Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours . Subsequently , they were pre - treated overnight with either type I or type III IFNs ( 1 ng / mL IFN - λ1 and 10 U / mL IFN - α2 , respectively ) before infection with Sendai virus ( SeV ) and influenza A virus ( IAV , strain PR8 ) for 6 hours as indicated . The column labelled c shows control samples collected directly after purification . Quantification of the presence of SeV or IAV . Each colour corresponds to a donor ; 9 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for the statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Alveolar macrophages from different donors were purified from BAL fluid by allowing them to attach to the culture dish for 2 hours. Subsequently, they were pre-treated overnight with either type I or type III IFNs (1 ng/mL IFN-λ1 and 10 U/mL IFN-α2, respectively) before infection with Sendai virus (SeV) and influenza A virus (IAV, strain PR8) for 6 hours as indicated. The column labelled c shows control samples collected directly after purification. Quantification of the presence of SeV or IAV. Each colour corresponds to a donor; 9 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for the statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( A ) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 ( Donors marked in orange and blue were not quantifiable in all test conditions ) . ( B ) ISG response measured by ISG15 , RSAD2 , and IFIT1 .

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Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (A) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1 (Donors marked in orange and blue were not quantifiable in all test conditions). (B) ISG response measured by ISG15, RSAD2, and IFIT1.


Alveolar macrophages from different donors were purified from BAL fluid and pre - treated with either Poly ( I : C ) or IFN - λ1 ( + ; 1 ng / mL , or + + ; 10 ng / mL ) before infection with SARS - CoV - 2 . ( C ) Quantification of the presence of SARS - CoV - 2 .

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Alveolar macrophages from different donors were purified from BAL fluid and pre-treated with either Poly(I:C) or IFN-λ1 (+; 1 ng/mL, or ++; 10 ng/mL) before infection with SARS-CoV-2. (C) Quantification of the presence of SARS-CoV-2.


Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( B ) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2 , IFNB1 , and IFNL1 . ( C ) ISG response measured by ISG15 , RSAD2 , and IFIT1 ( Donors marked in green and red were not quantifiable in all test conditions ) .

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Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (B) Total RNA was harvested and subjected to qPCR for quantification of the IFN response measured by IFNA2, IFNB1, and IFNL1. (C) ISG response measured by ISG15, RSAD2, and IFIT1 (Donors marked in green and red were not quantifiable in all test conditions).


Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( D ) Quantification of the presence of SARS - CoV - 2 .

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Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (D) Quantification of the presence of SARS-CoV-2.


Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS - CoV - 2 before treatment with either Poly ( I : C ) or IFN - λ1 ( 1 ng / mL ) . ( E ) Western blots were performed using samples from two donors . ( A - D ) Each colour corresponds to a donor ; 5 different donors were analysed . The dashed line represents the detection limit . An ordinary one - way - ANOVA test was used for statistical analysis : ns , not significant ; * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; * * * , P ≤ 0 . 001 ; * * * * , P ≤ 0 . 0001 .

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Alveolar macrophages from different donors were purified from BAL fluid and infected with SARS-CoV-2 before treatment with either Poly(I:C) or IFN-λ1 (1 ng/mL). (E) Western blots were performed using samples from two donors. (A-D) Each colour corresponds to a donor; 5 different donors were analysed. The dashed line represents the detection limit. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.


Alveolar macrophages from different donors were purified from BAL and treated with SARS - CoV - 2 for either 2 or 24 hours . ( A ) The amount of SARS - CoV - 2 nucleocapsid phosphoprotein was quantified using LC - MS / MS . ( B ) After subtraction of the background ( unchallenged AMs ) , the ratio between the amount of SARS - CoV - 2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated . Each colour corresponds to a donor ; 3 different donors were analyzed . An ordinary one - way - ANOVA test was used for statistical analysis : ns , not significant ; * , P ≤ 0 . 05 .

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Alveolar macrophages from different donors were purified from BAL and treated with SARS-CoV-2 for either 2 or 24 hours. (A) The amount of SARS-CoV-2 nucleocapsid phosphoprotein was quantified using LC-MS/MS. (B) After subtraction of the background (unchallenged AMs), the ratio between the amount of SARS-CoV-2 nucleocapsid phosphoprotein at 24 and 2 hours was calculated. Each colour corresponds to a donor; 3 different donors were analyzed. An ordinary one-way-ANOVA test was used for statistical analysis: ns, not significant; *, P ≤ 0.05.


( A ) Total RNA was harvested from AMs and primary human airway epithelial cells ( HAE ) and subjected to qPCR for quantification of the ACE2 receptor . Each colour corresponds to a donor . The dashed line represents the detection limit .

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(A) Total RNA was harvested from AMs and primary human airway epithelial cells (HAE) and subjected to qPCR for quantification of the ACE2 receptor. Each colour corresponds to a donor. The dashed line represents the detection limit.


( B ) The presence of nucleoprotein mRNA of SARS - CoV - 2 in AMs and primary HAE was also quantified after SARS - CoV - 2 treatment . Each colour corresponds to a donor . An unpaired t - test test was used for statistical analysis : * * * * , P ≤ 0 . 0001 .

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(B) The presence of nucleoprotein mRNA of SARS-CoV-2 in AMs and primary HAE was also quantified after SARS-CoV-2 treatment. Each colour corresponds to a donor. An unpaired t-test test was used for statistical analysis: ****, P≤0.0001.


( C ) Primer efficiency was tested using serial dilutions of positive samples using qPCR . The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points . This analysis revealed an amplification factor of 1 . 92 , 1 . 91 , and 1 . 85 for the SARS - CoV - 2 , IAV , and SeV primer set , respectively . The different colours correspond to different primer sets .

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(C) Primer efficiency was tested using serial dilutions of positive samples using qPCR. The Ct values were plotted on a logarithmic scale along with the corresponding dilutions before performing a linear regression in order to calculate the slope of the trend line through the data points. This analysis revealed an amplification factor of 1.92, 1.91, and 1.85 for the SARS-CoV-2, IAV, and SeV primer set, respectively. The different colours correspond to different primer sets.


( b ) Volcano plots of up ( red ) and down ( blue ) regulated genes ( compared to healthy control samples ) in S . Typhi and S . Paratyphi A positive individuals ( Nepal and Oxford ) . Black numbers indicate the up - and down - regulated genes compared to healthy controls .

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(b) Volcano plots of up (red) and down (blue) regulated genes (compared to healthy control samples) in S. Typhi and S. Paratyphi A positive individuals (Nepal and Oxford). Black numbers indicate the up- and down-regulated genes compared to healthy controls.


( d - e ) Scatter plots of BTMs enriched ( p > 0 . 05 ) in blood - culture positive samples in Nepal ( y - axis ) versus Oxford ( x - axis ) for typhoid fever ( d ) and paratyphoid fever ( e ) . For further details on BTMs refer to reference ( Chaussabel et al . , 2014 ) .

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(d-e) Scatter plots of BTMs enriched (p>0.05) in blood-culture positive samples in Nepal (y-axis) versus Oxford (x-axis) for typhoid fever (d) and paratyphoid fever (e). For further details on BTMs refer to reference (Chaussabel et al., 2014).


( a ) Single - sample GSEA Normalised Enrichment Scores ( NES ) of IFN and DC BTMs of individuals with blood - culture confirmed enteric fever in Nepal and Oxford .

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(a) Single-sample GSEA Normalised Enrichment Scores (NES) of IFN and DC BTMs of individuals with blood-culture confirmed enteric fever in Nepal and Oxford.


( b ) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort . Bar graph on top of the heatmap shows temperature of each individual at the time of sampling . Three samples labelled as ' 03NP - CONT ' are samples that grew bacterial contaminants and were thus excluded from the entire analysis .

(b) Heatmap of the 500 most variably expressed genes in samples of the Nepali cohort. Bar graph on top of the heatmap shows temperature of each individual at the time of sampling. Three samples labelled as '03NP-CONT' are samples that grew bacterial contaminants and were thus excluded from the entire analysis.


( d ) Combined expression score for samples based on the 5 - gene signature for samples in the discovery cohort ( top ) and validation cohort ( bottom ) . Ox . CTRL : Oxford controls ( D0 ) ; CTRL : Nepali control samples . PTB : pulmonary TB ; DENV : Dengue samples ; bsPf : blood - stage P . falciparum ; SPT : S . Paratyphi A ; ST : S . Typhi . ST and SPT samples are derived from the challenge models as well as from Nepal .

(d) Combined expression score for samples based on the 5-gene signature for samples in the discovery cohort (top) and validation cohort (bottom). Ox.CTRL: Oxford controls (D0); CTRL: Nepali control samples. PTB: pulmonary TB; DENV: Dengue samples; bsPf: blood-stage P. falciparum; SPT: S. Paratyphi A; ST: S. Typhi. ST and SPT samples are derived from the challenge models as well as from Nepal.


( b ) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5 - gene signature ( based on gene array data ) .

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(b) Dot plot of prediction probability of being class EF versus the expression score calculated on the bases of the 5-gene signature (based on gene array data).


( c ) qPCR gene expression scores of the 5 - gene signature ( ∆ ∆ CT over PPIA ) for CTRLs , 03NP - sEF , 03NP - SPT and 03NP - ST samples from Nepal . Yellow diamonds in the 03NP - sEF category represent the 9 patients classified as EF based on the random forest algorithm .

(c) qPCR gene expression scores of the 5-gene signature (∆∆CT over PPIA) for CTRLs, 03NP-sEF, 03NP-SPT and 03NP-ST samples from Nepal. Yellow diamonds in the 03NP-sEF category represent the 9 patients classified as EF based on the random forest algorithm.


( d ) qPCR expression values ( ∆ ∆ Ct over PPIA ) of the 5 - gene signature in control samples ( Oxford and Nepal ) , S . Paratyphi A ( 03NP - SPT ) or S . Typhi ( 03NP - ST ) in Nepal , samples at day 7 after challenge of participants who stayed well following challenge with S . Typhi ( nD7 ) , or typhoid diagnosis after challenge ( TD ) in the Vi - TCV

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(d) qPCR expression values (∆∆Ct over PPIA) of the 5-gene signature in control samples (Oxford and Nepal), S. Paratyphi A (03NP-SPT) or S. Typhi (03NP-ST) in Nepal, samples at day 7 after challenge of participants who stayed well following challenge with S. Typhi (nD7), or typhoid diagnosis after challenge (TD) in the Vi-TCV study


( e ) Combined qPCR expression score of the 5 - gene signature . Black arrows indicate outlier samples .

(e) Combined qPCR expression score of the 5-gene signature. Black arrows indicate outlier samples.


( f ) Temperature and CRP for samples of which data was available ( CRP was only measured in the Oxford CHIM ) . D0 = pre - challenge baseline Vi - TCV study ; nD7 = day 7 samples of participants who stayed well following challenge ( Vi - TCV study ) ; SPT = S . Paratyphi A ( 03NP ) ; ST = S . Typhi ( 03NP ) ; TD = Typhoid diagnosis ( Vi - TCV study ) . ( g ) Spearman ' s rank correlation of the 5 - gene combined expression score and ( left ) temperature ( only nD7 and TD samples from the Oxford CHIM - Vi - TCV and SPT and ST cases from Nepal at presentation to hospital were included ) and ( right ) CRP ( CRP was only available for Oxford CHIM - Vi - TCV samples and we excluded D0 baseline measures ) .

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(f) Temperature and CRP for samples of which data was available (CRP was only measured in the Oxford CHIM). D0 = pre-challenge baseline Vi-TCV study; nD7 = day 7 samples of participants who stayed well following challenge (Vi-TCV study); SPT = S. Paratyphi A (03NP); ST = S. Typhi (03NP); TD = Typhoid diagnosis (Vi-TCV study). (g) Spearman's rank correlation of the 5-gene combined expression score and (left) temperature (only nD7 and TD samples from the Oxford CHIM - Vi-TCV and SPT and ST cases from Nepal at presentation to hospital were included) and (right) CRP (CRP was only available for Oxford CHIM - Vi-TCV samples and we excluded D0 baseline measures).


IHC images for collagen The inserts in panel B represent the hematoxylin ( nucleus ) & eosin ( cytoplasm ) staining for the same tissue area of the collagen staining In all cases n = 4 control mice , and ≥ 3 mice for 2mg and 5 mg ) The quantification for each staining is shown in the histogram in the right

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IHC images for collagen The inserts in panel B represent the hematoxylin (nucleus) & eosin (cytoplasm) staining for the same tissue area of the collagen staining In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) The quantification for each staining is shown in the histogram in the right side


IHC images fo aSMA , CD68 , and CD204 For aSMA , CD68 , and CD204 the percentage of positive cells is relative to the control condition . In all cases n = 4 control mice , and ≥ 3 mice for 2mg and 5 mg ) Scale bar : 100 mm . The quantification for each staining is shown in the histogram in the right

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IHC images fo aSMA, CD68, and CD204 For aSMA, CD68, and CD204 the percentage of positive cells is relative to the control condition. In all cases n=4 control mice, and ≥3 mice for 2mg and 5 mg) Scale bar: 100 mm. The quantification for each staining is shown in the histogram in the right side


Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse , n = 27 control and n = 37

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Representative traces tracking bead displacements Histogram shows relative bead displacement for the first and last pulse, n=27 control and n=37 tamoxifen


Histogram shows percentage of gel contraction , n > 10 gels per

Histogram shows percentage of gel contraction, n>10 gels per condition


Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor ( ER antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all

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Representative mechanosensing trace of PSC treated with tamoxifen and Estrogen receptor (ER antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


Representative mechanosensing trace of PSC treated with tamoxifen an G coupled - protein estrogen receptor ( GPER ) antagonists Histogram shows relative bead displacement for the first and last pulse , n > 20 for all

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Representative mechanosensing trace of PSC treated with tamoxifen an G coupled-protein estrogen receptor (GPER) antagonists Histogram shows relative bead displacement for the first and last pulse, n>20 for all conditions


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Histogram shows percentage of gel contraction by PSCs treated with tamoxifen in the presence of indicated antagonist


Histogram shows percentage of gel contraction after treatment with indicated agonist , n > 10 gels per

Histogram shows percentage of gel contraction after treatment with indicated agonist, n>10 gels per conditio


Young " s modulus for matrices previously remodeled by PSCs ; control n = 126 and tamoxifen n = 145 . Lines and error bars indicate mean ± s . e . m . Four experimental

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Young"s modulus for matrices previously remodeled by PSCs; control n=126 and tamoxifen n=145. Lines and error bars indicate mean±s.e.m. Four experimental repl


Representative images of H & E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs ( top ) or tamoxifen treated PSCs ( bottom ) , scale bar 50 μm AsPC1 invasion quantification , the central box represents values from the lower to upper quartile . The middle line represents the mean . The vertical line extends from the minimum to the maximum value , n = 30 control and n = 40

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Representative images of H&E staining showing AsPC1 cancer cell invasions in matrices previously remodeled by control PSCs (top) or tamoxifen treated PSCs (bottom), scale bar 50 μm AsPC1 invasion quantification, the central box represents values from the lower to upper quartile. The middle line represents the mean. The vertical line extends from the minimum to the maximum value, n=30 control and n=40 tamoxifen


Immunofluorescence images of PSCs stained for YAP


Quantification of the nuclear / cytoplasm YAP in PSCs ( four experimental replicates )

Quantification of the nuclear/cytoplasm YAP in PSCs (four experimental replicates)


qPCR mRNA levels for YAP target genes connective tissue grow factor ( CTGF ) and Ankyrin Repeat Domain 1 ( ANRKD1 ) , ( three experimental replicates )

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qPCR mRNA levels for YAP target genes connective tissue grow factor (CTGF) and Ankyrin Repeat Domain 1 (ANRKD1), (three experimental replicates)


Western blot bands for YAP , pS127 YAP and total protein . ( E ) Quantification of YAP and pYAP Ser127 normalised to total protein , expressed relative to unstimulated control ( n = 8 )

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Western blot bands for YAP, pS127 YAP and total protein. (E) Quantification of YAP and pYAP Ser127 normalised to total protein, expressed relative to unstimulated control (n=8)


IHC images for YAP staining in KPC mice pancreatic tissues ( control and treated with 2 and 5 mg tamoxifen ) , YAP staining is brown . Scale bar : 100

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IHC images for YAP staining in KPC mice pancreatic tissues (control and treated with 2 and 5 mg tamoxifen), YAP staining is brown. Scale bar: 100 μm


Quantification of YAP positive nucleus . n = 5 control mice , and ≥ 3 mice for 2mg and 5

Quantification of YAP positive nucleus. n=5 control mice, and ≥3 mice for 2mg and 5 mg


IF images for total ( MLC - 2 ) and phosphorylated ( pMLC - 2 ) in PSCs , scale bar is 20

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IF images for total (MLC-2) and phosphorylated (pMLC-2) in PSCs, scale bar is 20 μm


Quantification of staining in A . Each data point represents a cell Western blot for total and phosphorylated mlc -

Quantification of staining in A. Each data point represents a cell Western blot for total and phosphorylated mlc-2


Quantification of total and active RhoA , expressed as percentage of the total RhoA in the control

Quantification of total and active RhoA, expressed as percentage of the total RhoA in the control condition


IHC images for phosphorylated mlc - 2 in mice pancreatic tissues ( n = 5 control , n = 5 2mg , and n = 3 5mg ) . Scale bar : 100 μm Quantification of staining in

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IHC images for phosphorylated mlc-2 in mice pancreatic tissues (n=5 control, n=5 2mg, and n=3 5mg). Scale bar: 100 μm Quantification of staining in E


B Light transmittance of tissue increased more rapidly with FEC than with CLARITY . Each datapoint plotted is the mean from three neighboring transmittance readings ( hence resulting in 10 datapoints from the 30 measurements / brain , technical replicates ) ( One - way ANOVA * * * p < 0 . 001 , * * * * p < 0 . 0001 ) ( n = 3 per group , biological replicates ) .

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B Light transmittance of tissue increased more rapidly with FEC than with CLARITY. Each datapoint plotted is the mean from three neighboring transmittance readings (hence resulting in 10 datapoints from the 30 measurements/brain, technical replicates) (One-way ANOVA *** p<0.001, **** p<0.0001) (n = 3 per group, biological replicates).


C Comparison of mouse brains cleared by FEC ( 6 hrs ) and passive ( 3 days ) . Passive clearing was incomplete even after 3 days .

C Comparison of mouse brains cleared by FEC (6 hrs) and passive (3 days). Passive clearing was incomplete even after 3 days.


D After whole - brain electrophoretic staining with polythiophenes , sagittal 500 µm slices were cut and plaques were counted . By passively re - staining the same slice , no additional plaques were detected ( " FEC + passive " ) . Focal shifts after slice reprocessing account for the slight differences between the images . One biological replicate . Scale bars : 100

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D After whole-brain electrophoretic staining with polythiophenes, sagittal 500 µm slices were cut and plaques were counted. By passively re-staining the same slice, no additional plaques were detected ("FEC + passive"). Focal shifts after slice reprocessing account for the slight differences between the images. One biological replicate. Scale bars: 100 µm


A , B The distinct significant effect of each of the strongest treatments on plaque size cluster , NB360 5 - month and LIN5044 14 - month , as well as their overlap , using either ( A ) neuroanatomical boundaries , or ( B ) SAVs .

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A, B The distinct significant effect of each of the strongest treatments on plaque size cluster, NB360 5-month and LIN5044 14-month, as well as their overlap, using either (A) neuroanatomical boundaries, or (B) SAVs.


A . Tunicamycin ( Tn , 2 µg / ml ) increased Nox4 protein levels in H9c2 cells . Tubulin was used as a loading control . n = 4 - 6 / group . * , significant compared to baseline .

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A. Tunicamycin (Tn, 2 µg/ml) increased Nox4 protein levels in H9c2 cells. Tubulin was used as a loading control. n=4-6/group. *, significant compared to baseline.


B . Thapsigargin ( Tp , 1 µg / ml ) increased Nox4 protein levels in H9c2 cells . n = 4 - 6 / group . * , significant compared to baseline .

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B. Thapsigargin (Tp, 1 µg/ml) increased Nox4 protein levels in H9c2 cells. n=4-6/group. *, significant compared to baseline.


C . Effect of Nox4 on the unfolded protein response . Nox4 was depleted in H9c2 cells by shRNA - mediated knockdown ( Ad . shNox4 ) or cells were treated with a control adenovirus ( Ad . Ctl ) . In cells with Nox4 knockdown , tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94 , Grp78 and calreticulin than in conrol cells . Nuclear protein levels of ATF4 were substantially lower in Nox4 - depleted cells than control cells but the levels of cleaved ATF6 ( ATF6c ) were similar . Histone was used as a loading control . The relative mRNA levels of Xbp1 - s ( a readout of IRE1 signaling ) were unaltered after Nox4 knockdown . Mean data are shown in Appendix Fig S1E . Similar results were obtained with an independent siRNA approach ( Appendix Fig S2A ) . D . Effect of adenoviral - mediated overexpression of Nox4 ( Ad . Nox4 ) or a control β - galactosidase protein ( Ad . β - Gal ) on tunicamycin responses of H9c2 cells . Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin - induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels . Mean data are shown in Appendix Fig S2B .

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C. Effect of Nox4 on the unfolded protein response. Nox4 was depleted in H9c2 cells by shRNA-mediated knockdown (Ad.shNox4) or cells were treated with a control adenovirus (Ad.Ctl). In cells with Nox4 knockdown, tunicamycin treatment resulted in lower increases in protein levels of the ER chaperones Grp94, Grp78 and calreticulin than in conrol cells. Nuclear protein levels of ATF4 were substantially lower in Nox4-depleted cells than control cells but the levels of cleaved ATF6 (ATF6c) were similar. Histone was used as a loading control. The relative mRNA levels of Xbp1-s (a readout of IRE1 signaling) were unaltered after Nox4 knockdown. Mean data are shown in Appendix Fig S1E. Similar results were obtained with an independent siRNA approach (Appendix Fig S2A).D. Effect of adenoviral-mediated overexpression of Nox4 (Ad.Nox4) or a control β-galactosidase protein (Ad.β-Gal) on tunicamycin responses of H9c2 cells. Nox4 enhanced the increase in cellular ER chaperones and nuclear ATF4 levels but did not affect tunicamycin-induced changes in nuclear ATF6c levels and caused minor reduction in Xbp1s mRNA levels. Mean data are shown in Appendix Fig S2B.


E , F . Effect of Nox4 knockdown or overexpression , respectively , on the tunicamycin - induced changes in mRNA levels of ATF4 target genes . n = 4 / group . Psat1 , phosphoserine aminotransferase ; Phgdh , 3 - phosphoglycerate dehydrogenase ; Asns , asparagine synthetase ; Slc6a9 , glycine transporter 1 .

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E,F. Effect of Nox4 knockdown or overexpression, respectively, on the tunicamycin-induced changes in mRNA levels of ATF4 target genes. n=4/group. Psat1, phosphoserine aminotransferase; Phgdh, 3-phosphoglycerate dehydrogenase; Asns, asparagine synthetase; Slc6a9, glycine transporter 1.


G . Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin - treated H9c2 cells . Scrambled siRNAs were used as a control ( Ctl ) . Representative immunoblots shown to the top ( captions at bottom of bar graphs refer also to the immunoblots ) ; tubulin was used as a loading control . n = 4 / group . * , significant compared to baseline ; # , significant comparing siATF4 versus corresponding siCtl .

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G. Effect of ATF4 silencing with two different siRNAs on Nox4 protein levels in tunicamycin-treated H9c2 cells. Scrambled siRNAs were used as a control (Ctl). Representative immunoblots shown to the top (captions at bottom of bar graphs refer also to the immunoblots); tubulin was used as a loading control. n=4/group. *, significant compared to baseline; #, significant comparing siATF4 versus corresponding siCtl.


H . Effect of ATF4 overexpression on Nox4 mRNA and protein levels . n = 3 / group .

H. Effect of ATF4 overexpression on Nox4 mRNA and protein levels. n=3/group.


A . The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin - induced eIF2a phosphorylation in H9c2 cells , with no change in phospho - Thr980 - PERK ( PERK - P ) levels . GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels . B . Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin - induced eIF2a phosphorylation , with minimal change in phospho - PERK levels . C , D . Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin - treated cells after Nox4 knockdown or overexpression , respectively . n = 3 / group . * , significant compared to baseline ; # , significant comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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A. The knockdown of endogenous Nox4 resulted in a substantial inhibition of tunicamycin-induced eIF2a phosphorylation in H9c2 cells, with no change in phospho-Thr980-PERK (PERK-P) levels. GADD34 levels were significantly decreased after Nox4 knockdown while there was no change in PP1 protein levels.B. Overexpression of Nox4 in H9c2 cells caused prolongation of tunicamycin-induced eIF2a phosphorylation, with minimal change in phospho-PERK levels.C,D. Mean levels of phosphorylated eIF2a relative to total eIF2a protein in tunicamycin-treated cells after Nox4 knockdown or overexpression, respectively. n=3/group. *, significant compared to baseline; #, significant comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


E , F . Effect of Nox4 knockdown or overexpression , respectively , on okadaic acid - resistant Ser / Thr phosphatase activity in membrane fractions of tunicamycin - treated H9c2 cells . n = 4 / group . * p < 0 . 05 , * * p < 0 . 01 cf . baseline ; # , p < 0 . 05 , # # , p < 0 . 01 comparing Nox4 knockdown ( Ad . shNox4 ) or overexpression ( Ad . Nox4 ) versus corresponding controls ( Ad . Ctl or Ad . β - Gal , respectively ) .

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E,F. Effect of Nox4 knockdown or overexpression, respectively, on okadaic acid-resistant Ser/Thr phosphatase activity in membrane fractions of tunicamycin-treated H9c2 cells. n=4/group. *p<0.05, **p<0.01 cf. baseline; #, p<0.05, ##, p<0.01 comparing Nox4 knockdown (Ad.shNox4) or overexpression (Ad.Nox4) versus corresponding controls (Ad.Ctl or Ad.β-Gal, respectively).


G , H . Nox4 knockdown or overexpression , respectively , had no effect on the phosphorylation of glycogen synthase at Ser641 ( GS - P ) or Histone H3 at Ser57 ( H3 - P ) in H9c2 cells .

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G,H. Nox4 knockdown or overexpression, respectively, had no effect on the phosphorylation of glycogen synthase at Ser641 (GS-P) or Histone H3 at Ser57 (H3-P) in H9c2 cells.


I . Nox4 - / - MEF cells ( KO ) showed blunted tunicamycin - induced increases in levels of phospho - eIF2a , ATF4 and ER chaperones as compared to wild - type ( WT ) MEFs , a response that was rescued by re - introduction of Nox4 ( KO + Nox4 ) . The latter had no effect on GS - P or H3 - P levels .

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I. Nox4-/- MEF cells (KO) showed blunted tunicamycin-induced increases in levels of phospho-eIF2a, ATF4 and ER chaperones as compared to wild-type (WT) MEFs, a response that was rescued by re-introduction of Nox4 (KO+Nox4). The latter had no effect on GS-P or H3-P levels.


A . Sub - cellular localization of Nox4 . Tunicamycin ( Tn 2 µg / ml , 6h ) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy ( scale bar , 10 µm ) . 3D SIM images ( scale bars , 2 µm ) showed localisation of Nox4 ( green ) to the ER , which was labeled with an anti - KDEL antibody ( red ) . At higher magnification ( right ) , yellow dots denote co - localization of Nox4 and KDEL signals . Cell nuclei were stained with DAPI ( blue ) . 1 Z slice from 3D stack is shown .

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A. Sub-cellular localization of Nox4. Tunicamycin (Tn 2 µg/ml, 6h) increased Nox4 levels in H9c2 cells as assessed by spinning disk confocal microscopy (scale bar, 10 µm).3D SIM images (scale bars, 2 µm) showed localisation of Nox4 (green) to the ER, which was labeled with an anti-KDEL antibody (red). At higher magnification (right), yellow dots denote co-localization of Nox4 and KDEL signals. Cell nuclei were stained with DAPI (blue). 1 Z slice from 3D stack is shown.


B . Progressive enrichment of GADD34 , PP1 , eIF2α and Nox4 in membrane fractions of tunicamycin - treated H9c2 cells .

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B. Progressive enrichment of GADD34, PP1, eIF2α and Nox4 in membrane fractions of tunicamycin-treated H9c2 cells.


C . After sucrose gradient fractionation of lysates of tunicamycin - treated H9c2 cells , GADD34 , PP1 and Nox4 co - eluted in fractions 12 and 13 ( F12 , F13 ) .

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C. After sucrose gradient fractionation of lysates of tunicamycin-treated H9c2 cells, GADD34, PP1 and Nox4 co-eluted in fractions 12 and 13 (F12, F13).


D . Immunoprecipitation ( IP ) of pooled fractions 12 / 13 with an anti - GADD34 antibody revealed the presence of both PP1 and Nox4 .

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D. Immunoprecipitation (IP) of pooled fractions 12/13 with an anti-GADD34 antibody revealed the presence of both PP1 and Nox4.


E . The association of Nox4 with GADD34 was validated in HEK293 cells co - transfected with Nox4 and either Flag - tagged or non - tagged GADD34 , followed by IP with an anti - Flag antibody .

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E. The association of Nox4 with GADD34 was validated in HEK293 cells co-transfected with Nox4 and either Flag-tagged or non-tagged GADD34, followed by IP with an anti-Flag antibody.


F . Co - transfection of HEK293 cells with GADD34 - Flag and different myc - tagged Nox4 constructs , followed by IP with an anti - myc antibody . GADD34 binds to full - length Nox4 ( FL ) and the Nox4 transmembrane domain ( TD ) but not the C - terminal domain ( CD ) .

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F. Co-transfection of HEK293 cells with GADD34-Flag and different myc-tagged Nox4 constructs, followed by IP with an anti-myc antibody. GADD34 binds to full-length Nox4 (FL) and the Nox4 transmembrane domain (TD) but not the C-terminal domain (CD).


G . Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto , respectively , in tunicamycin - treated MEF cells . Redox - insensitive mutant probes were used as negative controls and to exclude pH changes . Extracellular H2O2 ( 200 nM ) was added as a positive control . The pseudocolor scale is shown along the left vertical edge of each image . KO = Nox4 - / - . Scale bars , 2 µm .

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G. Representative pseudocolor images of simultaneous ER and cytosolic ROS measurement with HyPer ER and HyPerRed Cyto, respectively, in tunicamycin-treated MEF cells. Redox-insensitive mutant probes were used as negative controls and to exclude pH changes. Extracellular H2O2 (200 nM) was added as a positive control. The pseudocolor scale is shown along the left vertical edge of each image. KO = Nox4-/-. Scale bars, 2 µm.


H . Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity ( bar graph ) and decreased phospho - eIF2 levels ( immunoblots ) . Co - transfection of full - length Nox4 reduced PP1 activity and increased phospho - eIF2 levels ( captions at bottom refer both to the bar graphs and immunoblots ) . These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain ( TD ) were transfected . Nox4 did not affect phosphorylation of glycogen synthase ( GS - P ) or histone H3 ( H3 - P ) . All experiments were n = 3 / group . Values below the immunoblots are mean ± SEM levels for phospho - eIF2 / total - eIF2 . * , p < 0 . 05 comparing third and fourth lanes ; Levels of significance for comparisons of PP1 activities are shown above the bar columns . See also Fig EV1 and Appendix Fig S4 .

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H. Transfection of HEK293 cells with PP1 and GADD34 increased PP1 activity (bar graph) and decreased phospho-eIF2 levels (immunoblots). Co-transfection of full-length Nox4 reduced PP1 activity and increased phospho-eIF2 levels (captions at bottom refer both to the bar graphs and immunoblots). These effects were abrogated when either Nox4 P437H or the Nox4 transmembrane domain (TD) were transfected. Nox4 did not affect phosphorylation of glycogen synthase (GS-P) or histone H3 (H3-P). All experiments were n=3/group. Values below the immunoblots are mean ± SEM levels for phospho-eIF2/total-eIF2. *, p<0.05 comparing third and fourth lanes; Levels of significance for comparisons of PP1 activities are shown above the bar columns. See also Fig EV1 and Appendix Fig S4.


A . Recombinant PP1 was inhibited by H2O2 ( 0 . 2 mM ) and activity was not restored by glutathione ( GSH ) , cysteine ( Cys ) or dithiothreitol ( DTT ) . A Cys127Ser / Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild - type PP1 . Values above bars denote level of significance for the inhibitory effect of H2O2 .

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A. Recombinant PP1 was inhibited by H2O2 (0.2 mM) and activity was not restored by glutathione (GSH), cysteine (Cys) or dithiothreitol (DTT). A Cys127Ser/Cys273Ser PP1 mutant was inhibited by H2O2 similarly to wild-type PP1. Values above bars denote level of significance for the inhibitory effect of H2O2.


B . Ascorbate ( Asc ) dose - dependently restored PP1 activity . # , significant effect of Asc compared to H2O2 alone .

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B. Ascorbate (Asc) dose-dependently restored PP1 activity. #, significant effect of Asc compared to H2O2 alone.


C . EPR spectra of PP1 incubated with ascorbate ( 1 mM ) alone ( a ) or PP1 exposed to H2O2 followed by catalase treatment , then incubation with ascorbate ( b ) . Panel b shows a typical spectrum for the ascorbyl radical ( hyperfine splitting constant , aH = 1 . 8G ) , similar to the positive control obtained by exposing ascorbate to H2O2 ( c ) . Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1 , prior to ascorbate addition .

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C. EPR spectra of PP1 incubated with ascorbate (1 mM) alone (a) or PP1 exposed to H2O2 followed by catalase treatment, then incubation with ascorbate (b). Panel b shows a typical spectrum for the ascorbyl radical (hyperfine splitting constant, aH = 1.8G), similar to the positive control obtained by exposing ascorbate to H2O2 (c). Panel d shows that no ascorbyl radical is detected if H2O2 is degraded by catalase in the absence of PP1, prior to ascorbate addition.


D . Cartoon representation of the active site of H2O2 - treated PP1 as in Fig EV2D . 2mFo - DFc electron density map at the 2 . 2 Å resolution is shown in yellow at the 1 . 1 level . H2O2 - treatment causes an overall shrinkage of the PP1 coordination sphere by 0 . 12 Å compared to ascorbate - treated crystals consistent with the oxidation of the dinuclear center . This increases the energy barrier for the catalytic steps involving - OH - attack on the phosphorous center of the bridging phosphate and rupture of the P - O scissile bond with the assistance of H125 ( black arrows ) . Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit . See also Appendix Table S1 .

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D. Cartoon representation of the active site of H2O2-treated PP1 as in Fig EV2D. 2mFo-DFc electron density map at the 2.2 Å resolution is shown in yellow at the 1.1 level. H2O2-treatment causes an overall shrinkage of the PP1 coordination sphere by 0.12 Å compared to ascorbate-treated crystals consistent with the oxidation of the dinuclear center. This increases the energy barrier for the catalytic steps involving -OH- attack on the phosphorous center of the bridging phosphate and rupture of the P-O scissile bond with the assistance of H125 (black arrows). Reported coordination distances in Å are averaged over the two PP1 molecules in the crystallographic asymmetric unit. See also Appendix Table S1.


E . Effect of Nox4 on GADD34 / PP1 - mediated eIF2 dephosphorylation in transfected HEK293 cells . Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1 , and resulted in even higher phospho - eIF2α levels in cells transfected with N124D or D64N PP1 variants .

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E. Effect of Nox4 on GADD34/PP1-mediated eIF2 dephosphorylation in transfected HEK293 cells. Nox4 increased eIF2 phosphorylation in cells transfected with GADD34 and WT PP1, and resulted in even higher phospho-eIF2α levels in cells transfected with N124D or D64N PP1 variants.


F . Effect of ascorbate ( Asc , 0 . 5 mM ) on phosphatase inhibition in tunicamycin - treated H9c2 cells with overexpression or knockdown of Nox4 ( Ad . Nox4 and Ad . shNox4 , respectively ) . In control cells , ascorbate enhanced tunicamycin - stimulated increases in phosphatase activity . Phosphatase activity was lower in Nox4 - overexpressing than control cells but was normalized by ascorbate to the same level as in control cells . In Nox4 knockdown cells , tunicamycin - induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect .

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F. Effect of ascorbate (Asc, 0.5 mM) on phosphatase inhibition in tunicamycin-treated H9c2 cells with overexpression or knockdown of Nox4 (Ad.Nox4 and Ad.shNox4, respectively). In control cells, ascorbate enhanced tunicamycin-stimulated increases in phosphatase activity. Phosphatase activity was lower in Nox4-overexpressing than control cells but was normalized by ascorbate to the same level as in control cells. In Nox4 knockdown cells, tunicamycin-induced increases in phosphatase activity were enhanced and ascorbate had minimal additional effect.


A . H9c2 cells treated with tunicamycin ( 2 µg / ml , 12h ) showed significantly lower survival when endogenous Nox4 was silenced ( siNox4 ) as compared to cells treated with a scrambled siRNA ( siCtl ) . Cell survival was restored by treatment with either guanabenz ( Gbz , 5 µM ) or salubrinal ( Sal , 50 µM ) but was unaffected by clonidine ( Cld , 5 µM ) . n = 3 / group .

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A. H9c2 cells treated with tunicamycin (2 µg/ml, 12h) showed significantly lower survival when endogenous Nox4 was silenced (siNox4) as compared to cells treated with a scrambled siRNA (siCtl). Cell survival was restored by treatment with either guanabenz (Gbz, 5 µM) or salubrinal (Sal, 50 µM) but was unaffected by clonidine (Cld, 5 µM). n=3/group.


B . Nox4 - depleted cells had lower levels of phospho - eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz ( Gbz ) .

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B. Nox4-depleted cells had lower levels of phospho-eIF2α and ATF4 than control cells but these were restored in the presence of guanabenz (Gbz).


D . Hearts from Nox4 knockout ( KO ) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion ( I / R ) . Infarct size assessed by triphenyltetrazolium chloride ( TTC ) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz ( Gbz ) . In the representative heart sections shown at the top , white denotes infarct area and red the viable area . Scale bars , 1 mm . Numbers of hearts are indicated within the bars .

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D. Hearts from Nox4 knockout (KO) mice and WT controls were subjected to global ischemia followed by aerobic reperfusion (I/R). Infarct size assessed by triphenyltetrazolium chloride (TTC) staining was greater in Nox4 KO hearts compared to WT and was significantly reduced by guanabenz (Gbz). In the representative heart sections shown at the top, white denotes infarct area and red the viable area. Scale bars, 1 mm. Numbers of hearts are indicated within the bars.


E . Immunoblotting of heart homogenates after I / R showed lower levels of phospho - eIF2a , ATF4 and ER chaperones , and higher levels of cleaved caspase - 12 , in Nox4 KO compared to WT . Tubulin was used as a loading control . Treatment with guanabenz ( Gbz ) reversed these changes ( blots shown to the right ) .

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E. Immunoblotting of heart homogenates after I/R showed lower levels of phospho-eIF2a, ATF4 and ER chaperones, and higher levels of cleaved caspase-12, in Nox4 KO compared to WT. Tubulin was used as a loading control. Treatment with guanabenz (Gbz) reversed these changes (blots shown to the right).


A . Plasma urea levels were elevated to a greater extent in tunicamycin - treated Nox4 KO mice than WT . Co - treatment with guanabenz ( Gbz ) reduced urea levels in both groups . Numbers of animals are indicated within bars .

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A. Plasma urea levels were elevated to a greater extent in tunicamycin-treated Nox4 KO mice than WT. Co-treatment with guanabenz (Gbz) reduced urea levels in both groups. Numbers of animals are indicated within bars.


B . 48 hours after systemic tunicamycin treatment , kidneys of Nox4 KO mice showed a marked surface pallor ( bottom right ) .

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B. 48 hours after systemic tunicamycin treatment, kidneys of Nox4 KO mice showed a marked surface pallor (bottom right).


C . TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin - treated Nox4 KO mice . n = 4 / group .

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. TUNEL staining revealed a significantly higher number of apoptotic cells in tunicamycin-treated Nox4 KO mice. n=4/group.


D . Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase - 12 and cleaved PARP levels in tunicamycin - treated Nox4 KO mice compared to WT .

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

D. Immunoblotting of kidney homogenates showed significantly elevated cleaved caspase-12 and cleaved PARP levels in tunicamycin-treated Nox4 KO mice compared to WT.


E . Survival curves showed that a very high proportion of Nox4 KO mice died after AKI . Guanabenz ( Gbz ) treatment dramatically improved survival in tunicamycin - treated KO mice . Number of animals as indicated . Levels of significance by Kaplan Meier analysis are reported to the right .

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

E. Survival curves showed that a very high proportion of Nox4 KO mice died after AKI. Guanabenz (Gbz) treatment dramatically improved survival in tunicamycin-treated KO mice. Number of animals as indicated. Levels of significance by Kaplan Meier analysis are reported to the right.


B ( left ) SRM images of paraspeckles in HAP1 NEAT1 ∆ 3 ′ mutant cells ( ∆ 3 ′ ) detected by NEAT1 _ 5 ′ ( green ) and NEAT1 _ 15k ( magenta ) FISH probes in the presence of MG132 ( 5 μM for 6 h ) . Scale bar , 500 nm . ( right ) Graph showing the proportion of paraspeckles with localization of the NEAT1 3 ′ ends in the core or in the core and shell ( n = 44 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (left) SRM images of paraspeckles in HAP1 NEAT1 ∆3′ mutant cells (∆3′) detected by NEAT1_5′ (green) and NEAT1_15k (magenta) FISH probes in the presence of MG132 (5 μM for 6 h). Scale bar, 500 nm. (right) Graph showing the proportion of paraspeckles with localization of the NEAT1 3′ ends in the core or in the core and shell (n = 44).


B SRM images of the paraspeckles in ∆ 0 - 0 . 8kb and ∆ 0 - 1 . 9kb ( ∆ 5 ′ ) cells treated with MG132 ( 5 μM for 6 h ) detected by NEAT1 _ 5 ′ , 1k , and 2k ( green ) and NEAT1 _ 3 ′ ( magenta ) FISH probes . Scale bar , 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SRM images of the paraspeckles in ∆0-0.8kb and ∆0-1.9kb (∆5′) cells treated with MG132 (5 μM for 6 h) detected by NEAT1_5′, 1k, and 2k (green) and NEAT1_3′ (magenta) FISH probes. Scale bar, 500 nm.


D ( left ) EM observation of the paraspeckles in ∆ 5 ′ cells treated with MG132 ( 5 μM for 17 h ) using NEAT1 _ 5 ′ probes . Scale bar , 100 nm . ( middle ) Graph showing the proportion of localization of NEAT1 _ 5 ′ probes ( 347 gold particles ) within the paraspeckles in ∆ 5 ′ cells . ( right ) Graph showing the proportion of the localization of NEAT1 _ 5 ′ probes in each paraspeckle in ∆ 5 ′ cells ( n = 20 ) . The box plot shows the median ( inside line ) , 25 - 75 percentiles ( box bottom to top ) , and 10 - 90 percentiles ( whisker bottom to top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (left) EM observation of the paraspeckles in ∆5′ cells treated with MG132 (5 μM for 17 h) using NEAT1_5′ probes. Scale bar, 100 nm. (middle) Graph showing the proportion of localization of NEAT1_5′ probes (347 gold particles) within the paraspeckles in ∆5′ cells. (right) Graph showing the proportion of the localization of NEAT1_5′ probes in each paraspeckle in ∆5′ cells (n = 20). The box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


B SRM images of the paraspeckles in MG132 - treated ( 5 μM for 6 h ) HAP1 NEAT1 ∆ 5 ′ / ∆ 3 ′ cells detected by NEAT1 _ 2k ( green ) and NEAT1 _ 15k FISH probes ( left , magenta ) or NONO immunofluorescence ( IF ) ( right , magenta ) . Scale bar , 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 SRM images of the paraspeckles in MG132-treated (5 μM for 6 h) HAP1 NEAT1 ∆5′/∆3′ cells detected by NEAT1_2k (green) and NEAT1_15k FISH probes (left, magenta) or NONO immunofluorescence (IF) (right, magenta). Scale bar, 500 nm.


D ( upper ) EM observation of the paraspeckles in MG132 - treated ( 5 μM for 17 h ) ∆ 5 ′ / ∆ 3 ′ cells using NEAT1 _ 5 ′ ( left ) and NEAT1 _ D2 probes ( right ) . Scale bar , 100 nm . ( middle ) Graph showing the proportion of the localization of NEAT1 _ 5 ′ ( left , 310 gold particles ) and NEAT1 _ D2 ( right , 294 gold particles ) probes within the paraspeckles in HAP1 NEAT1 ∆ 5 ′ / ∆ 3 ′ cells . ( lower ) Graph showing the proportion of the localization of NEAT1 _ 5 ′ ( left , 19 cells ) and D2 ( right , 22 cells ) probes in each paraspeckle in ∆ 5 ′ / ∆ 3 ′ cells . Each box plot shows the median ( inside line ) , 25 - 75 percentiles ( box bottom to top ) , and 10 - 90 percentiles ( whisker bottom to top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 (upper) EM observation of the paraspeckles in MG132-treated (5 μM for 17 h) ∆5′/∆3′ cells using NEAT1_5′ (left) and NEAT1_D2 probes (right). Scale bar, 100 nm. (middle) Graph showing the proportion of the localization of NEAT1_5′ (left, 310 gold particles) and NEAT1_D2 (right, 294 gold particles) probes within the paraspeckles in HAP1 NEAT1 ∆5′/∆3′ cells. (lower) Graph showing the proportion of the localization of NEAT1_5′ (left, 19 cells) and D2 (right, 22 cells) probes in each paraspeckle in ∆5′/∆3′ cells. Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


C Diameters ( Sx ) of the paraspeckles in WT , ∆ 3 ′ , ∆ 5 ′ , and ∆ 5 ′ / ∆ 3 ′ cells treated with MG132 ( 5 μM for 6 h ) determined by SRM ( WT : n = 129 , ∆ 3 ′ : n = 106 , ∆ 5 ′ : n = 100 , ∆ 5 ′ / ∆ 3 ′ : n = 292 ) . WT ( mean size : 387 . 5 nm ) , ∆ 3 ′ ( mean size : 492 . 4 nm ) , ∆ 5 ′ ( mean size : 497 . 7 nm ) , and ∆ 5 ′ / ∆ 3 ′ mutant ( mean size : 753 . 3 nm ) . ( * * * * : P < 0 . 0001 , compared with WT : Kruskal - Wallis test with Dunn ' s multiple comparison test ) . Each box plot shows the median ( inside line ) , 25 - 75 percentiles ( box bottom to top ) , and 10 - 90 percentiles ( whisker bottom to top ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Diameters (Sx) of the paraspeckles in WT, ∆3′, ∆5′, and ∆5′/∆3′ cells treated with MG132 (5 μM for 6 h) determined by SRM (WT: n = 129, ∆3′: n = 106, ∆5′: n = 100, ∆5′/∆3′: n = 292). WT (mean size: 387.5 nm), ∆3′ (mean size: 492.4 nm), ∆5′ (mean size: 497.7 nm), and ∆5′/∆3′ mutant (mean size: 753.3 nm). (****: P < 0.0001, compared with WT: Kruskal-Wallis test with Dunn's multiple comparison test). Each box plot shows the median (inside line), 25-75 percentiles (box bottom to top), and 10-90 percentiles (whisker bottom to top).


B Quantitation of NEAT1 _ 2 by RT - qPCR in WT and ∆ 5 ′ / ∆ 3 ′ / ∆ PAS cells with or without MG132 treatment ( 6 h ) . Data are represented as mean ± SD ( 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 B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O

B Quantitation of NEAT1_2 by RT-qPCR in WT and ∆5′/∆3′/∆PAS cells with or without MG132 treatment (6 h). Data are represented as mean ± SD (n = 3).


C The paraspeckles in WT and ∆ 5 ′ / ∆ 3 ′ / ∆ PAS cells detected by SRM using NEAT1 _ 5 ′ and 2k FISH probes ( green ) , and NONO IF ( magenta ) under the conditions shown in ( B ) . Scale bar , 500 nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The paraspeckles in WT and ∆5′/∆3′/∆PAS cells detected by SRM using NEAT1_5′ and 2k FISH probes (green), and NONO IF (magenta) under the conditions shown in (B). Scale bar, 500 nm.


( b ) Chromatin immunoprecipitation using α - FLAG antibody was performed to confirm the pull - down of Sα chromatin from the Sα - engineered ( FNLDD - Sα - LexA ) CH12F3 - 2A cells . The a , b , and c bars indicate the primers used for ChIP - qPCR as shown in Fig . 1a ( n = 3 ; mean + sd ; 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chromatin immunoprecipitation using α-FLAG antibody was performed to confirm the pull-down of Sα chromatin from the Sα-engineered (FNLDD-Sα-LexA) CH12F3-2A cells. The a, b, and c bars indicate the primers used for ChIP-qPCR as shown in Fig. 1a (n=3; mean + sd; two tailed unpaired Student's t-test).


( e ) Bar graph showing the proteins with ≥ 2 - fold enrichment as calculated by the heavy to light SILAC ratio .

(e) Bar graph showing the proteins with ≥2-fold enrichment as calculated by the heavy to light SILAC ratio.


( f ) Effect of the siRNA - mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3 - 2A cells . Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA ( siCONT ) ( n = 2 ; 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

(f) Effect of the siRNA-mediated KD of histone chaperones and chromatin remodelers on IgM to IgA switching in CH12F3-2A cells. Data are presented as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=2; mean + sd).


( a ) Top : Scheme of the IgA - switching assay in CH12F3 - 2A cells . After electroporation of siRNAs , cells were cultured for 24 h , and then stimulated by CIT , cultured for another 24 h , and subjected to FACS analysis . Bottom : Confirmation of the siRNA - mediated KD of SAMHD1 in CH12F3 - 2A cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Top: Scheme of the IgA-switching assay in CH12F3-2A cells. After electroporation of siRNAs, cells were cultured for 24 h, and then stimulated by CIT, cultured for another 24 h, and subjected to FACS analysis. Bottom: Confirmation of the siRNA-mediated KD of SAMHD1 in CH12F3-2A cells.


( b ) FACS profile of the percentage of CH12F3 - 2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3 - 2A cells cultured with ( + ) or without ( - ) CIT stimulation for 24 h . The number in the FACS plot represents the percentage of cells expressing IgA on their surface .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FACS profile of the percentage of CH12F3-2A cells undergoing IgA switching after transfection of the indicated siRNAs into CH12F3-2A cells cultured with (+) or without (-) CIT stimulation for 24 h. The number in the FACS plot represents the percentage of cells expressing IgA on their surface.


( c ) Summary of the FACS data obtained from three independent experiments . Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA ( siCONT ) ( n = 3 ; mean + sd ; 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 O O O O O O O O O O O O O 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) Summary of the FACS data obtained from three independent experiments. Data are shown as the % IgA switching relative to the IgA switching in cells transfected with control siRNA (siCONT) (n=3; mean + sd; two tailed unpaired Student's t-test).


( d ) Quantitative RT - PCR analysis of the μGLT , αGLT , Aid , and Samhd1 expression in the indicated samples . The data were normalized to Gapdh , and represents as the mean of the two independent experiments with standard deviations . Statistical significance as determined by two - tailed unpaired Student ' s t - test is shown . ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative RT-PCR analysis of the μGLT, αGLT, Aid, and Samhd1 expression in the indicated samples. The data were normalized to Gapdh, and represents as the mean of the two independent experiments with standard deviations. Statistical significance as determined by two-tailed unpaired Student's t-test is shown. ns = not significant.


( e ) Confirmation of the CRISPR / Cas9 mediated KO of Samhd1 in CH12F3 - 2A cells . Beta - actin was used as a loading control .

O O O O O 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) Confirmation of the CRISPR/Cas9 mediated KO of Samhd1 in CH12F3-2A cells. Beta-actin was used as a loading control.


( f ) CSR time course analysis of WT and Samhd1 KO clones . The data are shown as percentage of IgA expressing cells ( n = 2 ; mean ± sd ; two tailed unpaired Student ' s t - test ; * * , p < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) CSR time course analysis of WT and Samhd1 KO clones. The data are shown as percentage of IgA expressing cells (n=2; mean ± sd; two tailed unpaired Student's t-test; **, p<0.01).


( g ) Proliferation of WT and Samhd1 KO clones after stimulation with CIT ( n = 2 ; mean ± sd ; two tailed unpaired Student ' s t - test ; ns = not significant ) .

O O O O O O O O O O O O O O O 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) Proliferation of WT and Samhd1 KO clones after stimulation with CIT (n = 2; mean ± sd; two tailed unpaired Student's t-test; ns = not significant).


( h ) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3 - 2A . Histograms show the CFSE dye dilution derived from the indicated samples . Aphidicolin ( 2 μg / mL ) was also used as a positive control .

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

(h) Analysis of the cell proliferation of WT or Samhd1 KO clones of CH12F3-2A. Histograms show the CFSE dye dilution derived from the indicated samples. Aphidicolin (2 μg/mL) was also used as a positive control.


( i ) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells . Bottom : Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice . Tubulin was used as a loading control .

(i) Schematic representation of the IgG1 or IgG3 switching assay in spleen B cells. Bottom: Western blot analysis of the SAMHD1 expression in spleen B cells derived from WT or Samhd1 KO mice. Tubulin was used as a loading control.


( j ) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments . The data are shown as percentage of IgG1 ( left ) or IgG3 ( right ) expressing cells ( n = 5 ; mean ± sd ; 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 O O O O O O O O O O O O O O O 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) Bar graphs show the compilation of IgG1 and IgG3 CSR assays from five independent experiments. The data are shown as percentage of IgG1 (left) or IgG3 (right) expressing cells (n=5; mean ± sd; two-tailed unpaired Student's t-test).


( b ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . CH12F3 - 2A cells were transfected with the indicated siRNAs . ( c ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

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

(b) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. CH12F3-2A cells were transfected with the indicated siRNAs. (c) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3 - 2A cells by SAMHD1 siRNA or by tetracycline ( 50 nM ) treatment , respectively . Expression of Tubulin is shown as a loading control ( e : left ) . Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig . 3e ( e : right ) .

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

Western blot analysis of the KD of SAMHD1 or TOP1 by the transfection of CH12F3-2A cells by SAMHD1 siRNA or by tetracycline (50 nM) treatment, respectively. Expression of Tubulin is shown as a loading control (e: left). Relative expression of TOP1 and SAMHD1 as determined by densitometric quantification of western blot images in Fig. 3e (e: right).


Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments ( f ) . Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) ( g ) .

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

Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments (f). Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test) (g).


( h - j ) Analysis of the IgH / c - Myc translocation frequency in WT and Samhd1 KO CH12F3 - 2A cells . ( h ) Schematic illustration of the experimental design for the translocation assay . ( i ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( j ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

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

(h-j) Analysis of the IgH/c-Myc translocation frequency in WT and Samhd1 KO CH12F3-2A cells. (h) Schematic illustration of the experimental design for the translocation assay. (i) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (j) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( k - m ) Analysis of the IgH / c - Myc translocation frequency in spleen B cell derived from WT ( Samhd1 + / + ) or Samhd1 KO ( Samhd1 - / - ) mice . ( k ) Schematic illustration of the experimental design for the translocation assay . ( l ) Southern blot analysis of PCR - amplified fragments with a Myc - specific probe from two independent experiments . ( m ) Frequency of IgH / c - Myc chromosomal translocations derived from two independent experiments ( mean ± sd ; Fisher ' s exact test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-m) Analysis of the IgH/c-Myc translocation frequency in spleen B cell derived from WT (Samhd1+/+) or Samhd1 KO (Samhd1-/-) mice. (k) Schematic illustration of the experimental design for the translocation assay. (l) Southern blot analysis of PCR-amplified fragments with a Myc-specific probe from two independent experiments. (m) Frequency of IgH/c-Myc chromosomal translocations derived from two independent experiments (mean ± sd; Fisher's exact test).


( b ) Mutation frequency in the 5 ′ Sµ region of WT or Samhd1 KO CH12F3 - 2A cells . Statistical significance as evaluated by Fisher ' s exact test is shown . ns = not significant .

(b) Mutation frequency in the 5′ Sµ region of WT or Samhd1 KO CH12F3-2A cells. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.


( d ) LM - PCR analysis of the AID - induced DNA double - strand breaks in the Sµ region of WT or Samhd1 KO CH12F3 - 2A cells . Wedges indicate a three - fold increase in the DNA amount . Amplification of Gapdh was used as an internal loading control .

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

(d) LM-PCR analysis of the AID-induced DNA double-strand breaks in the Sµ region of WT or Samhd1 KO CH12F3-2A cells. Wedges indicate a three-fold increase in the DNA amount. Amplification of Gapdh was used as an internal loading control.


( e ) Gamma - H2AX - ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA ( % IP ) normalized to the total amount of DNA used for the immunoprecipitation ( n = 3 ; mean + sd ; two - tailed unpaired Student ' s t - test ; ns = not significant ) .

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(e) Gamma-H2AX-ChIP signal in the Sµ region is shown as the fraction of immunoprecipitated DNA (% IP) normalized to the total amount of DNA used for the immunoprecipitation (n=3; mean + sd; two-tailed unpaired Student's t-test; ns = not significant).


( f ) Left : Scheme of long - range interactions between Sμ - Sα elements in the IgH locus before and after AID activation . Right : Representative gel picture of the 3C assay detecting the Sμ - Sα interaction in WT or Samhd1 KO CH12F3 - 2A cells with or without CIT stimulation for 24 h . Gapdh was amplified as a loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Left: Scheme of long-range interactions between Sμ-Sα elements in the IgH locus before and after AID activation. Right: Representative gel picture of the 3C assay detecting the Sμ-Sα interaction in WT or Samhd1 KO CH12F3-2A cells with or without CIT stimulation for 24 h. Gapdh was amplified as a loading control.


( a - d ) Analysis of Sμ - Sα recombination junctions from WT or CH12F3 - 2A cells after transfection with control ( siCONT ) or Ligase 4 ( siLIG4 ) siRNA . The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis . A total of 62 ( siCONT ) and 73 ( siLIG4 ) junctions from WT , and 60 ( siCONT ) and 60 ( siLIG4 ) junctions from Samhd1 KO cells were sequenced and analyzed . Statistical significance as evaluated by Fisher ' s exact test ( b ) or two - tailed unpaired Student ' s t - test ( d ) or is shown . Schematic representation of the Sμ - Sα junction analysis ( a ) . Summary of the characteristics of Sμ - Sα recombination junctions ( b ) . Sequences at Sμ - Sα recombination junctions with insertions ( c ) . Summary of the average length of insertions at Sμ - Sα recombination junctions ( d ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-d) Analysis of Sμ-Sα recombination junctions from WT or CH12F3-2A cells after transfection with control (siCONT) or Ligase 4 (siLIG4) siRNA. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to junction analysis. A total of 62 (siCONT) and 73 (siLIG4) junctions from WT, and 60 (siCONT) and 60 (siLIG4) junctions from Samhd1 KO cells were sequenced and analyzed. Statistical significance as evaluated by Fisher's exact test (b) or two-tailed unpaired Student's t-test (d) or is shown. Schematic representation of the Sμ-Sα junction analysis (a). Summary of the characteristics of Sμ-Sα recombination junctions (b). Sequences at Sμ-Sα recombination junctions with insertions (c). Summary of the average length of insertions at Sμ-Sα recombination junctions (d).


( e - i ) Analysis of the CRISPR / Cas9 induced chromosomal translocations between the Rosa26 ( Chr6 ) and H3f3b ( Chr 11 ) genes . The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis . Schematic representation of the chromosomal translocations ( e ) . Representative images of agarose gels after PCR - amplification of the Rosa26 / H3f3b translocation from WT or Samhd1 KO CH12F3 - 2A cells ( f ) . Relative frequency of Rosa26 / H3b3f chromosomal translocations ( g ) . Frequency of junctions containing insertions ( h ) and average length of insertions ( i ) . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test ( g , i ) or Fisher ' s exact test ( h ) is 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(e-i) Analysis of the CRISPR/Cas9 induced chromosomal translocations between the Rosa26 (Chr6) and H3f3b (Chr 11) genes. The genomic DNA isolated from WT or Samhd1 KO cells from three independent experiments were pooled prior to chromosomal translocation or junction analysis. Schematic representation of the chromosomal translocations (e). Representative images of agarose gels after PCR-amplification of the Rosa26/H3f3b translocation from WT or Samhd1 KO CH12F3-2A cells (f). Relative frequency of Rosa26/H3b3f chromosomal translocations (g). Frequency of junctions containing insertions (h) and average length of insertions (i). Statistical significance as evaluated by two-tailed unpaired Student's t-test (g, i) or Fisher's exact test (h) is shown.


( b ) Confirmation of the expression of GFP - tagged human SAMHD1 ( WT , and DN , DA , TA , and RE mutants ) . Histone H3 expression is shown as a loading control .

O O O O O O O O O O O O O O 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) Confirmation of the expression of GFP-tagged human SAMHD1 (WT, and DN, DA, TA, and RE mutants). Histone H3 expression is shown as a loading control.


( c ) CSR complementation assay in Samhd1 KO cells . Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector , and are derived from three independent experiments ( mean ± sd ; 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 O O O O O O O O O O O O O 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) CSR complementation assay in Samhd1 KO cells. Data are shown as the % IgA switching relative to the IgA switching in WT cells transfected with empty vector, and are derived from three independent experiments (mean ± sd; two-tailed unpaired Student's t-test).


( e ) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3 - 2A cells . Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved ( n = 3 ; mean ± sd ; two - tailed unpaired Student ' s t - test ; * * = p ≤ 0 . 01 ; * * * = p ≤ 0 . 001 ) .

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(e) Effect of deoxyribonucleoside supplementation individually or in combination on the rate of IgM to IgA switching in CH12F3-2A cells. Data are presented as % IgA switching relative to the IgA switching in cells treated with an equivalent volume of the solvent in which the deoxyribonucleosides were dissolved (n=3; mean ± sd; two-tailed unpaired Student's t-test; ** = p ≤ 0.01; *** = p ≤ 0.001).


( f ) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH / c - Myc translocations in CH12F3 - 2A cells ( n = 2 ; mean ± sd ; Fisher ' s exact test ; ns = not significant ; * * * = p ≤ 0 . 001 ) . The concentration dNTPs used for supplementation either individually or in combination were as follows : 2 ′ - deoxyguanosine ( dG ) , 0 . 15 mM ; 2 ′ - deoxyadenosine ( dA ) , 1 . 0 mM ; 2 ′ - deoxycytidine ( dC ) , 12 mM ; and 2 ′ - deoxythymidine ( dT ) , 1 . 6 mM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Effect of deoxyribonucleoside supplementation individually or in combination on the frequency of IgH/c-Myc translocations in CH12F3-2A cells (n = 2; mean ± sd; Fisher's exact test; ns = not significant; *** = p ≤ 0.001). The concentration dNTPs used for supplementation either individually or in combination were as follows: 2′-deoxyguanosine (dG), 0.15 mM; 2′-deoxyadenosine (dA), 1.0 mM; 2′-deoxycytidine (dC), 12 mM; and 2′-deoxythymidine (dT), 1.6 mM.


( b ) FACS profile of the percentage of WT and Samhd1 KO CH12F3 - 2A cells undergoing IgA switching following transfection with the indicated plasmids . The number in the FACS plot represents the percentage of cells expressing IgA on their surface . ( c ) Summary of the FACS data obtained from two independent experiments . The data are shown as percentage of IgA expressing cells upon CSR induced by blunt ( left ) or staggered ( right ) DSBs ( n = 2 ; mean + sd ; two - tailed unpaired Student ' s t - test ) ; ns = not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) FACS profile of the percentage of WT and Samhd1 KO CH12F3-2A cells undergoing IgA switching following transfection with the indicated plasmids. The number in the FACS plot represents the percentage of cells expressing IgA on their surface. (c) Summary of the FACS data obtained from two independent experiments. The data are shown as percentage of IgA expressing cells upon CSR induced by blunt (left) or staggered (right) DSBs (n = 2; mean + sd; two-tailed unpaired Student's t-test); ns = not significant.


( d ) Length of resected DNA at Sμ - Sα junctions obtained from the joining of DSBs with blunt ends . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test is shown . ( e ) Summary of the characteristics of Sμ - Sα recombination junctions resulting from the joining of DSBs with blunt ends .

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

(d) Length of resected DNA at Sμ-Sα junctions obtained from the joining of DSBs with blunt ends. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown. (e) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends.


( f ) Length of resected DNA at Sμ - Sα junctions obtained from the joining of staggered DSBs . Left : Length of Sμ and Sα overhangs were 35 nt and 98 nt , respectively . Right : Length of Sμ and Sα overhangs were 35 nt and 187 nt , respectively . ( g ) Summary of the characteristics of Sμ - Sα recombination junctions resulting from the joining of staggered DSBs . Statistical significance as evaluated by Fisher ' s exact test is shown .

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

(f) Length of resected DNA at Sμ-Sα junctions obtained from the joining of staggered DSBs. Left: Length of Sμ and Sα overhangs were 35 nt and 98 nt, respectively. Right: Length of Sμ and Sα overhangs were 35 nt and 187 nt, respectively. (g) Summary of the characteristics of Sμ-Sα recombination junctions resulting from the joining of staggered DSBs. Statistical significance as evaluated by Fisher's exact test is shown.


( h ) Summary of the average length of insertions at Sμ - Sα recombination junctions . Statistical significance as evaluated by two - tailed unpaired Student ' s t - test is 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

(h) Summary of the average length of insertions at Sμ-Sα recombination junctions. Statistical significance as evaluated by two-tailed unpaired Student's t-test is shown.


Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ - Sα recombination junctions resulting from the joining of DSBs with blunt ends . ( j ) Top : Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells . Bottom : Western blot analysis of the expression of TdT in WT and Samhd1 KO cells . Tubulin was used as a loading control .

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

Analysis of the effect of TdT expression on the frequency of CSR as well as characteristics of Sμ-Sα recombination junctions resulting from the joining of DSBs with blunt ends. (j) Top: Schematic representation of the experimental design to analyze the effect of TdT expression on CSR in WT and Samhd1 KO cells. Bottom: Western blot analysis of the expression of TdT in WT and Samhd1 KO cells. Tubulin was used as a loading control.


( k ) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3 - 2A cells with or without TdT expression . ( l ) Summary of the FACS data obtained from two independent experiments ( n = 2 ; mean + sd ; two - tailed unpaired Student ' s t - test ; * = p ≤ 0 . 05 ) .

(k) FACS profile of the percentage IgA switching in WT or Samhd1 KO CH12F3-2A cells with or without TdT expression. (l) Summary of the FACS data obtained from two independent experiments (n = 2; mean + sd; two-tailed unpaired Student's t-test; * = p ≤ 0.05).


( m ) Summary of the characteristics of Sμ - Sα recombination junctions . Statistical significance as evaluated by Fisher ' s exact test is shown . ns = not significant .

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

(m) Summary of the characteristics of Sμ-Sα recombination junctions. Statistical significance as evaluated by Fisher's exact test is shown. ns = not significant.


A B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice . The percentages of intratumoral CD11bHi GR1Hi MDSCs ( n = 10 mice ) , CD25 + Foxp3 + regulatory T cells ( Tregs ) , or CD25 + Foxp3 − activated T cells ( n = 5 mice per group ) among CD45 + cells were analyzed by fluorescence - activated cell sorting ( FACS ) and represented as box plots Data information : data are presented by Box plots Min to Max showing all points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; Mann - Whitney test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B16F10 melanoma cells with either mTRF2 overexpression or knockdown were inoculated into the backs of immunocompetent mice. The percentages of intratumoral CD11bHi GR1Hi MDSCs (n = 10 mice), CD25+ Foxp3+ regulatory T cells (Tregs), or CD25+ Foxp3− activated T cells (n = 5 mice per group) among CD45+ cells were analyzed by fluorescence-activated cell sorting (FACS) and represented as box plots Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test.


TRF2 - overexpressing or - knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel . Five days later , the percentage of immune cells infiltrating the Matrigel plugs of CD11bHi GR1Hi MDSCs ( C ) were determined by FACS ( n = 5 mice 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, the percentage of immune cells infiltrating the Matrigel plugs of CD11bHi GR1Hi MDSCs (C) were determined by FACS (n = 5 mice per group).


TRF2 - overexpressing or - knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel . Five days later , the intranuclear staining of pSTAT3 ( D ) were determined by FACS ( n = 5 mice 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 O O O O O O O O O O O O O O O O O O O O O O O

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later , the intranuclear staining of pSTAT3 (D) were determined by FACS (n = 5 mice per group).


TRF2 - overexpressing or - knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel . Five days later , CD3 − NKp46 + and IFN - γ + among CD3 − NKp46 + NK cells ( E ) were determined by FACS ( n = 5 mice 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

TRF2-overexpressing or -knockdown BJcl2 cells were inoculated into the backs of immunodeficient mice in the presence of Matrigel. Five days later, CD3− NKp46+ and IFN-γ+ among CD3− NKp46+ NK cells (E) were determined by FACS (n = 5 mice per group).


Linear correlation plot of the percentage of IFN - γ + NK cells , the mean of fluorescence intensity ( MFI ) of pSTAT3 for MDSCs , and the expression of TRF2 ( r2 = 0 , 5177 ; Pearson ' s correlation , p = 0 . 003 ) ( F ) Data information : data are presented by Box plots Min to Max showing all points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; Mann - Whitney test . Pearson correlation and p values are indicated .

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

Linear correlation plot of the percentage of IFN- γ + NK cells, the mean of fluorescence intensity (MFI) of pSTAT3 for MDSCs, and the expression of TRF2 (r2= 0,5177; Pearson's correlation, p = 0.003) (F) Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated.


the mRNA expression level in the matrigel plugs of arg1 , pSTAT3 MFI and TRF2 expression ( r2 = 0 , 7077 ; Pearson ' s correlation , p < 0 . 0001 ) ( G ) ; the mRNA expression level in the matrigel plugs of tgfβ , pSTAT3 MFI and TRF2 expression ( r2 = 0 , 3669 ; Pearson ' s correlation , p = 0 . 0046 ) ( H ) and the mRNA expression level in the matrigel plugs of il - 10 , pSTAT3 MFI and TRF2 expression ( r2 = 0 , 7418 ; Pearson ' s correlation , p < 0 . 0001 ) ( I ) . Data information : data are presented by Box plots Min to Max showing all points with median . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 ; Mann - Whitney test . Pearson correlation and p values are indicated .

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

the mRNA expression level in the matrigel plugs of arg1, pSTAT3 MFI and TRF2 expression (r2= 0,7077; Pearson's correlation, p < 0.0001) (G); the mRNA expression level in the matrigel plugs of tgfβ, pSTAT3 MFI and TRF2 expression (r2= 0,3669; Pearson's correlation, p = 0.0046) (H) and the mRNA expression level in the matrigel plugs of il-10, pSTAT3 MFI and TRF2 expression (r2= 0,7418; Pearson's correlation, p < 0.0001) (I). Data information: data are presented by Box plots Min to Max showing all points with median. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001; Mann-Whitney test. Pearson correlation and p values are indicated.


B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) . The tumor volume was determined every 2 days using hemi - ellipsoid formula ( B ) . Tumor volume are presented in mm3 along the time in days . All experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; Mann - Whitney test .

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

B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP). The tumor volume was determined every 2 days using hemi-ellipsoid formula (B). Tumor volume are presented in mm3 along the time in days. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti - GR1 antibody or isotypic control ( 200µg / IP ) . On day 19 of the tumor growth experiment , the percentages of GFP + cells infiltrating the lungs were determined by FACS and presented as the number of GFP + cells / mg lung tissue ( C ) . Data information : data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45 + cells or the indicated parent population or the number of lung nodules . All experiments are performed with n = 8 mice per group ; * p < 0 . 05 , * * p < 0 . 01 , and * * * p < 0 . 001 ; Mann - Whitney test .

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

B16F10 melanoma cells with or without mTRF2 overexpression were inoculated into the backs of immunocompetent mice and treated with anti-GR1 antibody or isotypic control (200µg/IP). On day 19 of the tumor growth experiment, the percentages of GFP+ cells infiltrating the lungs were determined by FACS and presented as the number of GFP+ cells/mg lung tissue (C). Data information: data are presented by Box plots Min to Max showing all points with median corresponding to the percentages of each cell population among CD45+ cells or the indicated parent population or the number of lung nodules. All experiments are performed with n = 8 mice per group; *p < 0.05, **p < 0.01, and ***p < 0.001; Mann-Whitney test.


On day 21 , lung metastasis was analyzed by counting macroscopic metastatic nodules ( E scale bars = 500µM .

On day 21, lung metastasis was analyzed by counting macroscopic metastatic nodules (E scale bars = 500µM.