1 pyramidal neurons. (n = 17 cells from 4 mice for WT and 18 (4) for ΔC, **P < 0.01, ns, not significant, Mann-Whitney U test, Student's t-test). The error bars represent SEM. (I) Reduced NMDA/AMPA ratio at synapses in layer II/III pyramidal neurons in the prelimbic region of the mPFC of PtenΔC/ΔC mice (P20-21). The NMDAR component was obtained 60 ms after stimulation. (n = 9 cells from 3 mice for WT and 10 (3) for ΔC, *P < 0.05, **P < 0.01, Student's t-test, Mann-Whitney U test). The error bars represent SEM. Data information: (C-F) Results from the last 10-minute recordings are shown in scatter plots.


A ) A volcano plot showing DEGs ( adjusted p value < 0 . 1 ) derived from RNA - Seq results for PtenΔC / ΔC and WT mice ( P21 ) . For the calculation of adjusted p value , we have employed the R package DESeq2 where the P ‐ values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method . ( n = 3 mice for WT and ΔC ) . See also Dataset EV1 for the full RNA - Seq results .

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

A) A volcano plot showing DEGs (adjusted p value < 0.1) derived from RNA-Seq results for PtenΔC/ΔC and WT mice (P21). For the calculation of adjusted p value, we have employed the R package DESeq2 where the P‐values obtained by the Wald test are corrected for multiple testing using the Benjamini and Hochberg method. (n = 3 mice for WT and ΔC). See also Dataset EV1 for the full RNA-Seq results.