Cntl or ALYREF siRNA treated Flag - Lsm11 stable expression were used for IPs with IgG or Flag antibody . immunoprecipitates were subjected to RT - qPCRs ( E ) * indicates antibody heavy chain . bars show relative IP efficiencies of histone pre - mRNAs to U7 snRNA . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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Cntl or ALYREF siRNA treated Flag-Lsm11 stable expression cells were used for IPs with IgG or the Flag antibody. The immunoprecipitates were subjected to RT-qPCRs (E) * indicates the antibody heavy chain. The bars show the relative IP efficiencies of histone pre-mRNAs to U7 snRNA. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( A ) ( Top ) Illustration of HIST2H2AA3 reporter construct . ( Bottom ) FISH to detect distribution of HIST2H2AA3 mRNA in HeLa depleted of Cntl , ALYREF , THOC2 ( THO ) UAP56 . DAPI served as a marker . N C indicate FISH signals , respectively . N / C ratios were determined for 20 in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(A) (Top) Illustration of the HIST2H2AA3 reporter construct. (Bottom) FISH to detect the distribution of the HIST2H2AA3 mRNA in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. DAPI staining served as a nuclear marker. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( B ) Distribution of endogenous HIST2H2AA3 mRNA was detected with transcript - specific probe in HeLa depleted of Cntl , ALYREF , THOC2 ( THO ) UAP56 . N C indicate FISH signals , respectively . N / C ratios were calculated Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(B) Distribution of the endogenous HIST2H2AA3 mRNA was detected with the transcript-specific probe in HeLa cells depleted of Cntl, ALYREF, THOC2 (THO) and UAP56. N and C indicate nuclear and cytoplasmic FISH signals, respectively. N/C ratios were calculated Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


Western to examine purities of fractions ( C ) in Cntl UAP56 KD . MTR4 Tubulin were used as marker , respectively .

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Western analysis to examine the purities of nuclear and cytoplasmic fractions (C) in Cntl and UAP56 KD cells. MTR4 and Tubulin were used as the nuclear and cytoplasmic marker, respectively.


RT - qPCRs to examine N / C distribution of each histone mRNA ( D ) in Cntl UAP56 KD . Data information : Error bars represent standard deviations from biological repeats ( n = 3 Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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RT-qPCRs to examine N/C distribution of each histone mRNA (D) in Cntl and UAP56 KD cells. Data information: Error bars represent standard deviations from biological repeats (n=3 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( F ) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl , ALYREF SLBP KD . Data information : Error bars represent standard deviations from biological repeats n = 2 Statistical was performed using Student ' s t - . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 .

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(F) Relative NXF1 iCLIP read populations on histone mRNAs in Cntl, ALYREF and SLBP KD cells. Data information: Error bars represent standard deviations from biological repeats n=2 Statistical analysis was performed using Student's t-test. *P < 0.05, **P < 0.01, ***P< 0.001.


( G ) NXF1 iCLIP signals along histone mRNA . histone mRNA is divided into 10 fractions on their mRNA . Each fraction represents 10 % of each histone mRNA . tenth fractions that contains SL is boxed by green dashed lines .

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(G) NXF1 iCLIP signals along the histone mRNA. The histone mRNA is divided into 10 fractions based on their mRNA size. Each fraction represents 10% of each histone mRNA. The tenth fractions that contains the SL is boxed by green dashed lines.


( A ) ( Top ) Illustration of reporter constructs . black red lines indicate H2AA3 - SL H2AA3 - SLD PCR products , respectively . dashed indicates FISH probe . ( Bottom ) FISH to detect distribution of corresponding mRNA at 2 hr after injection of H2AA3 - SL H2AA3 - SLD PCR products . C N indicate FISH signals , respectively . C / N ratios were determined for 20 in each experiment . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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(A) (Top) Illustration of the reporter constructs. The black and red lines indicate H2AA3-SL and H2AA3-SLD PCR products, respectively. The dashed line indicates the FISH probe. (Bottom) FISH to detect the distribution of the corresponding mRNA at 2 hr after injection of H2AA3-SL and H2AA3-SLD PCR products. C and N indicate cytoplasmic and nuclear FISH signals, respectively. C/N ratios were determined for 20 cells in each experiment. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( B ) ( Top ) Illustration of reporter constructs . dashed indicates FISH probe . FISH to detect mRNA distribution at 24 hr after transfection of H1C - SL - Rb ( cH1C ) H1C - SLD - Rb ( pH1C ) constructs . C / N ratios were quantified Data information Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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(B) (Top) Illustration of the reporter constructs. The dashed line indicates the FISH probe. FISH to detect mRNA distribution at 24 hr after transfection of the H1C-SL-Rb (cH1C) and H1C-SLD-Rb (pH1C) constructs. C/N ratios were quantified Data information Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( Top ) Illustration of reporter constructs . PCR products of cG - SL cG - SLD were used instead . cG , β - globin cDNA . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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(Top) Illustration of the reporter constructs. PCR products of cG-SL and cG-SLD were used instead. cG, β-globin cDNA. Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa , followed by IPs with IgG or ALYREF antibody at 24 hr post - transfection . immunoprecipitates were subjected to western to detect ALYREF ( D )

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis to detect ALYREF (D)


H1C - SL - Rb or H1C - SLD - Rb construct , together with VSV M were co - transfected into HeLa , followed by IPs with IgG or ALYREF antibody at 24 hr post - transfection . RT - qPCRs to detect RNAs ( E ) . bars in graph shows relative ALYREF IP efficiency , with that for H1C - SL - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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H1C-SL-Rb or H1C-SLD-Rb construct, together with VSV M were co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. RT-qPCRs to detect RNAs (E). The bars in the graph shows the relative ALYREF IP efficiency, with that for the H1C-SL-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( F ) H1C - SLD - Rb construct with wild - type ( H1C - SLDWT - Rb ) or mutant ( H1C - SLDmut - Rb ) U7 - binding , together with VSV M , was co - transfected into HeLa , followed by IPs with IgG or ALYREF antibody at 24 hr post - transfection . immunoprecipitates were subjected to western ( left panel ) RT - qPCRs ( right panel ) . bars in right graph show relative ALYREF IP efficiency , with that for H1C - SLDWT - Rb mRNA set as " 1 " . Data information : Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * P < 0 . 05 , * * * P < 0 . 001 .

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(F) H1C-SLD-Rb construct with wild-type (H1C-SLDWT-Rb) or mutant (H1C-SLDmut-Rb) U7-binding sequence, together with VSV M, was co-transfected into HeLa cells, followed by IPs with IgG or the ALYREF antibody at 24 hr post-transfection. The immunoprecipitates were subjected to western analysis (left panel) and RT-qPCRs (right panel). The bars in the right graph show the relative ALYREF IP efficiency, with that for the H1C-SLDWT-Rb mRNA set as "1". Data information: Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. *P < 0.05, ***P< 0.001.


( A ) Western blotting to examine expression of H2B H4 in Cntl , ALYREF ARS2 KD . GAPDH was used as a loading control . delineates boundary where irrelevant lanes have been removed from same

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(A) Western blotting to examine the expression levels of H2B and H4 in Cntl, ALYREF and ARS2 KD cells. GAPDH was used as a loading control. The white line delineates the boundary where irrelevant lanes have been removed from the same blot


( B ) Growth curve of Cntl ALYREF KD . Error bars represent standard deviations from biological repeats ( n = 3 ) . Statistical was performed using Student ' s t - . * * P < 0 . 01 .

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(B) Growth curve of Cntl and ALYREF KD cells. Error bars represent standard deviations from biological repeats (n=3). Statistical analysis was performed using Student's t-test. **P < 0.01.


( C ) cycle of Cntl , ALYREF ARS2 KD . Quantifications of in G0 / G1 ( 1N ) , G2 ( 2N ) S - phase ( intermediate ) of cycle are shown in upper right corner of each histogram .

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(C) Cell cycle analysis of Cntl, ALYREF and ARS2 KD cells. Quantifications of cells in G0/G1 (1N), G2 (2N) and S-phase (intermediate) of the cell cycle are shown in the upper right corner of each histogram.


Results from multiplex ACE2 competition are shown for three spike - derived antigens : RBD , S1 - domain ( S1 ) homotrimeric spike ( Spike ) . Color - coded beads coated with respective antigens were co - incubated with biotinylated ACE2 dilution series of NM1267 ( 8 pM to 126 nM ) followed by measuring residual binding of ACE2 . MFI signals were normalized to maximum detectable signal per antigen given by ACE2 - only control . IC50 values were calculated from a four - parametric sigmoidal model . Data are presented as mean ± s . d . of three technical replicates .

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Results from multiplex ACE2 competition assay are shown for the three spike-derived antigens: RBD, S1-domain (S1) and homotrimeric spike (Spike). Color-coded beads coated with the respective antigens were co-incubated with biotinylated ACE2 and dilution series of NM1267 (8 pM to 126 nM) followed by measuring residual binding of ACE2. MFI signals were normalized to the maximum detectable signal per antigen given by the ACE2-only control. IC50 values were calculated from a four-parametric sigmoidal model. Data are presented as mean ± s.d. of three technical replicates.


E Neutralization potency of NM1267 was analyzed in Caco ‑ 2 using SARS - CoV - 2 - mNG infectious clones . Infection rate normalized to - only infection control is illustrated as of infection ( % Infection ) . IC50 value was calculated from a four - parametric sigmoidal model data are presented as mean ± s . e . m . of three biological replicates ( n = 3 ) .

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E Neutralization potency of NM1267 was analyzed in Caco‑2 cells using the SARS-CoV-2-mNG infectious clones. Infection rate normalized to virus-only infection control is illustrated as percent of infection (% Infection). IC50 value was calculated from a four-parametric sigmoidal model and data are presented as mean ± s.e.m. of three biological replicates (n = 3).


B For NeutrobodyPlex , antigen - coated beads comprising RBD , S1 or spike were co - incubated with serum samples from 5 a dilution series of NM1267 ( 1 µM to 6 pM ) ( n = 1 ) . Mean intensities ( MFI ) derived from antigen - bound IgGs in presence of bipNb normalized to MFI values of IgGs in serum - only samples , illustrated as MFI ( % control ) , are shown .

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B For the NeutrobodyPlex, antigen-coated beads comprising RBD, S1 or spike were co-incubated with serum samples from 5 patients and a dilution series of NM1267 (1 µM to 6 pM) (n = 1). Mean fluorescent intensities (MFI) derived from antigen-bound IgGs in the presence of bipNb normalized to the MFI values of IgGs in the serum-only samples, illustrated as MFI (% control), are shown.


A , B Total IgGs derived from spike - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . C , D Total IgGs derived from N - binding IgGs were plotted against normalized MFI values from IgGs binding to RBD ( MFI RBD ( % control ) ) in presence of both concentrations of NM1267 ( 1 µM ; 1 nM , ) . F

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A ,B Total IgGs derived from spike-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). C, D Total IgGs derived from N-binding IgGs were plotted against normalized MFI values from IgGs binding to RBD (MFI RBD (%control)) in the presence of both concentrations of NM1267 (1 µM; 1 nM,). F


( A ) Graph derived from two published data available in PubMed GEO database ( GSE6919 GSE35988 ) . box charts depict relative expression of ESM1 in benign , , metastatic prostate cancer . centre donates median value while box contains 25th to 75th percentiles of dataset . whiskers mark 5th 95th percentiles , values beyond these upper lower bounds are considered outliers .

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(A) Graph derived from two published data available in the PubMed GEO database (GSE6919 and GSE35988). The box charts depict the relative expression of ESM1 in benign, primary, and metastatic prostate cancer patients. The centre line donates the median value while the box contains the 25th to 75th percentiles of dataset. The whiskers mark the 5th and 95th percentiles, and values beyond these upper and lower bounds are considered outliers.


( B ) Relative ESM1 mRNA expression ESM1 expression in two sets of human metastatic PCa lines . P : parental , M : metastatic .

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(B) Relative ESM1 mRNA expression and ESM1 protein expression in two sets of human metastatic PCa cell lines. P: parental cells, M: metastatic cells.


( C ) invasion assays of 22Rv1 - M PC3 - M transfected with either Scramble - shRNA or ESM1 - shRNA . data are shown as relative fold change of invasive compared with shScramble group . Scale bar : 100 μm .

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(C) Cell invasion assays of 22Rv1-M and PC3-M cells transfected with either Scramble-shRNA or ESM1-shRNA. The data are shown as the relative fold change of invasive cells compared with the shScramble group. Scale bar: 100 μm.


( D ) Representative of lungs , livers , intestines , kidneys from mice 8 after orthotopical injection of 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA . Scale bar : 0 . 5 cm . Tumors were removed weighed . Quantitative summary of luminescence in different metastases measured in photons / second are shown .

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(D) Representative images of lungs, livers, intestines, and kidneys from mice 8 weeks after orthotopical injection of 22Rv1-M cells transfected with either Scramble-shRNA or ESM1-shRNA. Scale bar: 0.5 cm. Tumors were removed and weighed. Quantitative summary of tumor luminescence in different metastases measured in photons/second are shown.


( E ) Formalin - fixed , paraffin - embedded tissue microarray sections of 59 normal prostate tissues 65 prostate adenocarcinoma tissues with without metastasis ( n = 16 n = 49 , respectively ) were stained with an anti - ESM1 antibody . Tissue - bound ESM1 is shown in brown . Scale bar : 50 μm . Arrowheads indicate positively stained nucleus of cancer . Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to prostate adenocarcinoma tissue with without metastasis . scores are calculated by ( score 1 - 3 ) x ( score 1 - 4 ) of stained . in middle of box is plotted at median . box extends from 25th to 75th percentiles . Whiskers above below box indicate 5th 95th percentiles .

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(E) Formalin-fixed, paraffin-embedded tissue microarray sections of 59 normal prostate tissues and 65 prostate adenocarcinoma tissues with and without metastasis (n=16 and n=49, respectively) were stained with an anti-ESM1 antibody. Tissue-bound ESM1 is shown in brown. Scale bar: 50 μm. Arrowheads indicate positively stained nucleus of cancer cells. Plot representation of scores according to immunohistochemical expression of ESM1 in normal prostate tissue related to the prostate adenocarcinoma tissue with and without metastasis. The scores are calculated by intensity (score 1-3) x percentage (score 1-4) of stained cells. The line in the middle of the box is plotted at the median. The box extends from the 25th to 75th percentiles. Whiskers above and below the box indicate the 5th and 95th percentiles.


( A ) Immunoblotting of ESM1 in supernatant ( S ) , nucleus ( N ) , cytoplasm ( C ) was performed . Ponceau S , Lamin A / C , α - tubulin were used as loading control in supernatant , nucleus , cytoplasm , respectively . P : parental , M : metastatic .

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(A) Immunoblotting analysis of ESM1 in supernatant (S), nucleus (N), and cytoplasm (C) was performed. Ponceau S, Lamin A/C, and α-tubulin were used as loading control in supernatant, nucleus, and cytoplasm, respectively. P: parental cells, M: metastatic cells.


( B ) Left , representative of in transwell invasion . Right , of that invaded through matrigel - coated following treatment with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody . Scale bar : 100 μm .

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(B) Left, representative images of cells in the transwell invasion assay. Right, quantification of cells that invaded through the matrigel-coated membrane following treatment with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody. Scale bar: 100 μm.


( D ) Elicitation of ESM1 overexpression using HA - tag antibody results in detectable ESM1 expression in nucleus cytoplasm of 22Rv1 - P . Lamin A / C α - tubulin were used as loading control in nucleus cytoplasm , respectively .

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(D) Elicitation of ESM1 overexpression using HA-tag antibody results in detectable ESM1 expression in the nucleus and cytoplasm of 22Rv1-P cells. Lamin A/C and α-tubulin were used as loading control in the nucleus and cytoplasm, respectively.


( E ) Immunoblotting of ESM1 expression with different expressing patterns in supernatant ( S ) of 22Rv1 - P .

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(E) Immunoblotting of ESM1 expression with different expressing patterns in the supernatant (S) of 22Rv1-P cells.


( F ) Left , representative of 22Rv1 - P in transwell invasion . Right , statistic data of invasive ability of ESM1 expressing . Scale bar : 100 μm .

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(F) Left, representative images of 22Rv1-P cells in the transwell invasion assay. Right, statistic data of invasive ability of ESM1 expressing cells. Scale bar: 100 μm.


( G ) Left , representative bioluminescence of lung metastasis in mice after tail vein injection of 22Rv1 - Luc with different ESM1 expression patterns . Scale bar : 0 . 5 cm . Right , of lung bioluminescence by photons / seconds .

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(G) Left, representative bioluminescence images of lung metastasis in mice after tail vein injection of 22Rv1-Luc cells with different ESM1 expression patterns. Scale bar: 0.5 cm. Right, quantification of the lung bioluminescence by photons/ seconds.


( A ) spheroid formation . Representative of spheres formed quantitative data comparing average number of spheres formed in indicated treated with PBS or human recombinant ESM1 ( rhESM1 ) or normal goat IgG control or anti - ESM1 neutralizing antibody .

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(A) Tumor spheroid formation. Representative images of tumor spheres formed and quantitative data comparing the average number of spheres formed in the indicated cells treated with PBS or human recombinant ESM1 (rhESM1) or normal goat IgG control or anti-ESM1 neutralizing antibody.


( B ) Representative of spheres . Spheres were cultured for 14 days before counting . Histogram shows mean numbers of spheres cultured . Scale bar : 100 μm . Bars are mean ± SD of three independent experiments .

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(B) Representative images of tumor spheres. Spheres were cultured for 14 days before counting. Histogram shows the mean numbers of spheres cultured. Scale bar: 100 μm. Bars are the mean ± SD of three independent experiments.


( C ) 22Rv1 - P stably expressing ESM1 were treated with docetaxel at indicated concentrations for 48 h . * *

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(C) 22Rv1-P cells stably expressing ESM1 were treated with docetaxel at the indicated concentrations for 48 h. **


( D ) with or without ESM1 - NLS overexpression were subcutaneously injected ( 10 , 100 , 1000 per mouse ) into NOD / SCID mice . formation ability were analyzed . Scale bar : 1 cm . Bars are mean ± SD of indicated ( n ) independent experiments .

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(D) Cells with or without ESM1-NLS overexpression were subcutaneously injected (10, 100, 1000 cells per mouse) into NOD/SCID mice. Tumor formation ability and tumor weight were analyzed. Scale bar: 1 cm. Bars are the mean ± SD of indicated (n) independent experiments.


( E ) Flow cytometry of of CD44 + CD133 + in indicated with different subcellular localization types of ESM1 overexpression were performed .

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(E) Flow cytometry analysis of the ratio of CD44+ and CD133+ cells in the indicated cells with different subcellular localization types of ESM1 overexpression were performed.


( F ) qRT - PCR of mRNA of indicated genes in with different types of ESM1 overexpression . Differences in mRNA compared with vector are shown as fold changes presented as mean ± SD of three independent experiments . *

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(F) qRT-PCR analysis of the mRNA levels of the indicated genes in cells with different types of ESM1 overexpression. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments. *


( A ) IPA of - seq data showing 14 top - predicted upstream regulators of differentially expressed genes . Positive Z - scores ( orange ) represent activated upstream regulators , negative Z - scores ( blue ) represent inhibited upstream regulators .

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(A) IPA analysis of RNA-seq data showing the 14 top-predicted upstream regulators of differentially expressed genes. Positive Z-scores (orange) represent activated upstream regulators, negative Z-scores (blue) represent inhibited upstream regulators.


( E ) transcriptional activity of β - catenin in 22Rv1 - P with different patterns of ESM1 overexpression . Bars are mean ± SD of three independent experiments .

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(E) The transcriptional activity of β-catenin in 22Rv1-P cells with different patterns of ESM1 overexpression. Bars are the mean ± SD of three independent experiments.


( F ) Manipulating ESM1 expression impacts transcriptional activity of NFκB . Bars are mean ± SD of three independent experiments .

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(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the mean ± SD of three independent experiments.


( A ) qRT - PCR of mRNA of genes related to β - catenin downstream signaling pathway . Differences in mRNA compared with vector are shown as fold changes presented as mean ± SD of three independent experiments .

(A) qRT-PCR analysis of the mRNA levels of the genes related to β-catenin downstream signaling pathway. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( B ) qRT - PCR of mRNA of NFκB targeting genes . Differences in mRNA compared with vector are shown as fold changes presented as mean ± SD of three independent experiments .

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(B) qRT-PCR analysis of the mRNA levels of NFκB targeting genes. Differences in mRNA levels compared with vector cells are shown as fold changes presented as the mean ± SD of three independent experiments.


( C ) Immunoblotting of 22Rv1 - P with different ESM1 overexpression in nucleus cytoplasm were performed with antibodies indicated .

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(C) Immunoblotting analysis of 22Rv1-P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the antibodies indicated.


( D ) Suppression of β - catenin or NFκB in 22Rv1 - P stably expressing ESM1 - NLS were treated with docetaxel at indicated concentrations for 48 h . * * P < 0 . 01 when compared to vector - shScramble by two - Student ' s t error bars represent standard deviation of three independent experiments .

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(D) Suppression of β-catenin or NFκB in 22Rv1-P cells stably expressing ESM1-NLS were treated with docetaxel at the indicated concentrations for 48 h. ** P < 0.01 when compared to vector-shScramble cells by two-tailed Student's t test and error bars represent the standard deviation of three independent experiments.


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

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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 quantitative data comparing invasion of indicated . Scale bar : 100 μm . Bars are mean ± SD of three independent experiments . # # P < 0 . 01 when compared to vector - shScramble , * * P < 0 . 01 when compared to ESM1 - NLS - shScramble by two - Student ' s t .

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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 ) extracts of 22Rv1 - M transfected with either shScramble or shCTNNB1 were prepared of indicated 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 ) or extracts of 22Rv1 - M 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 GST - β - catenin 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 were co - transfected with indicated β - catenin - Flag ESM1 - HA constructs . extracts were immunoprecipitated with Flag - M2 agarose beads . 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 were transfected with ESM1 - HA constructs 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 were transfected with indicated ESM1 - GFP constructs . 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 were transfected with indicated ESM1 point mutant constructs . 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 ) extracts of 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA were immunoprecipitated with an β - catenin antibody blotted with anti - TCF4 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 were transfected with ESM1 - HA constructs . extracts were treated with short peptides , pulled down with purified GST - ARM blotted with anti - HA 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 were transfected with indicated ESM1 point mutant constructs . extracts were pulled down with purified GST - ARM 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 CCND1 c - Myc promoter in 22Rv1 - M transfected with either Scramble - shRNA or ESM1 - shRNA . Bars are 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 CCND1 c - Myc transcription start sites ( TSSs ) . Bar graphs show β - catenin ESM1 binding at CCND1 c - Myc promoter regions . Differences in compared with IgG control are shown as fold changes presented as 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 ) effects of ESM1 targeting peptides on invasion spheroid formation . Scale bar : 100 μm . Bars are 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 ) effects of ESM1 point mutants on invasion spheroid formation . Scale bar : 100 μm . Bars are 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 ) dye release from mitochondria - mimic liposomes by indicated wild - type ( wt ) or mutant Bax in presence ( B ) or absence ( C ) of tBid or by tBid alone ( C ) was measured by quenching of released dyes by dye - specific antibodies outside liposomes during a time course . fraction of dye release was normalized to that by detergent . data shown were obtained from n = 3 independent replicates using same preparations of 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 - / - expressing SMAC - mCherry in mitochondria intermembrane space ( 50 μL , 500 , 000 total ) were incubated with 25 nM of Bax 2 nM cBid for indicated time at 37 ° C in a 96 - well plate . samples were centrifuged for 10 min separated into supernatant pellet fractions . SMAC - mCherry release was calculated as fraction of total ( supernatant + pellet ) mCherry coming from supernatant fraction . data was normalized to SMAC - mCherry release of wt Bax cBid at 60 min . Each symbol represents normalized SMAC - mCherry release for one single replicate ( n = 3 or more independent replicates ) . Where applicable data was fit with [ Agonist ] vs . response - Variable slope equation in Graphpad Prism 8 . 0 . 1 . dotted lines represent 95 % confidence interval of 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 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 were treated for 24 hrs with vehicle alone ( - ) , 1 µM MPA , 10 µM MPA , or combination of 10 µM MPA with 400 µM guanosine . Guanylate nucleotides were measured by LC - MS , normalized to content . Mean ± 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 .

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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 expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . mRNAs of IMPDH1 IMPDH2 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate 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 .

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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 expressing a stable tetracycline - inducible shRNA against IMPDH2 ( IM2iKD ) either a stable inducible non - targeting ( NT ) shRNA ( IM2iKD - shNT ) or an shRNA targeting IMPDH1 ( IM2iKD - shIM1 ) , were grown for 7 days in absence or presence of doxycycline ( 2 µg / mL ) ( - dox or + dox , respectively ) . Whole extracts were analyzed on Western blots with indicated antibodies . GAPDH was used as a loading control . of band of p53 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 .

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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 treated for 24 hrs with indicated 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 were transfected with either a NT or a siRNA against RPL11 for 24 hrs treated with vehicle alone ( - ) or indicated of MPA for 24 hrs . of p53 p21 were analyzed on Western blots . GAPDH was used as a loading control . of band of p53 p21 of four independent experiments is shown ( right panel ) . 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 .

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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 were transfected with indicated siRNA 24 hrs later treated with vehicle alone or 10 µM MPA for 6 hrs of p53 p21 were analyzed on Western blots . GAPDH was used as a loading control . of band of p53 of at least four independent experiments is shown ( right panel ) . 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 .

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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 were transfected with either a NT siRNA or a siRNA against RPL11 for 24 hrs were pretreated for 30 mins in absence ( - ) or presence ( + ) of combination of 10 µM of ATR inhibitor VE - 821 ( ATRi ) 10 µM ATM inhibitor KU - 55933 ( ATMi ) , followed by addition of vehicle alone ( - ) or MPA 10 µM for additional 24 hrs . of p53 were analyzed on Western blots . GAPDH was used as a loading control . of band of p53 of at least two independent experiments is shown ( right panel ) . 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 .

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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 were treated with either vehicle alone ( - ) , 1 µM MPA , 10 µM MPA or 5 nM ActD for 24 hrs mRNA of p21 were quantified by real - time qPCR in 2 independent experiments carried out in triplicate 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 .

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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 ) Ethidium Bromide ( EtBr ) - stained agarose gel ( lower panel ) of 18S 28S rRNA from HCT116 treated as indicated in 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 ) EtBr - stained TBE - urea polyacrylamide gel ( lower panel ) of 5S , 5 . 8S rRNA 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 were treated for 24 hrs . lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or IgG control ( IgG ) . D , of HDM2 , RPL5 , RPL11 , IP negative control mTOR in whole extract ( WCE ) , post - ribosomal lysates ( INPUT , left panel ) in HDM2 immunoprecipitated fraction ( right panel ) were analyzed on Western blots . results are representative of two independent experiments . of band of HDM2 , RPL5 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 were treated for 24 hrs . lysates were collected , subjected to ultracentrifugation , spiked with firefly luciferase mRNA immunoprecipitated with anti - HDM2 rabbit antibody ( IP HDM2 ) or IgG control ( IgG ) . E , of immunoprecipitated 5S rRNA were determined by real time qPCR normalized to firefly luciferase mRNA . Data are normalized presented as relative to untreated immunoprecipitated with anti - HDM2 . 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 .

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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 were pretreated with vehicle alone ( - ) , 1 µM , or 10 µM MPA for 24 hrs , of p53 p21 were followed on Western blots in presence of 100 µg / mL cycloheximide ( CHX ) for indicated time , in continued presence of treatment . GAPDH was used as a loading control . of band intensities is shown in 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 were pretreated as in ( F ) 10 µM of proteasome inhibitor MG132 was added for 4 hrs when indicated . of p53 p21 were analyzed on Western blots . GAPDH was used as a loading control . of p21 band of at least four independent experiments is shown in 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 .

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

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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 distribution , distribution of long fork / short fork IdU track ratios ( insert ) . IdU track of at least 300 was measured for each condition . For fork symmetry analyses , IdU track was measure in at least 38 bidirectional forks ( see methods ) . * * * p < 0 . 001 , * * * * p < 0 . 0001 by Kruskal - Wallis of means . D , Representative 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 , 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 permeabilization fixation ( see Method ) . were immunostained for native BrDU ( green ) , or RPA ( red ) . 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 treated with 10 µM MPA for increasing time were analyzed by Western blots for indicated . 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 were treated with either vehicle alone ( - ) , 1 µM MPA or 10 µM MPA for 24 hrs , before p21 immuno - labeling , propidium iodide FACS . cycle profiles of total population ( upper panel , left ) , total of p21 propidium iodide were determined in 20 , 000 plotted in a scatter diagram ( lower panel , left ) . specific cycle profiles are shown of p21high ( upper panel , right ) p21low ( lower panel , right ) populations . Mean ± SD of of in each cycle phase of at least two independent experiments is shown in 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 lines were transfected for 24 hrs with an empty plasmid ( EV ) or a plasmid encoding a p21wt cDNA ( p21 - OE ) , then were serum - deprived for 16 hrs before re - addition of serum for additional 24 hrs in absence or presence of 10 µM MPA . were subjected to p21 immunolabeling , propidium iodide FACS . 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 were transfected for 8 hrs with indicated siRNA then treated analyzed as in ( Fig 5B ) . Here are shown 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 ) . 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 transfected with a NT siRNA or a siRNA against RPL11 for 24 hrs were treated with 10 µM MPA for additional 72 hrs . 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 of immunostaining of γ - H2AX ( C ) in HCT116 treated Total intensities were obtained on > 6000 ( γ - H2AX ) for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis . 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 of immunostaining of 53BP1 ( D ) in HCT116 treated Total intensities were obtained on > 1000 ( 53BP1 ) for each condition from four or two independent biological replicates , respectively . * * p < 0 . 01 , * * * * p < 0 . 0001 by Kruskal - Wallis . 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 labeled with γ - H2AX ( green ) , 53BP1 ( red ) 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 treated were analyzed by comet . of tail / total was analyzed in a total of 300 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 . depicting mean expression of top 20 upregulated genes of fibroblast isolated from wound - induced tumours ( Pap ) compared to fibroblasts from wild - type ( WT ) 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 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 d30 post - wounding , n = 4 per condition ) skin . Nuclei were stained with Dapi ( 4 ' , 6 - diamidino - 2 - phenylindole ; blue ) . Dotted represents epidermal - dermal boundary . Scale bars : 50 μm . b . of 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 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 of PRSS35 in lysates from normal skin ( WT , n = 4 mice ) , inflamed skin ( InvEE , n = 4 mice ) , wounds ( d14pw , n = 4 mice ) wound - induced papillomas ( Pap , n = 5 mice ) . ( * p = 0 . 0159 ; Mann - Whitney ) . 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 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 ) ( c ) number of tumours per mouse ( d ) in wild - type ( WT , n = 17 ) PRSS35 - / - ( n = 22 ) mice treated with DMBA - TPA ( * p = 0 . 0171 ; Wilcoxon matched - paired rank ) . 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 of indicated ECM genes in fibroblast cultures ( col1a1 : collagen type 1 alpha 1 ; αSMA , alpha smooth muscle actin ; Cyr61 : Cysteine - rich angiogenic inducer 61 ( = CCN1 ) ) in fibroblast cultures isolated from WT 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 of indicated cytokines / growth factors in 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 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 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 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 of normal skin , day 14 post - wounding ( d14pw ) skin or wound - induced papillomas from WT PRSS35 KO mice . Red colouring indicates thick fibres , green colouring indicates thin fibres . Scale bars : 200 µm . e . of picrosirius red as total collagen density ( upper panel ) of red versus green picrosirius ( lower panel ) . Using QuPath Bioimage software pixels colouring red or green were determined per . ( 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 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

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 ) counterstained with Dapi ( n = 5 per condition ) . Boxed areas are magnifications as shown in lower panel dotted represents epidermal - dermal boundary . Scale bars : 100 μm . b . of PRSS35 positive signal by Volocity ( 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 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

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

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

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 error bar depicts 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

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 ) Rv as compared with Rv ∆ aosR under oxidative conditions ( h ) . Data were normalized with respect to 16s rRNA results are expressed as mean log2 fold change + SD of three independent replicates 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 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

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

O O O O O O B-CONTROLLED_VAR 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

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 intracellular viability was monitored 96 h p . i ( mean ± ; 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

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


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

O O B-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 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 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 1 / 10th IP was probed with α - FLAG 9 / 10th IP with α - AosR . 50 µg WCLs were probed with α - FLAG , α - AosR , α - SigA as controls .

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 B-MEASURED_VAR 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

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 GroEL1 ( control ) was analyzed in WCLs of indicated 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 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 ( lanes 1 6 ) or 4 μΜ BSA ( lanes 2 7 ) or 2 μΜ His - AosR + 2 μΜ BSA ( lanes 3 8 ) or , 2 μΜ His - SigH + 2 μΜ BSA ( lanes 4 9 ) or 2 μΜ His - AosR + 2 μΜ His - SigH ( lanes 5 10 ) . reactions were either incubated in presence of 10 mM reduced DTT or 10 mM diamide to reduce or oxidize thiols , respectively , resolved on 8 % PAGE gel . - complexes ( bound ) free probe are indicated by arrows . To confirm specificity of - interaction , EMSA reaction was performed in presence of 5 , 10 , 25 , 50 , 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 B-CONTROLLED_VAR 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-CONTROLLED_VAR O O O B-MEASURED_VAR 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 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 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 1 / 10th IP was probed with α - FLAG 9 / 10th IP with α - HA . 50 µg WCLs were probed with α - FLAG , α - HA 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 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

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 strains were subjected to oxidative stress with 50 µM CHP for 6 h 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 of reduced mycothiol , MSH ergothioneine , ERG were measured in Rv , Rv ∆ aosR , Rv ∆ aosR : : aosR in normal CHP stressed using LC - MRM MS / MS . absolute values obtained under normal conditions were normalized to 100 % for each relative values were calculated for samples processed from CHP treated . data indicates relative of metabolites ( mean ± SD ) of four replicates ( n = 4 ) .

O O O O O O O O O O B-CONTROLLED_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 B-MEASURED_VAR 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 log2 fold induction ( mean ± SD , n = 3 ) for indicated genes in Rv ∆ aosR Rv ∆ aosR treated with 1mM L - cysteine as compared with Rv . strains were treated with 50 μM CHP . Data were normalized with respect to 16s rRNA 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 B-CONTROLLED_VAR 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 B-CONTROLLED_VAR 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 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 I-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 PGRP - LE112 flies , as well as flies treated with RNAi targeting PGRP - LE with hml - Gal4 driver ( hml - Gal4 > PGRP - LE RNAi ) 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 ) : 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 average of four independent experiments with over 30 flies of each genotype examined at same time .

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 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 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 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

(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 infected with wild - type L . monocytogenes . Hemocyte nuclei ( filled arrowhead ) of L . monocytogenes ( open arrowhead ) are visualized by DAPI ( blue or ) ; actin cytoskeleton is stained with rhodamine - labeled phalloidin ( red ) . hml - Gal4 > Atg5IR , expression of an inverted repeat 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 B-CONTROLLED_VAR 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 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 - ) . 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 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

(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 ) 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 ) : 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 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 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

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.