Immunoblotting of His - GID1 proteins using constructs shown in ( D ) . Proteins were separated by SDS - PAGE , followed by immunoblotting with anti - His - tag antibodies . The PA binding activity of different truncation mutations were analyzed by fat - immunoblotting . The red arrowheads indicate truncated proteins with different molecular weights .

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Immunoblotting of His-GID1 proteins using constructs shown in (D). Proteins were separated by SDS-PAGE, followed by immunoblotting with anti-His-tag antibodies. The PA binding activity of different truncation mutations were analyzed by fat-immunoblotting. The red arrowheads indicate truncated proteins with different molecular weights.


Immunoblotting of His - GID1 mutants and lipid - immunoblotting of PA binding by GID1 mutant proteins on a filter .

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Immunoblotting of His-GID1 mutants and lipid-immunoblotting of PA binding by GID1 mutant proteins on a filter.


PLDα6 - GFP distribution in rice protoplasts with different concentrations of GA3 or IAA . Protoplasts from 12 - day old rice ( ZH11 background ) leaf sheath tissue were collected . Rice protoplasts were transfected with pM999 - PLDα6 for 12 hours . After that , GA3 or IAA was added to protoplasts and 2 hours later , confocal images of protoplasts are shown . pM999 - GFP refers to the empty vector with GFP only that was transformed as control and Ghd7 - RFP was a nucleus marker . Scale bar = 10 μm .

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PLDα6-GFP distribution in rice protoplasts with different concentrations of GA3 or IAA. Protoplasts from 12-day old rice (ZH11 background) leaf sheath tissue were collected. Rice protoplasts were transfected with pM999-PLDα6 for 12 hours. After that, GA3 or IAA was added to protoplasts and 2 hours later, confocal images of protoplasts are shown. pM999-GFP refers to the empty vector with GFP only that was transformed as control and Ghd7-RFP was a nucleus marker. Scale bar = 10 μm.


Immunoblotting of PLDα6 in subcellular fractions . Total ( T ) , soluble ( S ) and nuclear ( N ) proteins were isolated from PLDα6 : GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA . Equal amount of each sample was loaded for SDS - PAGE and blotting .

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Immunoblotting of PLDα6 in subcellular fractions. Total (T), soluble (S) and nuclear (N) proteins were isolated from PLDα6:GFP expressed in rice protoplasts treated with 10 μM GA3 or IAA. Equal amount of each sample was loaded for SDS-PAGE and blotting.


Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments . Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours . Bars = 10 μm .

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Subcellular location of GID1 in WT and pldα6 protoplasts with or without GA3 treatments. Cells for 12 hours after transformation were treated with or without 10 μM GA3 for two hours. Bars = 10 μm.


Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts . After 2 - hour treatment with 10 μM GA3 , total ( T ) , soluble ( S ) and nuclear ( N ) protein fractions were isolated from 10 samples of protoplasts . Equal amount of each sample was loaded for SDS - PAGE and immunoblotting .

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

Immunoblotting of subcellular fractions of GID1 expressed in WT and pldα6 protoplasts. After 2-hour treatment with 10 μM GA3, total (T), soluble (S) and nuclear (N) protein fractions were isolated from 10 samples of protoplasts. Equal amount of each sample was loaded for SDS-PAGE and immunoblotting.


Protoplasts of WT and pldα6 from 12 - day old stage leaf sheath were collected and transfected with a pM999 - SLR1 construct , incubated for 12 hours and then treated with 10 μM GA3 for 0 , 3 , and 9 hours . The first column , fluorescence from GFP ; the second , red fluorescence from the nucleus marker Ghd7 ; the third , bright field , and the fourth , overlay of the three channels . All confocal images were scanned using similar laser gain and offset settings . 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 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

Protoplasts of WT and pldα6 from 12-day old stage leaf sheath were collected and transfected with a pM999-SLR1 construct, incubated for 12 hours and then treated with 10 μM GA3 for 0, 3, and 9 hours. The first column, fluorescence from GFP; the second, red fluorescence from the nucleus marker Ghd7; the third, bright field, and the fourth, overlay of the three channels. All confocal images were scanned using similar laser gain and offset settings. Bars = 10 μm.


Immunoblotting of SLR1 - GFP proteins from protoplast cells . Equal amounts of proteins from each sample were used for SDS - PAGE , followed by immunoblotting with anti - GFP antibodies .

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Immunoblotting of SLR1-GFP proteins from protoplast cells. Equal amounts of proteins from each sample were used for SDS-PAGE, followed by immunoblotting with anti-GFP antibodies.


( 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 .

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

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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 ) 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 the supernatant ( S ) of 22Rv1 - P cells .

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


( 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 .

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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 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 .

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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 ) 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 .

(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 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 .

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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 cells stably expressing ESM1 were treated with docetaxel at the 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 ) 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 .

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

(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 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 . *

(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 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 .

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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 ) 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 .

(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 the mean ± SD of three independent experiments .

(F) Manipulating ESM1 expression impacts transcriptional activity of NFκB. Bars are the 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 .

(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 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 .

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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 analysis of 22Rv1 - P cells with different ESM1 overexpression in the nucleus and cytoplasm were performed with the 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 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 .

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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 ) 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 .

(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 .

(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 .

(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 .

(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 ) Correlation between replicate mock infections . Path - seq reads were recovered from samples of macrophage RNA spiked with 0 . 1 % MTB RNA . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 . Inset summarizes the mock infection replicates and their fraction of MTB reads ( of all aligned reads ) from Path - seq and standard RNA - seq methods .

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(B) Correlation between replicate mock infections. Path-seq reads were recovered from samples of macrophage RNA spiked with 0.1% MTB RNA. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001. Inset summarizes the mock infection replicates and their fraction of MTB reads (of all aligned reads) from Path-seq and standard RNA-seq methods.


( C ) Correlation between MTB RNA sequenced by RNA - seq ( Illumina TruSeq library prep ) and the same MTB RNA sample combined with macrophage RNA at 1 : 1000 ratio and processed using Path - seq method . Scatter plot of log2 RPKM values is shown with Pearson correlation , P - value < 0 . 0001 .

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(C) Correlation between MTB RNA sequenced by RNA-seq (Illumina TruSeq library prep) and the same MTB RNA sample combined with macrophage RNA at 1:1000 ratio and processed using Path-seq method. Scatter plot of log2 RPKM values is shown with Pearson correlation, P-value < 0.0001.


( A ) Path - seq profiles of desA1 , desA2 in MTB infected BMDMs . Error bars show the standard deviation from three biological samples . Representative results from two experiments are presented .

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(A) Path-seq profiles of desA1, desA2 in MTB infected BMDMs. Error bars show the standard deviation from three biological samples. Representative results from two experiments are presented.


( B ) Upper panel : dissolved oxygen curve over 120 h time course showing controlled depletion ( 1 ) , sustained hypoxia ( 2 ) and controlled reaeration ( 3 ) . Points represent the average of three biological replicates and were measured via fiber optic technology that non - invasively probes oxygen levels in the culture ( PreSens Precision Sensing GmbH ) . Lower panel : expression profiles ( RNA - seq ) of desA1 and desA2 over the time course and oxygen levels . Error bars show the standard deviation from three biological samples .

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(B) Upper panel: dissolved oxygen curve over 120 h time course showing controlled depletion (1), sustained hypoxia (2) and controlled reaeration (3). Points represent the average of three biological replicates and were measured via fiber optic technology that non-invasively probes oxygen levels in the culture (PreSens Precision Sensing GmbH). Lower panel: expression profiles (RNA-seq) of desA1 and desA2 over the time course and oxygen levels. Error bars show the standard deviation from three biological samples.


( B ) Heatmap of TFs with decreased ( purple ) or increased ( green ) activity at specific time points during in vitro or in vivo infection .

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(B) Heatmap of TFs with decreased (purple) or increased (green) activity at specific time points during in vitro or in vivo infection.


( C ) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity ( induced expression of target genes ) . ( D ) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity ( de - represses target genes ) . ( E ) Rv0691c regulon genes differentially expressed in vitro and in vivo ; evidence for increased Rv0691c activity ( increased up - and down - regulation of target genes ) at 24 h from both in vitro and in vivo infection .

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(C) Rv0681 regulon genes differentially expressed in vitro and the evidence for predicted high Rv0681 activity (induced expression of target genes). (D) Zur regulon genes differentially expressed in vitro and the evidence for decreased Zur activity (de-represses target genes). (E) Rv0691c regulon genes differentially expressed in vitro and in vivo; evidence for increased Rv0691c activity (increased up- and down-regulation of target genes) at 24 h from both in vitro and in vivo infection.


( B ) Plot of read pile - ups from MSM with inducible overexpression of MSMEG _ 0916 shows ChIP - binding in the promoters of desA1 and desA2 .

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(B) Plot of read pile-ups from MSM with inducible overexpression of MSMEG_0916 shows ChIP-binding in the promoters of desA1 and desA2.


Boxplots representing RPKM values from RNA - seq of MSM with inducible overexpression of MSMEG _ 0916 . Significant log2 fold change ( FC ) between uninduced ( - ATc ) and induced ( + ATc ) samples for MSMEG _ 0916 ( log2 FC = 4 . 99 ) , desA1 ( log2 FC = - 1 . 32 ) and desA2 ( log2 FC = - 1 . 8 ) with multiple hypothesis adjusted P - values < 0 . 0001 . Data is from three biological samples ( induced ) and nine biological samples ( uninduced ) .

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Boxplots representing RPKM values from RNA-seq of MSM with inducible overexpression of MSMEG_0916. Significant log2 fold change (FC) between uninduced (-ATc) and induced (+ATc) samples for MSMEG_0916 (log2 FC = 4.99), desA1 (log2 FC = -1.32) and desA2 (log2 FC = -1.8) with multiple hypothesis adjusted P-values < 0.0001. Data is from three biological samples (induced) and nine biological samples (uninduced).


( A ) Serial ten - fold dilutions of MTB H37Rv wild type ( wt ) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc .

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(A) Serial ten-fold dilutions of MTB H37Rv wild type (wt) and MTB with inducible overexpression of Rv0472c were spotted on 7H10 agar plates with or without ATc.


( B ) Argentation TLC of 14C - labeled methyl esters of mycolic acids ( MAMEs ) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG _ 0916 . The α , αʹ , epoxy ( e ) and cyclopropanated α - ( X1 ) MAMES species are labeled . Faster - migrating species that co - migrated with α - MAMES and accumulate with induced MSMEG _ 0916 overexpression are indicated as X2 and X3 .

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(B) Argentation TLC of 14C-labeled methyl esters of mycolic acids (MAMEs) obtained from apolar lipids and delipidated cell wall fractions of MSM wt and MSM with inducible overexpression of MSMEG_0916. The α, αʹ, epoxy (e) and cyclopropanated α- (X1) MAMES species are labeled. Faster-migrating species that co-migrated with α-MAMES and accumulate with induced MSMEG_0916 overexpression are indicated as X2 and X3.


( C ) BCG wt and BCG with inducible overexpression of Rv0472c cultures , labeled with 14C - acetate , were induced ( + ATc ) or uninduced ( - ATc ) for 4 h or 8 h . The total FAMEs and MAMEs were extracted and analyzed by autoradiography - TLC using equal counts ( 15 , 000 cpm ) for each lane .

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(C) BCG wt and BCG with inducible overexpression of Rv0472c cultures, labeled with 14C-acetate, were induced (+ATc) or uninduced (-ATc) for 4 h or 8 h. The total FAMEs and MAMEs were extracted and analyzed by autoradiography-TLC using equal counts (15,000 cpm) for each lane.


IF by confocal microscopy performed on KRAB - HA overexpressing cells . Staining was performed with anti - HA ( Alexa - 488 , green ) , anti - HSPA5 ( Alexa - 647 , red ) and DNA was stained with Dapi ( blue ) .

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IF by confocal microscopy performed on KRAB-HA overexpressing cells. Staining was performed with anti-HA (Alexa-488, green), anti-HSPA5 (Alexa-647, red) and DNA was stained with Dapi (blue).


HA immunoprecipitation of stably expressed ZNF282 - HA in cells previously transfected with ΔDUF3669 - ZNF398 - GFP and WT - ZNF398 - GFP followed by detection of ZNF398 constructs in the IPs through Western - Blot using an anti - GFP antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs . Input = Cellular lysate , IP =

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HA immunoprecipitation of stably expressed ZNF282-HA in cells previously transfected with ΔDUF3669-ZNF398-GFP and WT-ZNF398-GFP followed by detection of ZNF398 constructs in the IPs through Western-Blot using an anti-GFP antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs. Input = Cellular lysate, IP = Immunoprecipitate


Left : DUF3669 - only and KRAB domain constructs used . Right : HA immunoprecipitation in cells previously co - transfected with ZNF398 - DUF3669 - HA and either ZNF282 - KRAB - Flag or ZNF282 - DUF3669 - Flag followed by detection of either of these protein constructs in the IPs through Western - Blot using an anti - Flag antibody . Bottom : Western - Blot using an anti - HA antibody on the IPs .

Left: DUF3669-only and KRAB domain constructs used. Right: HA immunoprecipitation in cells previously co-transfected with ZNF398-DUF3669-HA and either ZNF282-KRAB-Flag or ZNF282-DUF3669-Flag followed by detection of either of these protein constructs in the IPs through Western-Blot using an anti-Flag antibody. Bottom: Western-Blot using an anti-HA antibody on the IPs.


Histograms representing the percentage of KZFPs ( identified in Fig . 3B ) binding sites falling in TEs ( red ) or TSSs ( blue ) .

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Histograms representing the percentage of KZFPs (identified in Fig. 3B) binding sites falling in TEs (red) or TSSs (blue).


KZFPs binding sites on L1PA5 and L1PA6 . Top : boxes containing the interactors of KZFPs found enriched on L1PA5 , 6 . Middle : plot showing the average ChIP - exo signals ( scaled between 0 and 1 ) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment ( MSA ) . Bottom : schematic representation of L1PA5 , 6 different domains .

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KZFPs binding sites on L1PA5 and L1PA6. Top: boxes containing the interactors of KZFPs found enriched on L1PA5,6. Middle: plot showing the average ChIP-exo signals (scaled between 0 and 1) for each selected KZFP plotted on top of L1PA5 and L1PA6 multiple sequence alignment (MSA). Bottom: schematic representation of L1PA5,6 different domains.


Human KZFPs KRAB domain A - boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme ( residues sharing common physicochemical properties display the same color : http : / / www . jal - view . org / help / html / colourSchemes / zappo . html ) . This figure also displays a zoom - in on the variant KRAB domain cluster ( right ) . On this zoom - in , the tested vKZFPs , whose interactomes were defined in our study , were marked by an asterisk . D . Box plot representing KAP1FC values in function of KZFPs age . On a superimposed swarm plot , the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts , by blue dots . Mann - Whitney two - sided rank test .

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Human KZFPs KRAB domain A-boxes protein phylogenetic tree associated with their corresponding amino acid sequences colored according to the Clustal ZAPPO color scheme (residues sharing common physicochemical properties display the same color: http://www.jal- view.org/ help/html/colourSchemes/zappo.html). This figure also displays a zoom-in on the variant KRAB domain cluster (right). On this zoom-in, the tested vKZFPs, whose interactomes were defined in our study, were marked by an asterisk. D. Box plot representing KAP1FC values in function of KZFPs age. On a superimposed swarm plot, the individual vKZFPs corresponding KAP1FC values were represented by red dots and for their sKZFPs counterparts, by blue dots. Mann-Whitney two-sided rank test.


Immunoprecipitations of HA - tagged KRAB domains in order to check interaction with cKAP1 . Co - transfection of Flag - tagged cKAP1 ( upper panel ) or Flag - tagged hKAP1 ( lower panel ) and HA - tagged ZNF398 DUF3669 domain negative control , H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA - immunoprecipitation . cKAP1 and hKAP1 presence was revealed by Western - Blot using an anti - Flag antibody . Input = Cellular lysate , IP = Immunoprecipitate . Western - Blot using an HA antibody on the IPs at the bottom .

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Immunoprecipitations of HA-tagged KRAB domains in order to check
 interaction with cKAP1. Co-transfection of Flag-tagged cKAP1 (upper panel) or Flag-tagged hKAP1 (lower panel) and HA-tagged ZNF398 DUF3669 domain negative control, H2ZYM4 and M3XKG1 cKRAB domains and ZNF93 hKRAB domain in KAP1 KO HAP1 cells followed by HA-immunoprecipitation. cKAP1 and hKAP1 presence was revealed by Western-Blot using an anti-Flag antibody. Input = Cellular lysate, IP = Immunoprecipitate. Western-Blot using an HA antibody on the IPs at the bottom.


( D ) smFISH images showing LAT RNA in Ntrk1 - positive neurons . SCG - derived cultures were infected with HSV - 1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV - 1 LAT ( green ) and neuronal marker Ntrk1 ( TrkA ) ( red ) or fibroblast marker Col3a1 ( red ) . Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ . The percentage of LAT - positive cells were quantified and displayed as bar graphs with mean ± SEM . Left : 3 biological replicates , signal intensities of 50 cells were quantified for each replicate . Middle : 3 biological replicates , signal intensities of 30 cells were quantified for each replicate . Right : the blue and red dots represent the number of biological replicates done for quantification of each cell type . Scale bar , 10 μm . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

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(D) smFISH images showing LAT RNA in Ntrk1-positive neurons. SCG-derived cultures were infected with HSV-1 in the presence of ACV and allowed to establish latency over 7 days before being processed for smFISH using probes for HSV-1 LAT (green) and neuronal marker Ntrk1 (TrkA) (red) or fibroblast marker Col3a1 (red). Signal intensities for the LAT probe were quantified in neurons and fibroblasts and plotted by ImageJ. The percentage of LAT-positive cells were quantified and displayed as bar graphs with mean ± SEM. Left: 3 biological replicates, signal intensities of 50 cells were quantified for each replicate. Middle: 3 biological replicates, signal intensities of 30 cells were quantified for each replicate. Right: the blue and red dots represent the number of biological replicates done for quantification of each cell type. Scale bar, 10 μm. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 ( immediate - early ) , UL30 ( early ) , UL36 ( true - late ) or cellular markers , Ntrk ( neurons ) , or Col3a1 ( fibroblasts ) . Nuclear DNA was visualized using DAPI ( blue ) . To induce reactivation , latently - infected SCG - derived cultures were simultaneously treated with LY294002 ( 20 μM ) and WAY - 150138 ( 20 μg / mL ) and cultured for 48 hrs prior to processing for smFISH . Scale bar , 10 μm .

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(A) smFISH images showing selected viral mRNAs in reactivating neurons probed for ICP27 (immediate-early), UL30 (early), UL36 (true-late) or cellular markers, Ntrk (neurons), or Col3a1 (fibroblasts). Nuclear DNA was visualized using DAPI (blue). To induce reactivation, latently-infected SCG-derived cultures were simultaneously treated with LY294002 (20 μM) and WAY-150138 (20 μg/mL) and cultured for 48 hrs prior to processing for smFISH. Scale bar, 10 μm.


( B ) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM . Latently - infected cultures were untreated ( latent ) or treated with LY294002 and WAY - 150138 for 18 , 48 , or 72 hrs . Each dot corresponds to the value from each biological replicate . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

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(B) The percentage of cells positive for a viral mRNA or for GFP fluorescence was quantified at different time points and displayed as bar graphs with mean ± SEM. Latently-infected cultures were untreated (latent) or treated with LY294002 and WAY-150138 for 18, 48, or 72 hrs. Each dot corresponds to the value from each biological replicate. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C - D ) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs . Signal overlap is evident as white or yellow nuclear foci in the merge . Bar , 10 μm . In ( D ) , the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

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(C-D) Reactivating neurons probed simultaneously for two separate viral productive cycle mRNAs. Signal overlap is evident as white or yellow nuclear foci in the merge. Bar, 10 μm. In (D), the percentage of positive nuclei with overlapping UL30 and UL36 smFISH signals were quantified as the mean ± SEM (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( B ) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units ( TU ) and a subset of cellular genes in neurons undergoing reactivation Three host transcripts were also downregulated . Gadd45b is highlighted with a green asterisk . Vertical hashed blue lines indicate Log2 fold changes > 1 . 5 and < - 1 . 5 , while the horizontal hashed blue line indicates an adjusted p - value of 0 . 01 . Significantly regulated genes are named and shown as magenta circles .

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(B) Differential gene expression analysis of cellular transcripts showed a significant upregulation of at least 19 viral transcript units (TU) and a subset of cellular genes in neurons undergoing reactivation Three host transcripts were also downregulated. Gadd45b is highlighted with a green asterisk. Vertical hashed blue lines indicate Log2 fold changes >1.5 and < -1.5, while the horizontal hashed blue line indicates an adjusted p-value of 0.01. Significantly regulated genes are named and shown as magenta circles.


( A ) smFISH images showing nuclear Gadd45b mRNA in latent ( UL36 - negative ) neurons and nuclear / cytoplasmic Gadd45b mRNA in viral UL36 - positive neurons . Latently - infected SCG neurons treated with LY294002 and WAY - 150138 for 48 hrs were processed using smFISH to detect Gadd45b ( green ) and viral UL36 ( red ) mRNA levels and colocalization . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm . ( B ) smFISH images processed as in ( A ) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002 . Scale bar , 10 μm .

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(A) smFISH images showing nuclear Gadd45b mRNA in latent (UL36-negative) neurons and nuclear/cytoplasmic Gadd45b mRNA in viral UL36-positive neurons. Latently-infected SCG neurons treated with LY294002 and WAY-150138 for 48 hrs were processed using smFISH to detect Gadd45b (green) and viral UL36 (red) mRNA levels and colocalization. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B) smFISH images processed as in (A) showing nuclear Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with LY294002. Scale bar, 10 μm.


( E ) The percentage of HSV - 1 latently - infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY - 150138 were scored for either predominantly nuclear or nuclear / cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM . Each purple dot represents a biological replicate experiment . ( F ) Quantification of nuclear or nuclear / cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO . Each light blue dot represents a biological replicate experiment . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * * , p < 0 . 0001 ) .

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(E) The percentage of HSV-1 latently-infected neurons or reactivating neurons at 48 hrs post treatment with LY294002 and WAY-150138 were scored for either predominantly nuclear or nuclear/cytoplasmic Gadd45b mRNA localization and displayed as mean ± SEM. Each purple dot represents a biological replicate experiment. (F) Quantification of nuclear or nuclear/cytoplasmic Gadd45b mRNA in uninfected neurons at 48 hrs post treatment with either LY294002 or in DMSO. Each light blue dot represents a biological replicate experiment. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ****, p<0.0001).


( H ) Combined smFISH and immunofluorescence ( IF ) images showing high GFP - expressing neurons ( GFP - positive ) or low GFP - expressing ( GFP absent ) neurons simultaneously probed for Gadd45b mRNA . Reactivation was induced by treatment with LY294002 and WAY - 150138 for 60 hrs and processed for simultaneous smFISH and IF staining . Right panel shows quantification ( as in G ) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY - 150138 ( n = 3 biological replicates ) . The signal intensity was quantified , plotted , and analysed by ImageJ . Bar , 10 μm . Data information : : R and R square values were calculated using Correlation analysis by Prism 8 .

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(H) Combined smFISH and immunofluorescence (IF) images showing high GFP-expressing neurons (GFP-positive) or low GFP-expressing (GFP absent) neurons simultaneously probed for Gadd45b mRNA. Reactivation was induced by treatment with LY294002 and WAY-150138 for 60 hrs and processed for simultaneous smFISH and IF staining. Right panel shows quantification (as in G) of the smFISH signal intensity of Gadd45b mRNA and IF signal intensity of GFP protein at 48 hrs post treatment with LY294002 and WAY-150138 (n=3 biological replicates). The signal intensity was quantified, plotted, and analysed by ImageJ. Bar, 10 μm. Data information: : R and R square values were calculated using Correlation analysis by Prism 8.


( A ) Viral DNA polymerase inhibitor phosphonoacetic acid ( PAA , 300 μg / ml ) treatment suppressed Gadd45b mRNA expression . Latently - infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time - points indicated . RNA was then collected and levels of Gadd45b mRNA quantified by RT - qPCR ( n = 3 biological replicates , the bars and error bars are mean ± SD ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Viral DNA polymerase inhibitor phosphonoacetic acid (PAA, 300 μg/ml) treatment suppressed Gadd45b mRNA expression. Latently-infected SCG neurons were treated with LY294002 for 18 hrs to induce reactivation then treated with or without PAA until the termination time-points indicated. RNA was then collected and levels of Gadd45b mRNA quantified by RT-qPCR (n=3 biological replicates, the bars and error bars are mean ± SD). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( C ) Immunofluorescence images showing nuclear - enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002 . Scale bar , 20 μm . ( D ) The signal intensity quantification showing the ratio of nuclear / cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY - 150138 neurons . The signal intensity was quantified by ImageJ . 3 biological replicates , 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate . The horizonal line and error bars are mean ± SD . ( E ) The percentage of Gadd45b nuclear puncta - localized neurons were quantified from latent neurons treated with LY294002 and WAY - 150138 for 72 hrs . Each dot represents each biological replicate ; 20 cells were quantified for each replicate . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test .

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 B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR 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 O O O O O O O O O O O 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) Immunofluorescence images showing nuclear-enriched Gadd45b in uninfected neurons at 72 hrs post treatment with LY294002. Scale bar, 20 μm. (D) The signal intensity quantification showing the ratio of nuclear/cytoplasmic Gadd45b at 72 hrs post treatment with LY294002 and WAY-150138 neurons. The signal intensity was quantified by ImageJ. 3 biological replicates, 10 EdU positive cells and 10 EdU negative cells were quantified for each replicate. The horizonal line and error bars are mean ± SD. (E) The percentage of Gadd45b nuclear puncta-localized neurons were quantified from latent neurons treated with LY294002 and WAY-150138 for 72 hrs. Each dot represents each biological replicate; 20 cells were quantified for each replicate. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test.


( G ) Latent SCG neurons treated with LY294002 and WAY - 150138 for 72 hrs , the Gadd45b nuclear puncta - presenting neurons were ICP4 negative ( none observed in over 60 Gadd45b nuclear puncta - positive neurons ) , but a small population ( ~ 5 % ) in Gadd45b nuclear - enriched neurons were ICP4 positive . Scale bar , 20 μm .

(G) Latent SCG neurons treated with LY294002 and WAY-150138 for 72 hrs, the Gadd45b nuclear puncta-presenting neurons were ICP4 negative (none observed in over 60 Gadd45b nuclear puncta-positive neurons), but a small population (~5%) in Gadd45b nuclear-enriched neurons were ICP4 positive. Scale bar, 20 μm.


( A ) Depletion of Gadd45b expression using lentiviral - transduced shRNAs in latently - infected SCG neurons case cause HSV - 1 reactivation . HSV1 - GFP - Us11 latently - infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non - silencing shRNA ( NS ) . Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days . Knockdown efficiencies for individual shRNAs in latently - infected SCG neurons were confirmed by RT - qPCR using RNA collected in parallel . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates . RT - qPCR were quantified from 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Depletion of Gadd45b expression using lentiviral-transduced shRNAs in latently-infected SCG neurons case cause HSV-1 reactivation. HSV1-GFP-Us11 latently-infected cultures were infected with lentiviruses expressing either two different shRNAs against Gadd45b mRNA or a non-silencing shRNA (NS). Reactivation was quantified by scoring the percentage of wells expressing GFP after 5 days. Knockdown efficiencies for individual shRNAs in latently-infected SCG neurons were confirmed by RT-qPCR using RNA collected in parallel. Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates. RT-qPCR were quantified from 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( B ) smFISH shows that depletion of Gadd45b leads to increased UL36 expression . smFISH was performed 3 days post - lentiviral transduction . smFISH signals were quantified and plotted using ImageJ and Prism . Left : 3 biological replicates , 30 cells were quantified for each replicate . The horizonal lines are mean . Right : 3 biological replicates . The horizonal lines and error bars are mean ± SD .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) smFISH shows that depletion of Gadd45b leads to increased UL36 expression. smFISH was performed 3 days post-lentiviral transduction. smFISH signals were quantified and plotted using ImageJ and Prism. Left: 3 biological replicates, 30 cells were quantified for each replicate. The horizonal lines are mean. Right: 3 biological replicates. The horizonal lines and error bars are mean ± SD.


( C ) Ectopic expression of Gadd45b - Myc - Flag inhibits viral mRNA expression . Lentivirus - delivered ectopic expression of Gadd45b - Myc - Flag in SCG neurons was confirmed by immunoblotting . Latently - infected SCG neurons were transduced with Gadd45b - Myc - Flag for 3 days and then treated with LY294002 and WAY - 150138 to induce reactivation . RNA was collected at the indicated time - points ( LY treatment for 18 or 72 hours ) and levels of viral UL36 mRNA quantified by RT - qPCR . 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ectopic expression of Gadd45b-Myc-Flag inhibits viral mRNA expression. Lentivirus-delivered ectopic expression of Gadd45b-Myc-Flag in SCG neurons was confirmed by immunoblotting. Latently-infected SCG neurons were transduced with Gadd45b-Myc-Flag for 3 days and then treated with LY294002 and WAY-150138 to induce reactivation. RNA was collected at the indicated time-points (LY treatment for 18 or 72 hours) and levels of viral UL36 mRNA quantified by RT-qPCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( D ) Ectopic expression of Gadd45b - Myc - Flag antagonizes LY294002 - induced HSV - 1 reactivation . Latently - infected SCG neurons were either transduced with vector control ( Ctrl ) or Gadd45b - Myc - Flag from lentivirus for 3 days prior to addition of LY294002 . Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days . The percentage of wells expressing GFP - positive was scored as the mean ± SEM ( n = 3 biological replicates ) . Reactivation rates were quantified from 30 wells for each condition , 3 biological replicates .

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

(D) Ectopic expression of Gadd45b-Myc-Flag antagonizes LY294002-induced HSV-1 reactivation. Latently-infected SCG neurons were either transduced with vector control (Ctrl) or Gadd45b-Myc-Flag from lentivirus for 3 days prior to addition of LY294002. Reactivation was quantified by scoring the percentage of wells expressing GFP fluorescence at the indicated days. The percentage of wells expressing GFP-positive was scored as the mean ± SEM (n=3 biological replicates). Reactivation rates were quantified from 30 wells for each condition, 3 biological replicates.


( F ) Ectopic expression of Gadd45b - Myc - Flag suppressed viral ICP4 mRNA expression . Latently - infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY - 150138 for 72 hrs . Viral ICP4 mRNA was quantified by qRT - PCR . 3 biological replicates . The bars and error bars are mean ± SD . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

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

(F) Ectopic expression of Gadd45b-Myc-Flag suppressed viral ICP4 mRNA expression. Latently-infected SCG neurons were transduced with Gadd45b for 3 days and then treated with LY294002 and WAY-150138 for 72 hrs. Viral ICP4 mRNA was quantified by qRT-PCR. 3 biological replicates. The bars and error bars are mean ± SD. Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( G ) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY - 150138 in the presence or absence of Gadd45b - Myc - Flag was quantified by ImageJ ( n = 3 biological replicates ) . Data information : P values equal to or less than 0 . 05 were considered significant , asterisks denote statistical significance ( * , p < 0 . 05 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ) . P values are calculated using two - tailed unpaired Student ' s t test . P values > 0 . 05 were not significant ( ns ) .

O O O O O O O O O O O O O O B-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Immunoblots for viral ICP4 protein in latent SCG neurons treated with LY294002 and WAY-150138 in the presence or absence of Gadd45b-Myc-Flag was quantified by ImageJ (n=3 biological replicates). Data information: P values equal to or less than 0.05 were considered significant, asterisks denote statistical significance (*, p<0.05; ***, p<0.001; ****, p<0.0001). P values are calculated using two-tailed unpaired Student's t test. P values > 0.05 were not significant (ns).


( A ) PKH26 ( PE ) - labelled RM1 ( V500 / eCFP ) cells were injected intracardiac ( IC ) into C57BL / 6 mice and FACS - isolated from bones with evident tumor burden ( 8 individual mice across 5 independent experiments ) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA - seq . Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH + ( PE / V500 ) and PKH - ( V500 ) RM1 cells and bone marrow cells ( grey ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PKH26 (PE)-labelled RM1 (V500/eCFP) cells were injected intracardiac (IC) into C57BL/6 mice and FACS-isolated from bones with evident tumor burden (8 individual mice across 5 independent experiments) from day 16 onward and individual dormant and proliferating cells were isolated for scRNA-seq. Representative tumor burden at the whole mouse level and in bone shown by bioluminescence with representative FACS plots of PKH+ (PE/V500) and PKH- (V500) RM1 cells and bone marrow cells (grey).


( B ) goana gene ontology ( GO ) analysis ( limma ) of all DE genes enriched in proliferating ( PKH - ; n = 32 ) cells compared to dormant cells ( PKH + ; n = 28 ) . Gene sets appear in order of significance ( p value ) with color representing fold enrichment and bar width indicating the number of genes in each process .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) goana gene ontology (GO) analysis (limma) of all DE genes enriched in proliferating (PKH-; n = 32) cells compared to dormant cells (PKH+; n = 28). Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.


( C ) goana GO analysis ( limma ) of all DE genes uniquely enriched in PKH + compared to PKH - cells . Gene sets appear in order of significance ( p value ) with color representing fold enrichment and bar width indicating the number of genes in each process .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) goana GO analysis (limma) of all DE genes uniquely enriched in PKH+ compared to PKH- cells. Gene sets appear in order of significance (p value) with color representing fold enrichment and bar width indicating the number of genes in each process.


( E ) Heatmap of relative BASiCS - derived log2 ( denoised counts + 1 ) of type I IRGs ( upregulated > 2 - fold in the INTERFEROME in at least one dataset ) that are differentially expressed between all dormant and active cells with no residual over - dispersion . Mice IDs for each sample are indicated , along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets . Genes are displayed if detected in at least 10 samples per population . Samples with zero counts for individual genes are represented in white .

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

(E) Heatmap of relative BASiCS-derived log2(denoised counts + 1) of type I IRGs (upregulated > 2-fold in the INTERFEROME in at least one dataset) that are differentially expressed between all dormant and active cells with no residual over-dispersion. Mice IDs for each sample are indicated, along with type I IRGs that are identified as such in at least 30 INTERFEROME datasets. Genes are displayed if detected in at least 10 samples per population. Samples with zero counts for individual genes are represented in white.


( F ) Dot plot of BASiCS - derived log2 ( denoised counts + 1 ) of key DE IRGs expressed in > 5 dormant cells associated with immune - activatory processes ( among one of the most enriched types of biological pathway determined by GO analyses ) present for all single cells ranked by ExpLogFC ( Fig . 1c ) . Open circles are zero counts . Bars indicate mean expression .

(F) Dot plot of BASiCS-derived log2(denoised counts + 1) of key DE IRGs expressed in > 5 dormant cells associated with immune-activatory processes (among one of the most enriched types of biological pathway determined by GO analyses) present for all single cells ranked by ExpLogFC (Fig. 1c). Open circles are zero counts. Bars indicate mean expression.


( A Bulk RNA - seq analysis of DE genes ( DEG ) significantly suppressed ( FDR < 0 . 05 ; generalized linear model extraction ( GLM ) by edgeR ) in proliferating RM1 bone - derived ( BD ) cells compared to parental RM1 and lung metastases . Tumor - intrinsic type I IFN signaling is suppressed in proliferating bone lesions ( n = 1 ) compared to parental RM1 ( n = 2 ) cells as shown by ( A ) camera analysis of Hallmark gene set responses associated with bone metastasis ( bar length is the - log10FDR ; false discovery rate ) . Color indicates mean log2fold change ( FC ) of all genes in gene set . Bar width is relative gene set size . Dashed line shows FDR = 0 . 05 ; ( B ) barcode plot showing enrichment of the Hallmark IFN - α response gene set in genes suppressed in bone metastases , using the signed log - likelihood ratio statistic ( EdgeR ) . Bars indicate value of the statistic for each gene in the gene set ;

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Bulk RNA-seq analysis of DE genes (DEG) significantly suppressed (FDR < 0.05; generalized linear model extraction (GLM) by edgeR) in proliferating RM1 bone-derived (BD) cells compared to parental RM1 and lung metastases. Tumor-intrinsic type I IFN signaling is suppressed in proliferating bone lesions (n = 1) compared to parental RM1 (n = 2) cells as shown by (A) camera analysis of Hallmark gene set responses associated with bone metastasis (bar length is the -log10FDR; false discovery rate). Color indicates mean log2fold change (FC) of all genes in gene set. Bar width is relative gene set size. Dashed line shows FDR = 0.05; (B) barcode plot showing enrichment of the Hallmark IFN-α response gene set in genes suppressed in bone metastases, using the signed log-likelihood ratio statistic (EdgeR). Bars indicate value of the statistic for each gene in the gene set;


( C ) log2FC values of key downregulated IRGs , with genes retained in dormant cells and classical downstream IFN signaling targets indicated . Altered IRGs directly involved in IFN - α / β production ( light blue ) compared to downstream IFN targets ( dark blue ) are segregated . p values represented as * < 0 . 05 , * * < 0 . 005 and * * * < 0 . 0005 ; GLM by edgeR .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) log2FC values of key downregulated IRGs, with genes retained in dormant cells and classical downstream IFN signaling targets indicated. Altered IRGs directly involved in IFN-α/β production (light blue) compared to downstream IFN targets (dark blue) are segregated. p values represented as * < 0.05, ** < 0.005 and *** < 0.0005; GLM by edgeR.


( E ) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases ( n = 1 ) compared to lung metastases ( n = 1 ) and parental cells ( n = 2 ) .

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(E) Heatmap of mean normalized voom expression of IRGs suppressed in RM1 bone metastases (n = 1) compared to lung metastases (n = 1) and parental cells (n = 2).


( F ) qRT - PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases ( RMI BD ) compared to parental RM1 cells , lung metastases ( RM1 lung ) and naïve bone marrow ( BM ) ( n = 3 mice per group ) . p values represented as * * < 0 . 005 and * * * < 0 . 0005 ( Student ' s t test ) .

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(F) qRT-PCR validation of Irf7 and Irf9 downregulation in RM1 cells from bone metastases (RMI BD) compared to parental RM1 cells, lung metastases (RM1 lung) and naïve bone marrow (BM) (n = 3 mice per group). p values represented as ** < 0.005 and *** < 0.0005 (Student's t test).


( G ) qRT - PCR of Irf7 and Irf9 downregulation in parental RM1 cells , RM1 cells from bone metastases ( RM1 BD ) in WT and Ifnar1 deficient ( - / - ) mice , with naïve BM from WT and Ifnar1 ( - / - ) animals for reference . ( n = 3 mice per group ; n = 1 for - / - BM control ) . p values represented as * < 0 . 05 and * * < 0 . 005 ( Student ' s t test ) .

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(G) qRT-PCR of Irf7 and Irf9 downregulation in parental RM1 cells, RM1 cells from bone metastases (RM1 BD) in WT and Ifnar1 deficient (-/-) mice, with naïve BM from WT and Ifnar1 (-/-) animals for reference. (n = 3 mice per group; n = 1 for -/- BM control). p values represented as * < 0.05 and ** < 0.005 (Student's t test).


( A ) Stability of Irf7 and Irf9 mRNA suppression by qRT - PCR in ex vivo bone - derived cells ( RM1 BD Irf - ; n = 7 ) in culture compared to a bone - derived line that showed initial loss during early passage ( RM1 BD REV ( EP ) ; n = 3 ) then reverted to parental expression levels at late passage ( RM1 BD REV ( LP ) ; n = 4 ) compared to parental RM1 ( 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Stability of Irf7 and Irf9 mRNA suppression by qRT-PCR in ex vivo bone-derived cells (RM1 BD Irf-; n = 7) in culture compared to a bone-derived line that showed initial loss during early passage (RM1 BD REV (EP); n = 3) then reverted to parental expression levels at late passage (RM1 BD REV (LP); n = 4) compared to parental RM1 (n = 3).


( B ) ELISA of IFN - α production by RM1 parental ( n = 3 ) , RM1 bone - derived Irf low ( RM1 BD Irf - ; n = 4 ) and RM1 BD REV cells ( n = 3 ) subsequent to poly I : C stimulation .

(B) ELISA of IFN-α production by RM1 parental (n = 3), RM1 bone-derived Irf low (RM1 BD Irf-; n = 4) and RM1 BD REV cells (n = 3) subsequent to poly I:C stimulation.


( C ) qRT - PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf - cells ± 48 h treatment with MS275 ( 1 μM ) ( n = 7 - 9 ) .

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

(C) qRT-PCR analysis of Irf7 and Irf9 expression in RM1 BD Irf- cells ± 48 h treatment with MS275 (1 μM) (n = 7-9).


( e ) qRT - PCR analysis of Irf7 and Irf9 expression in parental RM1 cells ( n = 4 ) 48 h post - contact culture with naïve BM ( n = 6 ) .

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(e) qRT-PCR analysis of Irf7 and Irf9 expression in parental RM1 cells (n = 4) 48 h post-contact culture with naïve BM (n = 6).


( F ) qRT - PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co - culture with naïve BM under contact ( non - transwell ; NT ) and transwell ( 0 . 4 μm filters that prevent cell contact ) conditions ( n = 6 - 8 per condition ) .

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(F) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h co-culture with naïve BM under contact (non-transwell; NT) and transwell (0.4 μm filters that prevent cell contact) conditions (n = 6-8 per condition).


( G ) qRT - PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co - culture with naïve BM ± MS275 ( 1 μM ) ( n = 3 - 6 per condition ) .

O 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

(G) qRT-PCR analysis of Irf9 expression in parental RM1 cells ± 48 h contact co-culture with naïve BM ± MS275 (1 μM) (n = 3-6 per condition).


( A ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 parental , RM1 BD Irf - and RM1 BD REV ( LP ) tumors ( n = 5 per group ; * * p = 0 . 0043 by log - rank ( Mantel - Cox ) test ) .

(A) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 parental, RM1 BD Irf- and RM1 BD REV (LP) tumors (n = 5 per group; **p = 0.0043 by log-rank (Mantel-Cox) test).


( C ) ELISA of IFN - α production by RM1 parental , RM1 BD Irf - base vector ( BV ) and RM1 BD Irf7 over - expressing ( OE ) with 24 h poly I : C stimulation ( n = 4 ) .

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

(C) ELISA of IFN-α production by RM1 parental, RM1 BD Irf- base vector (BV) and RM1 BD Irf7 over-expressing (OE) with 24 h poly I:C stimulation (n = 4).


( D ) Bone metastasis - free survival in WT C57BL / 6 mice subsequent to IC inoculation of RM1 BD Irf - BV ( n = 9 ) and RM1 BD Irf7 OE ( n = 7 ) cells ( * * p = 0 . 0028 by log - rank ( Mantel - Cox ) test ) .

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(D) Bone metastasis-free survival in WT C57BL/6 mice subsequent to IC inoculation of RM1 BD Irf- BV (n = 9) and RM1 BD Irf7 OE (n = 7) cells (**p = 0.0028 by log-rank (Mantel-Cox) test).


( E ) Bone metastasis - free survival in Ifnar1 - / - mice harboring RM1 BD Irf - BV and RM1 BD Irf7 OE tumors ( n = 4 per group ) and representative bioluminescent imaging of tumor burden in leg bones ( also shown in Appendix Fig S4F ) .

(E) Bone metastasis-free survival in Ifnar1 -/- mice harboring RM1 BD Irf- BV and RM1 BD Irf7 OE tumors (n = 4 per group) and representative bioluminescent imaging of tumor burden in leg bones (also shown in Appendix Fig S4F).


( F ) Immunohistochemical ( IHC ) staining for cerulean ( anti - eCFP ; green ) on naïve WT bone , RM1 parental , RM1 BD Irf - BV and RM1 BD Irf7 OE tumor - bearing bones derived from WT animals ; and RM1 BD Irf7 OE tumor - bearing bones derived from Ifnar1 - / - animals at survival assay endpoints . Blue represents DAPI nuclear staining . Scale bar , 100 μm .

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

(F) Immunohistochemical (IHC) staining for cerulean (anti-eCFP; green) on naïve WT bone, RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE tumor-bearing bones derived from WT animals; and RM1 BD Irf7 OE tumor-bearing bones derived from Ifnar1 -/- animals at survival assay endpoints. Blue represents DAPI nuclear staining. Scale bar, 100 μm.


( G - I ) FACS analysis of dormant ( claret + ) RM1 BD Irf - and RM1 BD Irf7 OE cells from bone at ( G ) day 11 post - IC injection ( n = 4 per group ) and ( h ) day 17 ( n = 4 - 6 mice group / time point ) with ( I ) D17 active ( claret - ) colony quantitation ( mean + 1 ) determined by multiphoton imaging and IMARIS interrogation ( n = 9 bones from 3 mice per condition ) . Median shown . Upper and low box hinges denote first and third quartiles . Whiskers mark value limits .

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

(G-I) FACS analysis of dormant (claret+) RM1 BD Irf- and RM1 BD Irf7 OE cells from bone at (G) day 11 post-IC injection (n = 4 per group) and (h) day 17 (n = 4-6 mice group/time point) with (I) D17 active (claret-) colony quantitation (mean +1) determined by multiphoton imaging and IMARIS interrogation (n = 9 bones from 3 mice per condition). Median shown. Upper and low box hinges denote first and third quartiles. Whiskers mark value limits.


( J ) FACS analysis of IFN - γ + and TNF - α + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from tumor - bearing mice ) activation by RM1 parental , RM1 BD Irf - BV and RM1 BD Irf7 OE cells ( n = 6 per condition / 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) FACS analysis of IFN-γ+ and TNF-α+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation by RM1 parental, RM1 BD Irf- BV and RM1 BD Irf7 OE cells (n = 6 per condition/group).


( K ) Mean quantitation of TRAP - stained osteoclasts differentiated with M - CSF ± RANKL in co - culture with RM1 BD Irf - BV and RM1 BD Irf7 OE cells with representative wells shown ( n = 3 per condition M - CSF ; n = 6 per condition M - CSF + RANKL ; also shown in Appendix Fig S4J ) . Scale bar represents 100 μm .

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

(K) Mean quantitation of TRAP-stained osteoclasts differentiated with M-CSF ± RANKL in co-culture with RM1 BD Irf- BV and RM1 BD Irf7 OE cells with representative wells shown (n = 3 per condition M-CSF; n = 6 per condition M-CSF + RANKL; also shown in Appendix Fig S4J). Scale bar represents 100 μm.


( A ) ELISA of IFN - α production by RM1 BD Irf - cells ± single agent MS275 and poly I : C treatment or 48 h pre - treatment with MS275 ( pMS ) prior to poly I : C stimulation ( n = 6 ) .

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

(A) ELISA of IFN-α production by RM1 BD Irf- cells ± single agent MS275 and poly I:C treatment or 48 h pre-treatment with MS275 (pMS) prior to poly I:C stimulation (n = 6).


( B ) Mean fluorescence intensity of H2 - Kb staining on RM1 BD Irf - cells by FACS ± MS275 , poly I : C or combination treatment ( n = 3 ) .

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

(B) Mean fluorescence intensity of H2-Kb staining on RM1 BD Irf- cells by FACS ± MS275, poly I:C or combination treatment (n = 3).


( C ) FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from RM1 BD Irf - tumor - bearing mice ) activation upon re - stimulation with RM1 cells and MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting cells ) and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 )

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

(C) FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1)


FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from RM1 BD Irf - tumor - bearing mice ) activation upon re - stimulation with RM1 cells and MS275 , poly I : C or combination treatment , with NAC ( no antigen - presenting cells ) and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) with ( D ) representative FACS plots shown .

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

FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from RM1 BD Irf- tumor-bearing mice) activation upon re-stimulation with RM1 cells and MS275, poly I:C or combination treatment, with NAC (no antigen-presenting cells) and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1) with (D) representative FACS plots shown.


( E ) Bone metastasis - free survival in WT C57BL / 6 mice harboring RM1 BD Irf - cells ± MS275 , poly I : C or combination treatment ( n = 6 per group ; log - rank ( Mantel - Cox ) test ) .

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 I-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

(E) Bone metastasis-free survival in WT C57BL/6 mice harboring RM1 BD Irf- cells ± MS275, poly I:C or combination treatment (n = 6 per group; log-rank (Mantel-Cox) test).


( F ) Mean total tumor burden in bone ( femurs , tibias , spine and humerus ) at endpoint by bioluminescent intensity score ( log2 p / sec / cm2 / sr ) with values < 4 . 0x104 representing zero burden ( n = 24 bones per group ; 6 mice per group ) and representative bioluminescent imaging of tumor burden in spine ( all shown in Appendix Figure S5E ) .

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

(F) Mean total tumor burden in bone (femurs, tibias, spine and humerus) at endpoint by bioluminescent intensity score (log2 p/sec/cm2/sr) with values < 4.0x104 representing zero burden (n = 24 bones per group; 6 mice per group) and representative bioluminescent imaging of tumor burden in spine (all shown in Appendix Figure S5E).


( A , FACS analysis of peripheral blood ( PB ) T and NK lymphocyte representation and activation

(A, FACS analysis of peripheral blood (PB) T and NK lymphocyte representation and activation status


B ) FACS analysis of FoxP3 + CD4 + and CD4 + effector memory T cell status at days 4 and 10 post - IC tumor cell inoculation across treatment settings ( n = 4 - 6 per 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O

B) FACS analysis of FoxP3+ CD4+ and CD4+ effector memory T cell status at days 4 and 10 post-IC tumor cell inoculation across treatment settings (n = 4-6 per group).


( C , Bioluminescent imaging ( C ) of a combination group mouse with tumor clearance from day 7 to 41 ( full experimental cohort shown in Appendix Fig S5E )

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(C, Bioluminescent imaging (C) of a combination group mouse with tumor clearance from day 7 to 41 (full experimental cohort shown in Appendix Fig S5E)


D ) post - IC inoculation with associated specific CD8 + memory T cell response . This is represented by FACS analysis of IFN - γ + CD8 + T cells ( % ) post - ICS induction of T cell ( spleen - derived from tumor - bearing mice ) activation upon re - stimulation with RM1 BD Irf - cells and MS275 , poly I : C or combination treatments in vitro , with NAC and RM1 BD Irf7 OE controls ( n = 3 ; Irf7 OE control ( C ) , n = 1 ) . Representative FACS plots shown on right .

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 B-CONTROLLED_VAR O B-CONTROLLED_VAR I-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

D) post-IC inoculation with associated specific CD8+ memory T cell response. This is represented by FACS analysis of IFN-γ+ CD8+ T cells (%) post-ICS induction of T cell (spleen-derived from tumor-bearing mice) activation upon re-stimulation with RM1 BD Irf- cells and MS275, poly I:C or combination treatments in vitro, with NAC and RM1 BD Irf7 OE controls (n = 3; Irf7 OE control (C), n = 1). Representative FACS plots shown on right.


Compositional analysis of RNA - seq data ( FDR < 0 . 05 ; GLM by edgeR ) from bone metastases ( n = 9 ) and primary tumors ( n = 12 ) revealed ( A ) immune cells and ( B ) tumor cells were relative ( % of total ) across all samples , with a mean tumor purity > 85 % as determined by ARMET . Median shown . Upper and low box hinges denote first and third quartiles . Upper and lower whiskers mark the values ± 1 . 5IQR from the hinge . Data points beyond the whiskers are shown individually . p values not significant by Wilcoxon rank sum test ( R ) .

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

Compositional analysis of RNA-seq data (FDR < 0.05; GLM by edgeR) from bone metastases (n = 9) and primary tumors (n = 12) revealed (A) immune cells and (B) tumor cells were relative (% of total) across all samples, with a mean tumor purity >85% as determined by ARMET. Median shown. Upper and low box hinges denote first and third quartiles. Upper and lower whiskers mark the values ± 1.5IQR from the hinge. Data points beyond the whiskers are shown individually. p values not significant by Wilcoxon rank sum test (R).


Hallmark gene set responses associated with bone metastasis by camera analysis shows ( C ) enrichment of type I ( IFN - α ) IFN responses in suppressed genes ( barcode plot ) .

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

Hallmark gene set responses associated with bone metastasis by camera analysis shows (C) enrichment of type I (IFN-α) IFN responses in suppressed genes (barcode plot).


( D ) Heatmap of type I IFN DE IRGs ( normalized log2CPM ; counts per million ) significantly suppressed in bone metastases ( n = 9 ) compared to primary ( n = 12 ) and benign ( n = 3 ) tumors , grouped by type I IFN specific , and type II and III IFN co - regulation status . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

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(D) Heatmap of type I IFN DE IRGs (normalized log2CPM; counts per million) significantly suppressed in bone metastases (n = 9) compared to primary (n = 12) and benign (n = 3) tumors, grouped by type I IFN specific, and type II and III IFN co-regulation status. Scale truncated at ± 5 relative log2CPM. GLM by edgeR.


( E ) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated . IRF9 indicated by brown ( DAB ) staining . Scale bar represents 100 μm . Full - face slides shown in Appendix Fig S7C .

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(E) IHC for IRF9 expression in primary prostate tumors and bone metastases with matched samples indicated. IRF9 indicated by brown (DAB) staining. Scale bar represents 100 μm. Full-face slides shown in Appendix Fig S7C.


( F ) Heatmap of HLA genes ( normalized log2CPM ) suppressed ( p < 0 . 05 ; FDR < 0 . 05 ) in bone metastases ( n = 9 ) , primary tumors ( n = 12 ) and benign tumors ( n = 3 ) . Scale truncated at ± 5 relative log2CPM . GLM by edgeR .

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(F) Heatmap of HLA genes (normalized log2CPM) suppressed (p <0.05; FDR <0.05) in bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). Scale truncated at ±5 relative log2CPM. GLM by edgeR.


( G ) CIBERSORT analysis of absolute leukocyte proportions ( arbitrary units , AU ) in mRNA samples of bone metastases ( n = 9 ) , primary tumors ( n = 12 ) and benign tumors ( n = 3 ) . p values by Mann - Whitney U .

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(G) CIBERSORT analysis of absolute leukocyte proportions (arbitrary units, AU) in mRNA samples of bone metastases (n = 9), primary tumors (n = 12) and benign tumors (n = 3). p values by Mann-Whitney U.