( E ) Tumor spheres formed by 22Rv1 - P cells stably expressing ESM1 - NLS with either β - catenin or NFκB shRNAs . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(E) Tumor spheres formed by 22Rv1-P cells stably expressing ESM1-NLS with either β-catenin or NFκB shRNAs. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( F ) Representative images and quantitative data comparing cell invasion of the indicated cells . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble cells , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble cells by two - tailed Student ' s t test .

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(F) Representative images and quantitative data comparing cell invasion of the indicated cells. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments. ## P< 0.01 when compared to vector-shScramble cells, ** P< 0.01 when compared to ESM1-NLS-shScramble cells by two-tailed Student's t test.


( A ) Nuclear and cytosolic extracts of 22Rv1 - M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting .

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(A) Nuclear and cytosolic extracts of 22Rv1-M cells transfected with either shScramble or shCTNNB1 were prepared and the levels of indicated proteins were detected by immunoblotting.


( B ) Nuclear or cytosolic extracts of 22Rv1 - M cells were immunoprecipitated with an ESM1 antibody .

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(B) Nuclear or cytosolic extracts of 22Rv1-M cells were immunoprecipitated with an ESM1 antibody.


( C ) Human recombinant His - ESM1 and GST - β - catenin protein were pull down with either Ni sepharose or Glutathione sepharose .

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(C) Human recombinant His-ESM1 and GST-β-catenin protein were pull down with either Ni sepharose or Glutathione sepharose.


( E ) HEK293T cells were co - transfected with the indicated β - catenin - Flag and ESM1 - HA constructs . Cell extracts were immunoprecipitated with Flag - M2 agarose beads . Light chain was labeled as l . c .

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(E) HEK293T cells were co-transfected with the indicated β-catenin-Flag and ESM1-HA constructs. Cell extracts were immunoprecipitated with Flag-M2 agarose beads. Light chain was labeled as l.c.


( F ) HEK293T cells were transfected with ESM1 - HA constructs and pull - down was carried out with different purified GST - ARM fragments .

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(F) HEK293T cells were transfected with ESM1-HA constructs and pull-down was carried out with different purified GST-ARM fragments.


( G ) HEK293T cells were transfected with the indicated ESM1 - GFP constructs . Cell extracts were pulled down with purified GST - ARM .

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(G) HEK293T cells were transfected with the indicated ESM1-GFP constructs. Cell extracts were pulled down with purified GST-ARM.


( B ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM .

(B) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM.


( C ) Cell extracts of 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA were immunoprecipitated with an β - catenin antibody and blotted with anti - TCF4 and anti - BCL9 antibody .

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(C) Cell extracts of 22Rv1-M transfected with either Scramble-shRNA or ESM1-shRNA were immunoprecipitated with an β-catenin antibody and blotted with anti-TCF4 and anti-BCL9 antibody.


( E ) Upper , Schematic diagram of ESM1 - targeting short peptides . Lower , HEK293T cells were transfected with ESM1 - HA constructs . Cell extracts were treated with the short peptides , pulled down with purified GST - ARM and blotted with anti - HA and anti - TCF4 antibody .

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(E) Upper, Schematic diagram of ESM1-targeting short peptides. Lower, HEK293T cells were transfected with ESM1-HA constructs. Cell extracts were treated with the short peptides, pulled down with purified GST-ARM and blotted with anti-HA and anti-TCF4 antibody.


( F ) HEK293T cells were transfected with the indicated ESM1 point mutant constructs . Cell extracts were pulled down with purified GST - ARM and blotted with an anti - TCF4 antibody .

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(F) HEK293T cells were transfected with the indicated ESM1 point mutant constructs. Cell extracts were pulled down with purified GST-ARM and blotted with an anti-TCF4 antibody.


( G ) ChIP - qPCR validation of β - catenin binding to the CCND1 and c - Myc promoter in 22Rv1 - M cells transfected with either Scramble - shRNA or ESM1 - shRNA . Bars are the mean ± SD of three independent experiments .

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(G) ChIP-qPCR validation of β-catenin binding to the CCND1 and c-Myc promoter in 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Bars are the mean ± SD of three independent experiments.


( H ) Upper , Schematic diagram of ChIP - qPCR primers targeting regions upstream of the CCND1 and c - Myc transcription start sites ( TSSs ) . Bar graphs show β - catenin and ESM1 binding at the CCND1 and c - Myc promoter regions . Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments .

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(H) Upper, Schematic diagram of ChIP-qPCR primers targeting regions upstream of the CCND1 and c-Myc transcription start sites (TSSs). Bar graphs show β-catenin and ESM1 binding at the CCND1 and c-Myc promoter regions. Differences in DNA levels compared with IgG control are shown as fold changes presented as the mean ± SD of three independent experiments.


( I ) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

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(I) The effects of ESM1 targeting peptides on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( J ) The effects of ESM1 point mutants on tumor invasion and spheroid formation . Scale bar : 100 μm . Bars are the mean ± SD of three independent experiments .

(J) The effects of ESM1 point mutants on tumor invasion and spheroid formation. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( B - C ) Fluorescent dye release from the mitochondria - mimic liposomes by the indicated wild - type ( wt ) or mutant Bax in the presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching the fluorescence of the released dyes by the dye - specific antibodies outside the liposomes during a time course . The fraction of dye release was normalized to that by detergent . The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes .

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(B-C) Fluorescent dye release from the mitochondria-mimic liposomes by the indicated wild-type (wt) or mutant Bax in the presence (B) or absence (C) of tBid or by tBid alone (C) was measured by quenching the fluorescence of the released dyes by the dye-specific antibodies outside the liposomes during a time course. The fraction of dye release was normalized to that by detergent. The data shown were obtained from n = 3 independent replicates using the same preparations of the proteins and liposomes.


( D ) Digitonin permeabilized BMK Bax - / - / Bak - / - cells expressing SMAC - mCherry in the mitochondria intermembrane space ( 50 μL , 500 , 000 total cells ) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37 ° C in a 96 - well plate . The samples were centrifuged for 10 min and separated into supernatant and pellet fractions . SMAC - mCherry release was calculated as the fraction of total ( supernatant + pellet ) mCherry fluorescence coming from the supernatant fraction . The data was normalized to the percent SMAC - mCherry release of wt Bax and cBid at 60 min . Each symbol represents the normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable the data was fit with the [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . The dotted lines represent the 95 % confidence interval of the fit .

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(D) Digitonin permeabilized BMK Bax-/-/Bak-/- cells expressing SMAC-mCherry in the mitochondria intermembrane space (50 μL, 500,000 total cells) were incubated with 25 nM of Bax and 2 nM cBid for the indicated time at 37°C in a 96-well plate. The samples were centrifuged for 10 min and separated into supernatant and pellet fractions. SMAC-mCherry release was calculated as the fraction of total (supernatant + pellet) mCherry fluorescence coming from the supernatant fraction. The data was normalized to the percent SMAC-mCherry release of wt Bax and cBid at 60 min. Each symbol represents the normalized SMAC-mCherry release for one single replicate (n = 3 or more independent replicates). Where applicable the data was fit with the [Agonist] vs. response - Variable slope equation in Graphpad Prism 8.0.1. The dotted lines represent the 95% confidence interval of the fit.


A , Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA , or 5 nM Actinomycin D ( ActD ) . GAPDH served as a loading control .

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A, Western blots in HCT116 cells treated for 24 hrs with increasing concentrations of MPA, or 5 nM Actinomycin D (ActD). GAPDH served as a loading control.


B , HCT116 cells were treated for 24 hrs with vehicle alone ( - ) , 1 µM MPA , 10 µM MPA , or the combination of 10 µM MPA with 400 µM guanosine . Guanylate nucleotides levels were measured by LC - MS , and normalized to protein content . Mean ± SEM is representative of 3 independent experiments carried out in triplicate . Data information data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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B, HCT116 cells were treated for 24 hrs with vehicle alone (-), 1 µM MPA, 10 µM MPA, or the combination of 10 µM MPA with 400 µM guanosine. Guanylate nucleotides levels were measured by LC-MS, and normalized to protein content. Mean ± SEM is representative of 3 independent experiments carried out in triplicate. Data information data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . mRNAs of IMPDH1 and IMPDH2 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to β - actin mRNA ( C ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). mRNAs of IMPDH1 and IMPDH2 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to β-actin mRNA (C). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


Parental HCT116 cells expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) and either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in the absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . Whole cell extracts were analyzed on Western blots with the indicated antibodies . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 is shown . Mean ± SD is representative of four independent experiments . ( right panel ) ( D ) . Data information : data are presented as relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 by two tail T - Student ' s test .

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Parental HCT116 cells expressing a stable tetracycline-inducible shRNA against IMPDH2 (IM2iKD) and either a stable inducible non-targeting (NT) shRNA (IM2iKD-shNT) or an shRNA targeting IMPDH1 (IM2iKD - shIM1), were grown for 7 days in the absence or presence of doxycycline (2 µg/mL) (- dox or + dox, respectively). Whole cell extracts were analyzed on Western blots with the indicated antibodies. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 is shown. Mean ± SD is representative of four independent experiments. (right panel) (D). Data information: data are presented as relative to control. *p<0.05, **p<0.01, ***p<0.001 by two tail T-Student's test.


E , Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA , in presence of dimethyl sulfoxide ( DMSO ) or 400 µM guanosine . GAPDH was used as a loading control .

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E, Western blots in HCT116 cells treated for 24 hrs with the indicated concentration of MPA, in presence of dimethyl sulfoxide (DMSO) or 400 µM guanosine. GAPDH was used as a loading control.


A , HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone ( - ) or the indicated concentration of MPA for 24 hrs . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 and p21 of four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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A, HCT116 cells were transfected with either a NT or a siRNA against RPL11 for 24 hrs and treated with the vehicle alone (-) or the indicated concentration of MPA for 24 hrs. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 and p21 of four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least four independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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B, HCT116 cells were transfected with the indicated siRNA and 24 hrs later treated with the vehicle alone or 10 µM MPA for 6 hrs and the levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least four independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


C , HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence ( - ) or the presence ( + ) of the combination of 10 µM of the ATR inhibitor VE - 821 ( ATRi ) and 10 µM the ATM inhibitor KU - 55933 ( ATMi ) , followed by addition of the vehicle alone ( - ) or MPA 10 µM for additional 24 hrs . The levels of p53 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of band intensity of p53 of at least two independent experiments is shown ( right panel ) . Data information : All data are presented as Mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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C, HCT116 cells were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs and were pretreated for 30 mins in the absence (-) or the presence (+) of the combination of 10 µM of the ATR inhibitor VE-821 (ATRi) and 10 µM the ATM inhibitor KU-55933 (ATMi), followed by addition of the vehicle alone (-) or MPA 10 µM for additional 24 hrs. The levels of p53 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of band intensity of p53 of at least two independent experiments is shown (right panel). Data information: All data are presented as Mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 cells were treated with either vehicle alone ( - ) , 1 µM MPA , 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA . Data information : , data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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A, HCT116 cells were treated with either vehicle alone (-), 1 µM MPA, 10 µM MPA or 5 nM ActD for 24 hrs and mRNA levels of p21 were quantified by real-time qPCR in 2 independent experiments carried out in triplicate and normalized to 28S rRNA. Data information: , data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


B , Autoradiogram ( upper panel ) and Ethidium Bromide ( EtBr ) - stained agarose gel ( lower panel ) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section .

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B, Autoradiogram (upper panel) and Ethidium Bromide (EtBr)-stained agarose gel (lower panel) of 18S and 28S rRNA from HCT116 cells treated as indicated in the Methods section.


C , Autoradiogram ( upper panel ) and EtBr - stained TBE - urea polyacrylamide gel ( lower panel ) of 5S , 5 . 8S rRNA and tRNAs in

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C, Autoradiogram (upper panel) and EtBr-stained TBE-urea polyacrylamide gel (lower panel) of 5S, 5.8S rRNA and tRNAs in lysates


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . D , Levels of HDM2 , RPL5 , RPL11 , and the IP negative control mTOR in the whole cell extract ( WCE ) , the post - ribosomal lysates ( INPUT , left panel ) and in the HDM2 immunoprecipitated fraction ( right panel ) were analyzed on Western blots . The results are representative of two independent experiments . Quantification of band intensity of HDM2 , RPL5 and RPL11 of at least two independent experiments is shown ( lower right panel ) .

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HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). D, Levels of HDM2, RPL5, RPL11, and the IP negative control mTOR in the whole cell extract (WCE), the post-ribosomal lysates (INPUT, left panel) and in the HDM2 immunoprecipitated fraction (right panel) were analyzed on Western blots. The results are representative of two independent experiments. Quantification of band intensity of HDM2, RPL5 and RPL11 of at least two independent experiments is shown (lower right panel).


HCT116 cells were treated for 24 hrs . Cell lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA and immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or the IgG control ( IgG ) . E , the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA . Data are normalized and presented as relative to untreated cells immunoprecipitated with anti - HDM2 . The results are representative of three independent experiments carried out in triplicates . Data information : data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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HCT116 cells were treated for 24 hrs. Cell lysates were collected, subjected to ultracentrifugation, spiked with firefly luciferase mRNA and immunoprecipitated with anti-HDM2 rabbit antibody (IP HDM2) or the IgG control (IgG). E, the Levels of immunoprecipitated 5S rRNA were determined by real time qPCR and normalized to the firefly luciferase mRNA. Data are normalized and presented as relative to untreated cells immunoprecipitated with anti-HDM2. The results are representative of three independent experiments carried out in triplicates. Data information: data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


F , HCT116 cells were pretreated with vehicle alone ( - ) , 1 µM , or 10 µM MPA for 24 hrs , and the levels of p53 and p21 were followed on Western blots in presence of 100 µg / mL cycloheximide ( CHX ) for the indicated time , in the continued presence of the treatment . GAPDH was used as a loading control . Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment .

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F, HCT116 cells were pretreated with vehicle alone (-), 1 µM, or 10 µM MPA for 24 hrs, and the levels of p53 and p21 were followed on Western blots in presence of 100 µg/mL cycloheximide (CHX) for the indicated time, in the continued presence of the treatment. GAPDH was used as a loading control. Quantification of band intensities is shown in the lower panels as relative to time 0 of CHX treatment.


G , HCT116 cells were pretreated as in ( F ) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated . The levels of p53 and p21 were analyzed on Western blots . GAPDH was used as a loading control . Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel . Data information : In panels data are presented as mean ± SD , relative to control . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , by two tail T - Student ' s test .

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G, HCT116 cells were pretreated as in (F) and 10 µM of the proteasome inhibitor MG132 was added for 4 hrs when indicated. The levels of p53 and p21 were analyzed on Western blots. GAPDH was used as a loading control. Quantification of p21 band intensity of at least four independent experiments is shown in the lower panel. Data information: In panels data are presented as mean ± SD, relative to control. *p<0.05, **p<0.01, ***p<0.001, by two tail T-Student's test.


A , HCT116 were treated as indicated for 24 hrs , subjected to propidium iodide staining and analyzed by fluorescence - activated cell sorting ( FACS ) . Data are representative of at least three independent experiments .

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A, HCT116 were treated as indicated for 24 hrs, subjected to propidium iodide staining and analyzed by fluorescence-activated cell sorting (FACS). Data are representative of at least three independent experiments.


B , Colon adenocarcinoma cell lines HCT116 ( circles ) , LoVo ( squares ) , LS174 ( triangles ) , or RKO ( inversed triangles ) were treated with the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis . Data are shown as the fold induction of the cell number in G0 / G1 phases ( blue ) , S phase ( red ) , and G2 / M phases upon treatment with 1 µM MPA ( dark ) or 10 µM MPA ( clear ) over vehicle treated cells .

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B, Colon adenocarcinoma cell lines HCT116 (circles), LoVo (squares), LS174 (triangles), or RKO (inversed triangles) were treated with the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs and subjected to propidium iodide staining and FACS analysis. Data are shown as the fold induction of the cell number in G0/G1 phases (blue), S phase (red), and G2/M phases upon treatment with 1 µM MPA (dark) or 10 µM MPA (clear) over vehicle treated cells.


C , IdU track length distribution , and distribution of long fork / short fork IdU track length ratios ( insert ) . IdU track length of at least 300 fibers was measured for each condition . For fork symmetry analyses , IdU track length was measure in at least 38 bidirectional forks ( see methods ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis test of the means . D , Representative images from ( C ) .

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C, IdU track length distribution, and distribution of long fork / short fork IdU track length ratios (insert). IdU track length of at least 300 fibers was measured for each condition. For fork symmetry analyses, IdU track length was measure in at least 38 bidirectional forks (see methods). ***p<0.001, ****p<0.0001 by Kruskal-Wallis test of the means. D, Representative images from (C).


E , HCT116 cells , pre - incubated with 10 µM BrdU for 48 hrs were treated with Vehicle ( - ) , 10µM MPA for 6 or 24 hrs , or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation ( see Method ) . Cells were immunostained for native BrDU ( green ) , or RPA ( red ) . DNA was counterstained with DAPI ( blue ) . One representative z - confocal stack is shown per condition . Scale bars correspond to 5 μm .

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E, HCT116 cells, pre-incubated with 10 µM BrdU for 48 hrs were treated with Vehicle (-), 10µM MPA for 6 or 24 hrs, or 20 µM Hydroxyurea for 4 hrs before cell permeabilization and fixation (see Method). Cells were immunostained for native BrDU (green), or RPA (red). DNA was counterstained with DAPI (blue). One representative z-confocal stack is shown per condition. Scale bars correspond to 5 μm.


F , HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins . α - tubulin was used as a loading control .

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F, HCT116 cells treated with 10 µM MPA for increasing time were analyzed by Western blots for the indicated proteins. α-tubulin was used as a loading control.


A , HCT116 cells were treated with either the vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs , before p21 immuno - labeling , propidium iodide staining and FACS analysis . Cell cycle profiles of the total cell population ( upper panel , left ) , the total intensity of p21 and propidium iodide were determined in 20 , 000 cells and plotted in a scatter diagram ( lower panel , left ) . The specific cell cycle profiles are shown of p21high ( upper panel , right ) and p21low ( lower panel , right ) cell populations . Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels .

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A, HCT116 cells were treated with either the vehicle alone (-), 1 µM MPA or 10 µM MPA for 24 hrs, before p21 immuno-labeling, propidium iodide staining and FACS analysis. Cell cycle profiles of the total cell population (upper panel, left), the total intensity of p21 and propidium iodide were determined in 20,000 cells and plotted in a scatter diagram (lower panel, left). The specific cell cycle profiles are shown of p21high (upper panel, right) and p21low (lower panel, right) cell populations. Mean ± SD of the percentage of cells in each cell cycle phase of at least two independent experiments is shown in the right panels.


B , HCT116 p21 - / - isogenic cell lines were transfected for 24 hrs with an empty plasmid ( EV ) or a plasmid encoding a p21wt cDNA ( p21 - OE ) , then cells were serum - deprived for 16 hrs before the re - addition of serum for additional 24 hrs in absence or presence of 10 µM MPA . Cells were subjected to p21 immunolabeling , propidium iodide staining and FACS analysis . The results are representative of two independent experiments .

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B, HCT116 p21-/- isogenic cell lines were transfected for 24 hrs with an empty plasmid (EV) or a plasmid encoding a p21wt cDNA (p21-OE), then cells were serum-deprived for 16 hrs before the re-addition of serum for additional 24 hrs in absence or presence of 10 µM MPA. Cells were subjected to p21 immunolabeling, propidium iodide staining and FACS analysis. The results are representative of two independent experiments.


A , HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in ( Fig 5B ) . Here are shown the cell cycle profiles after 16 hrs of serum deprivation ( top panels ) , or after an additional 24 hrs of serum re - addition in absence ( middle panels ) , or presence of 10 µM MPA ( bottom panels ) . The results are representative of at least three independent experiments .

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A, HCT116 cells were transfected for 8 hrs with the indicated siRNA then treated and analyzed as in (Fig 5B). Here are shown the cell cycle profiles after 16 hrs of serum deprivation (top panels), or after an additional 24 hrs of serum re-addition in absence (middle panels), or presence of 10 µM MPA (bottom panels). The results are representative of at least three independent experiments.


B , HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs . The levels of γ - H2AX were analyzed on Western blots . GAPDH was used as a loading control .

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B, HCT116 cells transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs. The levels of γ-H2AX were analyzed on Western blots. GAPDH was used as a loading control.


Statistical analysis of the immunostaining of γ - H2AX ( C ) in HCT116 cells treated Total nuclear intensities were obtained on > 6000 ( γ - H2AX ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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Statistical analysis of the immunostaining of γ-H2AX (C) in HCT116 cells treated Total nuclear intensities were obtained on >6000 (γ-H2AX) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


Statistical analysis of the immunostaining of 53BP1 ( D ) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 ( 53BP1 ) individual cells for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis test . A . U . Arbitrary Units .

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Statistical analysis of the immunostaining of 53BP1 (D) in HCT116 cells treated Total nuclear intensities were obtained on > 1000 (53BP1) individual cells for each condition from four or two independent biological replicates, respectively. **p<0.01, ****p<0.0001 by Kruskal-Wallis test. A.U. Arbitrary Units.


E , Representative images cells labeled with γ - H2AX ( green ) , 53BP1 ( red ) and DAPI ( blue ) . Scale bars correspond to 10

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E, Representative images cells labeled with γ-H2AX (green), 53BP1 (red) and DAPI (blue). Scale bars correspond to 10 μm


F , HCT116 cells treated were analyzed by the comet assay . The percentage of tail DNA / total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates . * * * * p < 0 . 0001 by Kruskal - Wallis

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F, HCT116 cells treated were analyzed by the comet assay. The percentage of tail DNA/total DNA was analyzed in a total of 300 cells for each condition from three independent biological replicates. ****p<0.0001 by Kruskal-Wallis test


b . Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound - induced tumours ( Pap ) compared to fibroblasts from wild - type ( WT ) and inflamed ( InvEE ) back skin ( n = 3 per condition ) . PRSS35 is indicated in red as a top upregulated gene in CAFs .

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b. Heatmap depicting mean expression of the top 20 upregulated genes of fibroblast isolated from wound-induced tumours (Pap) compared to fibroblasts from wild-type (WT) and inflamed (InvEE) back skin (n=3 per condition). PRSS35 is indicated in red as a top upregulated gene in CAFs.


a . Immunofluorescent images of skin sections from PDGFRα - eGFP reporter mice with normal ( WT , n = 4 ) , inflamed ( InvEE , n = 4 ) , wounded ( d14 post - wounding ; wound , n = 4 ) , or tumour - associated ( Papilloma , Pap d17 and d30 post - wounding , n = 4 per condition ) skin . Nuclei were stained with Dapi ( 4 ' , 6 - diamidino - 2 - phenylindole ; blue ) . Dotted line represents epidermal - dermal boundary . Scale bars : 50 μm . b . Quantification of the number of fibroblasts present in different skin conditions ( n = 4 mice per condition ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ; One - way ANOVA testing ) . Data represent means of multiple microscopic fields ± S . E . M .

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a. Immunofluorescent images of skin sections from PDGFRα-eGFP reporter mice with normal (WT, n=4), inflamed (InvEE, n=4), wounded (d14 post-wounding; wound, n=4), or tumour-associated (Papilloma, Pap d17 and d30 post-wounding, n=4 per condition) skin. Nuclei were stained with Dapi (4', 6-diamidino- 2- phenylindole; blue). Dotted line represents epidermal-dermal boundary. Scale bars: 50 μm. b. Quantification of the number of fibroblasts present in different skin conditions (n=4 mice per condition; ** p<0.01; *** p<0.001; **** p<0.0001; One-way ANOVA testing). Data represent means of multiple microscopic fields ± S.E.M.


c . Herovici staining of sections of normal ( WT ) , inflamed ( InvEE ) , wound , or tumour - associated ( Papilloma ) skin . Scale bars : 100 μm .

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c. Herovici staining of sections of normal (WT), inflamed (InvEE), wound, or tumour-associated (Papilloma) skin. Scale bars: 100 μm.


c . Relative mRNA levels of PRSS35 in lysates from normal skin ( WT , n = 4 mice ) , inflamed skin ( InvEE , n = 4 mice ) , wounds ( d14pw , n = 4 mice ) and wound - induced papillomas ( Pap , n = 5 mice ) . ( * p = 0 . 0159 ; Mann - Whitney test ) . Data represent means of three technical replicates ± S . E . M .

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c. Relative mRNA levels of PRSS35 in lysates from normal skin (WT, n=4 mice), inflamed skin (InvEE, n=4 mice), wounds (d14pw, n=4 mice) and wound-induced papillomas (Pap, n=5 mice). (* p=0.0159; Mann-Whitney test). Data represent means of three technical replicates ± S.E.M.


f . H & E - stained skin sections of WT and PRSS35 - / - wounds at different days post - wounding . Scale bars : 100 µm .

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f. H&E-stained skin sections of WT and PRSS35-/- wounds at different days post-wounding. Scale bars: 100 µm.


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 .

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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 .

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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 .

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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 .

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

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

(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 B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 .

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(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 .

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(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 .

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(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 .

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(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 ) .

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(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 .

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(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 ) ) .

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(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 ) .

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(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 . ) .

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(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 ) .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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 .

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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.