( b ) HeLa were transduced with AMBRA1 lentiviral shRNA transfected or not with FLAG - TRAF6 . of LC3II p62 bands is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 001 P = 0 . 0018 for LC3 P = 0 . 004 for p62 .

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(b) HeLa cells were transduced with AMBRA1 lentiviral shRNA and transfected or not with FLAG-TRAF6. Quantification of LC3II and p62 bands is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001 and P = 0.0018 for LC3 and P = 0.004 for p62.


( c ) of LC3 in HeLa transduced as in b . Five different confocal microscopy for each experiment were randomly chosen GFP - positive dots were examined on with identical brightness contrast settings . results are represented as average number of LC3 dots with 100 analysed per sample . Confocal are shown in Supplementary Fig . S3b .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Count of LC3 puncta in HeLa cells transduced as in b. Five different confocal microscopy images for each experiment were randomly chosen and GFP-positive dots were examined on the images with identical brightness and contrast settings. The results are represented as the average number of LC3 dots with 100 cells analysed per sample. Confocal images are shown in Supplementary Fig. S3b.


( d ) HEK293 were transfected with AMBRA1 FLAG - TRAF6 as indicated . extracts were immunoprecipitated using an anti - FLAG antibody .

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) HEK293 cells were transfected with AMBRA1 and FLAG-TRAF6 as indicated. Protein extracts were immunoprecipitated using an anti-FLAG antibody.


( e ) HeLa were transfected as in b FLAG - TRAF6 was purified as in d . of immunoprecipitated TRAF6 is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 0003 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected as in b and FLAG-TRAF6 was purified as in d. Quantification of immunoprecipitated TRAF6 is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.0003.


( f ) In HeLa , TRAF6 was downregulated by RNAi . AMBRA1 , ULK1 , TRAF6 , ATG13 actin were analysed by western blotting .

O O 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) In HeLa cells, TRAF6 was downregulated by RNAi. AMBRA1, ULK1, TRAF6, ATG13 and actin were analysed by western blotting.


( g ) HeLa were co - transfected with vectors encoding ULK1 - Myc , FLAG - TRAF6 Ub - Lys - 63 - HA , as indicated . extracts were immunoprecipitated using an anti - Myc antibody .

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(g) HeLa cells were co-transfected with vectors encoding ULK1-Myc, FLAG-TRAF6 and Ub-Lys-63-HA, as indicated. Protein extracts were immunoprecipitated using an anti-Myc antibody.


( h ) HeLa stably interfered for AMBRA1 were transfected with FLAG - TRAF6 Ub - Lys - 63 - HA as indicated . extracts were immunoprecipitated using anti - ULK1 antibody .

O O O O O O O O O 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) HeLa cells stably interfered for AMBRA1 were transfected with FLAG-TRAF6 and Ub-Lys-63-HA as indicated. Protein extracts were immunoprecipitated using anti-ULK1 antibody.


( i ) Endogenous ULK1 is immunoprecipitated from HeLa transfected as in b . resulting precipitates were subjected to a ULK1 kinase using MBP as substrate . Purified complexes were analysed by autoradiography by western blotting using an anti - ULK1 antibody .

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

(i) Endogenous ULK1 is immunoprecipitated from HeLa cells transfected as in b. The resulting precipitates were subjected to a ULK1 kinase assay using MBP as the substrate. Purified complexes were analysed by autoradiography and by western blotting using an anti-ULK1 antibody.


( a - c ) Two canonical TRAF6 - binding sites in AMBRA1 are essential for its interaction with TRAF6 . HEK293 were co - transfected with vectors encoding FLAG - TRAF6 together with AMBRA1WT mutant constructs . extracts were immunoprecipitated using an anti - FLAG antibody . AMBRA1 FLAG - TRAF6 were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-c) Two canonical TRAF6-binding sites in AMBRA1 are essential for its interaction with TRAF6. HEK293 cells were co-transfected with vectors encoding FLAG-TRAF6 together with AMBRA1WT and mutant constructs. Protein extracts were immunoprecipitated using an anti-FLAG antibody. AMBRA1 and FLAG-TRAF6 were analysed by western blotting.


( d ) HeLa were transfected with AMBRA1 RNAi oligonucleotide ( siRNA AMBRA1 ) . Then , were co - transfected with vectors encoding ubiquitin - HA AMBRA1WT or AMBRA1AA , respectively . extracts were immunoprecipitated using anti - ULK1 antibody ; ubiquitin - HA , ULK1 AMBRA1 were analysed by western blotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells were transfected with AMBRA1 RNAi oligonucleotide (siRNA AMBRA1). Then, cells were co-transfected with vectors encoding ubiquitin-HA and AMBRA1WT or AMBRA1AA, respectively. Protein extracts were immunoprecipitated using anti-ULK1 antibody; ubiquitin-HA, ULK1 and AMBRA1 were analysed by western blotting.


( e ) HeLa stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β - galactosidase ( negative control ) AMBRA1WT or AMBRA1AA respectively ; extracts were analysed by western blotting using anti - ULK1 , anti - AMBRA1 anti - ATG13 , anti - pATG13 anti - actin antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably interfered for AMBRA1 were transduced with lentiviral vectors encoding β-galactosidase (negative control) AMBRA1WT or AMBRA1AA respectively; protein extracts were analysed by western blotting using anti-ULK1, anti-AMBRA1 anti-ATG13, anti-pATG13 and anti-actin antibodies.


( f ) HeLa were transfected as in e some of them were treated with chloroquine for 1 h . LC3 , p62 , AMBRA1 actin were analysed by western blotting . of LC3II p62 are shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 03 for LC3 P = 0 . 045 for p62 .

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

(f) HeLa cells were transfected as in e and some of them were treated with chloroquine for 1 h. LC3, p62, AMBRA1 and actin were analysed by western blotting. Quantification of LC3II and p62 are shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.03 for LC3 and P = 0.045 for p62.


( g ) GFP - LC3 MEF from wild - type ( Ambra1 + / + ) homozygous ( Ambra1gt / gt ) were transduced with indicated lentiviral vectors of LC3 per is shown on right . Bars represent mean ± s . e . m . of triplicate samples with 50 analysed per sample ; n = 3 independent experiments . Significance is P = 0 . 001 P = 0 . 003 ( n = 3 ) . Scale bar , 10 μm .

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(g) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with the indicated lentiviral vectors and quantification of LC3 puncta per cells is shown on the right. Bars represent mean±s.e.m. of triplicate samples with 50 cells analysed per sample; n = 3 independent experiments. Significance is P = 0.001 and P = 0.003 (n = 3). Scale bar, 10 μm.


( a ) HEK293 were co - transfected with vectors encoding ULK1 - HA ULK1 - Myc then grown either in normal ( Ctrl ) or in starvation ( EBS ) medium for 1 h . extracts were immunoprecipitated using an anti - Myc antibody , analysed by western blotting using anti - HA , anti - Myc antibodies . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 001 .

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(a) HEK293 cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and then grown either in normal (Ctrl) or in starvation (EBS) medium for 1 h. Protein extracts were immunoprecipitated using an anti-Myc antibody, and analysed by western blotting using anti-HA, and anti-Myc antibodies. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.001.


( b ) HEK293 were transfected with TRAF6 RNAi oligonucleotide ( TRAF6 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , were co - transfected with vectors encoding ULK1 - HA ULK1 - Myc complexes were immunoprecipitated using anti - Myc antibody . extracts were analysed by western blotting using anti - HA , anti - Myc anti - TRAF6 antibodies . of ULK1 - HA relative to ULK1 - Myc is reported on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 037 .

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(b) HEK293 cells were transfected with TRAF6 RNAi oligonucleotide (TRAF6 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected with vectors encoding ULK1-HA and ULK1-Myc and protein complexes were immunoprecipitated using anti-Myc antibody. Protein extracts were analysed by western blotting using anti-HA, anti-Myc and anti-TRAF6 antibodies. Quantification of ULK1-HA relative to ULK1-Myc is reported on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.037.


( c ) HEK293 were transfected with AMBRA1 RNAi oligonucleotide ( AMBRA1 siRNA ) or unrelated oligonucleotides as a control ( Ctrl siRNA ) . Then , were co - transfected analysed as in a . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 044 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were transfected with AMBRA1 RNAi oligonucleotide (AMBRA1 siRNA) or unrelated oligonucleotides as a control (Ctrl siRNA). Then, cells were co-transfected and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.044.


( d ) HEK293 were co - transfected with vectors encoding for ULK1 - HA , ULK1 - Myc AMBRA1WT or AMBRA1AA respectively . complexes were immunoprecipitated analysed as in a . is shown on right . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 014 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) HEK293 cells were co-transfected with vectors encoding for ULK1-HA, ULK1-Myc and AMBRA1WT or AMBRA1AA respectively. Protein complexes were immunoprecipitated and analysed as in a. Quantification is shown on the right. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.014.


( a ) Highly purified mTOR was incubated with radiolabelled ATP without substrate , with purified FLAG - AMBRA1 purified FLAG - AMBRA1S52A analysed by autoradiography . lower panels show a FLAG mTOR western blot ( WB ) .

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

(a) Highly purified mTOR was incubated with radiolabelled ATP without substrate, with purified FLAG-AMBRA1 and purified FLAG-AMBRA1S52A and analysed by autoradiography. The lower panels show a FLAG and mTOR western blot (WB).


( b ) HEK293 expressing FLAG - RAPTOR were subjected to FLAG immunoprecipitation . complexes were analysed by western blotting using anti - FLAG anti - AMBRA1 antibodies .

O O O O O 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) HEK293 cells expressing FLAG-RAPTOR were subjected to FLAG immunoprecipitation. Protein complexes were analysed by western blotting using anti-FLAG and anti-AMBRA1 antibodies.


( c ) HEK293 were treated with Torin1 for 3 h or cultured in starvation medium for 2 h extracts were analysed by western blotting using anti - pSer - 52 anti - AMBRA1 antibodies .

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

(c) HEK293 cells were treated with Torin1 for 3 h or cultured in starvation medium for 2 h and protein extracts were analysed by western blotting using anti-pSer-52 and anti-AMBRA1 antibodies.


( d ) HeLa stably interfered for AMBRA1 were transduced with indicated lentiviral vectors encoding for β - galactosidase , AMBRA1WT or AMBRA1S52A respectively some of them were treated with chloroquine for 1 h . extracts were analysed by western blotting using anti - LC3 , anti - p62 anti - actin antibodies . of LC3II p62 bands is shown below . n = 3 extracts prepared from independent experiments . Data are presented as means ± s . d . significance is P = 0 . 038 .

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

(d) HeLa cells stably interfered for AMBRA1 were transduced with the indicated lentiviral vectors encoding for β-galactosidase, AMBRA1WT or AMBRA1S52A respectively and some of them were treated with chloroquine for 1 h. Protein extracts were analysed by western blotting using anti-LC3, anti-p62 and anti-actin antibodies. Quantification of LC3II and p62 bands is shown below. n = 3 extracts prepared from independent experiments. Data are presented as means±s.d. and significance is P = 0.038.


( e ) GFP - LC3 MEF from wild - type ( Ambra1 + / + ) homozygous ( Ambra1gt / gt ) were transduced with indicated lentiviral vectors of LC3 per is shown on right . Bars represent mean ± s . e . m . of triplicate samples 50 analysed per sample . n = 3 independent experiments . Significance is P = 0 . 001 P = 0 . 003 . Scale bar , 10

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) GFP-LC3 MEF cells from wild-type (Ambra1+/+) and homozygous (Ambra1gt/gt) embryos were transduced with indicated lentiviral vectors and quantification of LC3 puncta per cell is shown on the right. Bars represent mean±s.e.m. of triplicate samples and 50 cells analysed per sample. n = 3 independent experiments. Significance is P = 0.001 and P = 0.003. Scale bar, 10 μm


( f ) HeLa stably interfered for AMBRA1 were co - transfected with vectors encoding Ub - Lys - 63 - HA AMBRA1WT or AMBRA1S52A , respectively . extracts were immunoprecipitated using an anti - ULK1 antibody ubiquitin Lys - 63 - HA , ULK1 , AMBRA1 actin were analysed by western blotting .

(f) HeLa cells stably interfered for AMBRA1 were co-transfected with vectors encoding Ub-Lys-63-HA and AMBRA1WT or AMBRA1S52A, respectively. Protein extracts were immunoprecipitated using an anti-ULK1 antibody and ubiquitin Lys-63-HA, ULK1, AMBRA1 and actin were analysed by western blotting.


( g ) HeLa stably interfered for AMBRA1 were transduced as in d ; extracts were analysed by western blotting using anti - AMBRA1 , anti - ATG13 , anti - pATG13 anti - actin antibodies . of p - ATG13 band is shown below .

O O O O O O O O O O O O O O O O O O O O O O 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) HeLa cells stably interfered for AMBRA1 were transduced as in d; protein extracts were analysed by western blotting using anti-AMBRA1, anti-ATG13, anti-pATG13 and anti-actin antibodies. Quantification of the p-ATG13 band is shown below.


( h ) HeLa transfected with ULK1 - HA , ULK1 - Myc different AMBRA1 constructs , respectively . complexes were immunoprecipitated using anti - HA antibody analysed by western blotting using anti - HA , anti - Myc anti - AMBRA1 antibodies .

O O O O O O O O O O O O O O O O O O O O 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) HeLa cells transfected with ULK1-HA, ULK1-Myc and different AMBRA1 constructs, respectively. Protein complexes were immunoprecipitated using anti-HA antibody and analysed by western blotting using anti-HA, anti-Myc and anti-AMBRA1 antibodies.


( B ) Phase - contrast of wild - type or PABPC4KO HeLa depleted of PABPC1 by siRNA - mediated knockdown . Scale bar , 100 μm . ( C ) Total numbers of viable in ( B ) were quantified with acridine orange propidium iodide graphed as a of wild - type HeLa . Error bars represent of three biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Phase-contrast images of wild-type or PABPC4KO HeLa cells depleted of PABPC1 by siRNA-mediated knockdown. Scale bar, 100 μm. (C) Total numbers of viable cells in (B) were quantified with acridine orange and propidium iodide staining and graphed as a percentage of wild-type HeLa cells. Error bars represent the SEM of three biological replicates.


( C ) Phase - contrast of PABPDHFR , or PABPDHFR transfected with a plasmid coding for wild - type PABPC1 , 48 hours post - TMP removal . Scale bar , 100 μm .

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

(C) Phase-contrast images of PABPDHFR cells, or PABPDHFR cells transfected with a plasmid coding for wild-type PABPC1, 48 hours post-TMP removal. Scale bar, 100 μm.


( D ) Western blot of lysates derived from imaged in ( C ) .

(D) Western blot analysis of lysates derived from cells imaged in (C).


( B ) SUnSET of PABPDHFR grown in presence or absence of TMP for 12 hours to maintain or deplete PABP . were subsequently pulsed with either puromycin or puromycin cycloheximide ( control ) , lysed equal amounts were resolved by SDS - PAGE . Western blot was performed using a monoclonal antibody against puromycin or actin ( loading control ) .

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

(B) SUnSET assay of PABPDHFR cells grown in the presence or absence of TMP for 12 hours to maintain or deplete PABP. Cells were subsequently pulsed with either puromycin or puromycin and cycloheximide (control), lysed and equal protein amounts were resolved by SDS-PAGE. Western blot analysis was performed using a monoclonal antibody against puromycin or actin (loading control).


( D ) Scatter plot comparing log2 fold - changes [ ( - ) PABP vs ( + ) PABP ] in polysome - associated mRNA ( y - axis ) to corresponding changes in total mRNA . Transcripts identified as regulated via altered translation efficiency ( orange red ) or abundance ( green ) according to anota2seq are visualized together with non - regulated transcripts ( grey ) .

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

(D) Scatter plot comparing log2 fold-changes [(-) PABP vs (+) PABP] in polysome-associated mRNA (y-axis) to corresponding changes in total cytoplasmic mRNA. Transcripts identified as regulated via altered translation efficiency (orange and red) or abundance (light and dark green) according to anota2seq are visualized together with non-regulated transcripts (grey).


Boxplots of 5 ´ UTR ( A ) , 3 ´ UTR ( B ) lengths for mRNAs whose abundance decreased ( ' DOWN ' , green ) or increased ( ' UP ' , green ) in PABP - depleted as compared to expressing PABP . Solid horizonal middle lines in box plots denote median ; lower upper box limits correspond to first third quartiles ; whiskers extend from box limits to most extreme values . Wilcoxon - Mann - Whitney ( two - sided ) were used to determine differences between upregulated mRNAs downregulated mRNAs relative to each other to background ( mRNAs whose abundance did not change in absence of PABP relative to PABP - expressing ) .

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

Boxplots of 5´UTR (A), 3´UTR (B) lengths for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP. Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values. Wilcoxon-Mann-Whitney tests (two-sided) were used to determine differences between upregulated mRNAs and downregulated mRNAs relative to each other and to the background (mRNAs whose abundance did not change in the absence of PABP relative to PABP-expressing cells).


( A ) Boxplots of mRNA half - lives ( hrs ) assessed by metabolic labeling of normal human umbilical vein endothelial for mRNAs whose abundance decreased ( ' DOWN ' , green ) or increased ( ' UP ' , green ) in PABP - depleted as compared to expressing PABP comparing ( Tiana et al . , 2020 ) . Solid horizonal middle lines in box plots denote median ; lower upper box limits correspond to first third quartiles ; whiskers extend from box limits to most extreme values but not further than 1 . 5 × inter - quartile range ( IQR ) . ( B - H ) PABPDHFR were cultured in presence or absence of TMP for 12 hours . stabilities of select mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for indicated amounts of time . Total was isolated , reverse transcribed with random hexamer oligonucleotide primers quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in with zero time point set at 1 . Error bars represent of three independent experiments . First order exponential decay trend lines were generated by non - linear regression .

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

(A) Boxplots of mRNA half-lives (hrs) assessed by metabolic labeling of normal human umbilical vein endothelial cells for mRNAs whose abundance decreased ('DOWN', dark green) or increased ('UP', light green) in PABP-depleted cells as compared to cells expressing PABP comparing (Tiana et al., 2020). Solid horizonal middle lines in box plots denote the median; lower and upper box limits correspond to first and third quartiles; whiskers extend from box limits to the most extreme values but not further than 1.5× the inter-quartile range (IQR). (B- H) PABPDHFR cells were cultured in the presence or absence of TMP for 12 hours. The stabilities of select mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( B ) PABPDHFR were transfected with plasmids encoding RL - 6xB or RL - 6xBMUT . 24 hours after transfections , were split cultured for an additional 12 hours in presence or absence of TMP . stabilities of reporter mRNAs was assessed by using actinomycin D ( 5 μg / ml ) for indicated amounts of time . Total was isolated , reverse transcribed with random hexamer oligonucleotide primers quantified by qPCR . mRNA decay rates were normalized to an in vitro synthesized spike - in with zero time point set at 1 . Error bars represent of three independent experiments . First order exponential decay trend lines were generated by non - linear regression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PABPDHFR cells were transfected with plasmids encoding RL-6xB or RL-6xBMUT. 24 hours after transfections, the cells were split and cultured for an additional 12 hours in the presence or absence of TMP. The stabilities of reporter mRNAs was assessed by using actinomycin D (5 μg/ml) for the indicated amounts of time. Total RNA was isolated, reverse transcribed with random hexamer oligonucleotide primers and quantified by qPCR. mRNA decay rates were normalized to an in vitro synthesized spike-in RNA with the zero time point set at 1. Error bars represent the SEM of three independent experiments. First order exponential decay trend lines were generated by non-linear regression analysis.


( A ) Western blot PABPDHFR depleted of LSM1 , DCP2 or XRN1 by siRNA - mediated knockdown . siGFP represents a negative control .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1, DCP2 or XRN1 by siRNA-mediated knockdown. siGFP represents a negative control.


( B ) Phase - contrast of PABPDHFR previously transfected with siRNAs targeting GFP ( control ) , LSM1 , DCP2 or XRN1 cultured in presence or absence of TMP for 48 hours . Total numbers of viable , denoted in upper right corner of , were quantified with acridine orange propidium iodide calculated as a of PABPDHFR grown in presence of TMP , along with . Scale bar , 100 μm .

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(B) Phase-contrast images of PABPDHFR cells previously transfected with siRNAs targeting GFP (control), LSM1, DCP2 or XRN1 and cultured in the presence or absence of TMP for 48 hours. Total numbers of viable cells, denoted in the upper right hand corner of images, were quantified with acridine orange and propidium iodide and calculated as a percentage of PABPDHFR cells grown in the presence of TMP, along with the SEM. Scale bar, 100 μm.


( A ) Western blot PABPDHFR depleted of LSM1 by siRNA - mediated knockdown . Transfected were subsequently cultured in presence or absence of TMP to maintain or deplete endogenous PABP .

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(A) Western blot analysis PABPDHFR cells depleted of LSM1 by siRNA-mediated knockdown. Transfected cells were subsequently cultured in the presence or absence of TMP to maintain or deplete endogenous PABP.


( C D ) Poly ( A ) tail of GAPDH ACTB mRNAs from total isolated from PABPDHFR described in ( B ) was determined by extension poly ( A ) tail ( ePAT ) . PCR products were run on high - resolution agarose gels . ' A12 ' represents of ePAT RT - PCR amplicons derived from a mRNA with a fixed ( A12 ) - tail .

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(C and D) Poly(A) tail analysis of GAPDH and ACTB mRNAs from total RNA isolated from PABPDHFR cells described in (B) was determined by extension poly(A) tail (ePAT). PCR products were run on high-resolution agarose gels. 'A12' represents the size of ePAT RT-PCR amplicons derived from a mRNA with a fixed (A12)-tail.


( D - G ) mechanisms of C3H BMDMs transfected with miR - 342 - 3p mimic ( D ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( F ) , followed by Mtb infection for 36 hours . viabilities of C3H BMDMs transfected with miR - 342 - 3p mimic ( E ) , or B6 BMDMs transfected with miR - 342 - 3p inhibitor ( G ) , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . Data are shown as mean ± s . e . m . of n = 3 biological replicates .

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(D-G) Cell death mechanisms of C3H BMDMs transfected with miR-342-3p mimic (D), or B6 BMDMs transfected with miR-342-3p inhibitor (F), followed by Mtb infection for 36 hours. Cell viabilities of C3H BMDMs transfected with miR-342-3p mimic (E), or B6 BMDMs transfected with miR-342-3p inhibitor (G), followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 + / + C3H mice their wild - type littermate controls ( C3H ) ( n = 10 ) ( A ) , or Mir342 - / - B6 mice their wild - type littermate controls ( B6 ) ( n = 10 ) ( B ) after aerosol infection with around 400 CFU Mtb . Data are shown as Kaplan - Meier curves .

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(A, B) Survival of Mir342+/+C3H mice and their wild-type littermate controls (C3H) (n=10) (A), or Mir342-/-B6 mice and their wild-type littermate controls (B6) (n=10) (B) after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( G , H ) Secretion of cytokines TNF - α , IL - 1 , IL - 6 CXCL15 in BMDMs obtained from Mir342 + / + C3H their ( C3H ) ( G ) , or from Mir342 - / - B6 their ( B6 ) ( H ) after Mtb stimulation , was detected by ELISA . Data are shown as mean ± s . e . m . of n = 3 biological replicates .

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(G, H) Secretion of cytokines TNF-α, IL-1, IL-6 and CXCL15 in BMDMs obtained from Mir342+/+C3H and their littermates (C3H) (G), or from Mir342-/-B6 and their littermates (B6) (H) after Mtb stimulation, was detected by ELISA. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( C , D ) MiR - 342 - 3p mimic or inhibitor was transfected to NIH - 3T3 fibroblasts . After 48 hours , were collected for qRT - PCR ( C , data are shown as mean ± s . e . m . of n = 3 biological replicates ) western blotting ( D , representative blots from n = 3 biological replicates are shown ) to detect SOCS6 expression .

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(C, D) MiR-342-3p mimic or inhibitor was transfected to NIH-3T3 fibroblasts. After 48 hours, cells were collected for qRT-PCR (C, data are shown as the mean ± s.e.m. of n = 3 biological replicates) and western blotting (D, representative blots from n = 3 biological replicates are shown) to detect SOCS6 expression.


( F - H ) C3H BMDMs were transfected with miR - 342 - 3p mimic or SOCS6 - overexpressing lentivirus , followed by stimulation with Mtb or z - VAD ( 20 μM ) for 24 hours . mechanisms ( F ) , viabilities ( G ) Mtb growth rates ( H ) were analyzed respectively . Data are shown as mean ± s . e . m . of n = 3 biological replicates .

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(F-H) C3H BMDMs were transfected with miR-342-3p mimic or SOCS6-overexpressing lentivirus, followed by stimulation with Mtb or z-VAD (20 μM) for 24 hours. Cell death mechanisms (F), cell viabilities (G) and Mtb growth rates (H) were analyzed respectively. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( A , B ) Survival of Mir342 - / - B6 Mir342 - / - B6 mice supplemented with Socs6 siRNA ( n = 10 ) ( A ) , or Mir342 + / + C3H Mir342 + / + C3H mice supplemented with SOCS6 - overexpressing vector ( n = 10 ) ( B ) , after aerosol infection with around 400 CFU Mtb . Data are shown as Kaplan - Meier curves .

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(A, B) Survival of Mir342-/-B6 and Mir342-/-B6 mice supplemented with Socs6 siRNA (n=10) (A), or Mir342+/+C3H and Mir342+/+C3H mice supplemented with SOCS6-overexpressing vector (n=10) (B), after aerosol infection with around 400 CFU Mtb. Data are shown as the Kaplan-Meier curves.


( C - F ) Relative expressions of chemokines CCL5 , CXCL10 , ICAM1 ( C ) , Caspase 3 , Caspase 7 , Caspase 8 ( F ) in Mtb - stimulated Socs6 + / + or Socs6 - / - BMDMs were detected by qRT - PCR . Data are shown as mean ± s . e . m . of n = 3 biological replicates . Cluster of clinical samples .

(C-F) Relative expressions of chemokines CCL5, CXCL10, ICAM1 (C), and Caspase 3, Caspase 7, Caspase 8 (F) in Mtb-stimulated Socs6+/+ or Socs6-/- BMDMs were detected by qRT-PCR. Data are shown as the mean ± s.e.m. of n = 3 biological replicates. Cluster analysis of clinical samples.


( C ) Socs6 - / - BMDMs were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant for 24 hours . Afterwards , transfected were stimulated with Mtb for 12 hours , lysates were collected immunoprecipitated using an anti - RIPK1 antibody . recruitment of Caspase 8 , RIPK3 , FADD MLKL was analyzed by immunoblot . lower panel represents immunoblot of whole lysates . Representative blots from n = 3 biological replicates are shown .

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(C) Socs6-/- BMDMs were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant for 24 hours. Afterwards, transfected cells were stimulated with Mtb for 12 hours, cell lysates were collected and immunoprecipitated using an anti-RIPK1 antibody. The recruitment of Caspase 8, RIPK3, FADD and MLKL was analyzed by immunoblot. The lower panel represents the immunoblot analysis of whole cell lysates. Representative blots from n = 3 biological replicates are shown.


( D - F ) viabilities ( D ) , mechanisms ( E ) , or Mtb growth rates ( F ) of Mtb - infected Socs6 - / - BMDMs that were transfected with plasmids expressing Myc - RIPK3 or Myc - RIPK3 K51A mutant as indicated . Z - VAD treatment was 20 μM treatment time was 24 hours . Data are shown as mean ± s . e . m . of n = 3 biological replicates .

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(D-F) Cell viabilities (D), cell death mechanisms (E), or Mtb growth rates (F) of Mtb-infected Socs6-/- BMDMs that were transfected with plasmids expressing Myc-RIPK3 or Myc-RIPK3 K51A mutant as indicated. Z-VAD treatment concentration was 20 μM and the treatment time was 24 hours. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( E - G ) RAW264 . 7 were transfected with plasmids expressing A20 RIPK3 ( E ) , or A20 RIPK3 mutants ( G ) as indicated , lysates were collected for immunoprecipitation immunoblot . Myc - A20 or Flag - RIPK3 purified from transfected HEK - 293T was incubated with ATP , E1 , E2 ubiquitin as indicated . in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti - Ub antibody ( F ) . Representative blots from n = 3 biological replicates are shown .

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(E-G) RAW264.7 cells were transfected with plasmids expressing A20 and RIPK3 (E), or A20 and RIPK3 mutants (G) as indicated, cell lysates were collected for immunoprecipitation and immunoblot. Myc-A20 or Flag-RIPK3 purified from transfected HEK-293T cells was incubated with ATP, E1, E2 and ubiquitin as indicated. The in vitro ubiquitination of RIPK3 was analyzed by immunoblot using an anti-Ub antibody (F). Representative blots from n = 3 biological replicates are shown.


( A , B ) Production of cytokines TNF - α , IL - 1 , IL - 6 , CXCL15 ( A ) , or chemokines Ccl5 , Cxcl10 , Icam1 ( B ) in A20 - / - Socs6 + / + , A20 + / + Socs6 + / + , A20 - / - Socs6 - / - A20 + / + Socs6 - / - BMDMs after Mtb stimulation . Data are shown as mean ± s . e . m . of n = 3 biological replicates .

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(A, B) Production of cytokines TNF-α, IL-1, IL-6, CXCL15 (A), or chemokines Ccl5, Cxcl10, Icam1 (B) in the A20-/-Socs6+/+, A20+/+Socs6+/+, A20-/-Socs6-/- and A20+/+Socs6-/- BMDMs after Mtb stimulation. Data are shown as the mean ± s.e.m. of n = 3 biological replicates.


( B ) infectivity of mouse tail fibroblasts ( MTFs ) infected with WT , RH , RHEV EV CHIKV ( MOI 10 , n = 3 per group ) harvested at 12 hpi . Data are presented as means ± .

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(B) Percentage infectivity of mouse tail fibroblasts (MTFs) infected with WT, RH, RHEV and EV CHIKV (MOI 10, n=3 per group) harvested at 12 hpi. Data are presented as the means ± SEM.


( C ) Viremia progression in - infected mice was monitored over 2 ( n = 11 per group ) . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

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(C) Viremia progression in virus-infected mice was monitored over 2 weeks (n=11 per group). Viremia is detected with CHIKV nsP1 probe via qRT-PCR. Statistical


( D ) joint inflammation in - infected mice was monitored over 2 ( n = 11 per group ) .

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(D) joint inflammation in virus-infected mice was monitored over 2 weeks (n=11 per group).


( E ) Compiled percentages of CHIKV - infected ( ZsGreen positive ) leukocytes at 2 days post infection ( dpi ) for respective groups ( n = 5 per group ) . Background ZsGreen signals are indicated by dotted .

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(E) Compiled percentages of CHIKV-infected (ZsGreen positive) leukocytes at 2 days post infection (dpi) for the respective groups (n=5 per group). Background ZsGreen signals are indicated by the dotted line.


( F ) Dot - plots of localized type - I interferon ( IFN - α IFN - β ) concentrations at 15 hpi . Data are expressed as means ± SD .

(F) Dot-plots of the localized type-I interferon (IFN-α and IFN-β) concentrations at 15 hpi. Data are expressed as the means ± SD.


lysates were collected from joints of mice infected with WT CHIKV or CHIKV nsP - mutants at 15 hpi , 2 dpi or 6 dpi ( n = 5 per group ) . concentrations of 36 immune mediators were quantified using a 36 - plex microbead - immunoassay . Immune mediators are grouped on function . Each color represents relative of a particular analyte . Blue red indicates low high , respectively .

Cell lysates were collected from the joints of mice infected with WT CHIKV or CHIKV nsP-mutants at 15 hpi, 2 dpi or 6 dpi (n=5 per group). The concentrations of 36 immune mediators were quantified using a 36-plex microbead-based immunoassay. Immune mediators are grouped based on function. Each color represents the relative concentration of a particular analyte. Blue and red indicates low and high concentration, respectively.


Luminex analytes from WT CHIKV - infected joint lysates are categorized on fold change states ( up , down at 2 dpi relative to mock , 6 dpi relative to 2 dpi . numbers in represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from - sequencing ( - seq ) data appeared predominantly in ( A ) up then down state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) ,

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Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states (up, down at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (A) up then down state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively),


study analytes transcripts in - seq data from wild - type CHIKV - infected mock - infected samples in same states were used . ( C ,

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The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. (C,


Luminex analytes from WT CHIKV - infected joint lysates are categorized on fold change states no difference ( ND ) ) at 2 dpi relative to mock , 6 dpi relative to 2 dpi . numbers in represent Log2 fold changes of analytes or genes . Analytes in complete or partial agreement with corresponding gene expression values derived from - sequencing ( - seq ) data appeared predominantly in ( D ) up then ND state ( Mock to 2 dpi , then 2 dpi to 6 dpi , respectively ) . † indicates analyte involvement in canonical pathways . Differences in analyte concentrations during attenuated CHIKV infection are indicated in checkbox . Yellow represents lower while green represents higher concentrations relative to WT CHIKV .

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Luminex analytes from WT CHIKV-infected joint cell lysates are categorized based on fold change states no difference (ND)) at 2 dpi relative to mock, and 6 dpi relative to 2 dpi. The numbers in the heatmap represent Log2 fold changes of analytes or genes. Analytes in complete or partial agreement with corresponding gene expression values derived from RNA-sequencing (RNA-seq) data appeared predominantly in the (D) up then ND state (Mock to 2 dpi, then 2 dpi to 6 dpi, respectively). † indicates analyte involvement in the canonical pathways. Differences in analyte concentrations during attenuated CHIKV infection are indicated in the checkbox. Yellow represents lower while green represents higher concentrations relative to WT CHIKV.


study analytes transcripts in - seq data from wild - type CHIKV - infected mock - infected samples in same states were used . F )

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The study analytes and transcripts in the RNA-seq data from wild-type CHIKV-infected and mock-infected samples in the same states were used. F)


Joints from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV mock - infected WT C57BL / 6 mice were harvested at 6 dpi . ( A ) Absolute numbers of leukocytes present in joints as determined by immune - phenotyping . Data are presented as means ± SD ( n = 5 per group ) . No significance was obtained between groups by Kruskal - Wallis with Dunn ' s multiple comparisons .

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Joints from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected WT C57BL/6 mice were harvested at 6 dpi. (A) Absolute numbers of leukocytes present in the joints as determined by immune-phenotyping. Data are presented as the means ± SD (n=5 per group). No significance was obtained between the groups by Kruskal-Wallis test with Dunn's multiple comparisons.


( B ) Jensen - Shannon divergence showing level of divergence between infected groups .

(B) Jensen-Shannon divergence heatmap showing the level of divergence between infected groups.


( C ) Superimposed PhenoGraphs of UMAP transformed FACs data from immune - phenotyping of joint from WT - CHIKV , RH - CHIKV , RHEV - CHIKV , EV - CHIKV mock - infected ( n = 5 per group ) .

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(C) Superimposed PhenoGraphs of UMAP transformed FACs data from immune-phenotyping of joint cells from WT-CHIKV, RH-CHIKV, RHEV-CHIKV, EV-CHIKV and mock-infected animals (n=5 per group).


( E ) Cluster ID annotation with . Cluster IDs are indicated in rows , while surface marker expression are indicated by columns . color represents relative mean ( MFI ) of a surface marker . Blue red represents low high MFI , respectively . Red square ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi .

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(E) Cluster ID annotation with heatmap. Cluster IDs are indicated in rows, while surface marker expression levels are indicated by the columns. The color represents the relative mean fluorescence intensity (MFI) of a surface marker. Blue and red represents low and high MFI, respectively. Red square and ovals highlight differences of several PhenoGraph clusters in infected joints at 6 dpi.


( A ) Anti - CHIKV IgG titers from vaccination phase were determined with a CHIKV virion - ELISA . Sera from four were pooled diluted 1 : 500 for . Statistical analyses were performed using two - paired T - ( * P = 0 . 029 RH - CHIKV 6 dpv , * P = 0 . 029 RH - CHIKV 90 dpv , * P = 0 . 029 RHEV - CHIKV 6 dpv , * P = 0 . 029 RHEV - CHIKV 90 dpv , * P = 0 . 029 EV - CHIKV 90 dpv ) .

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(A) Anti-CHIKV IgG titers from the vaccination phase were determined with a CHIKV virion-based ELISA. Sera from four animals were pooled and diluted 1:500 for the assay. Statistical analyses were performed using two-tailed paired T-test (*P =0.029 RH-CHIKV 6 dpv, *P =0.029 RH-CHIKV 90 dpv, *P =0.029 RHEV-CHIKV 6 dpv, *P =0.029 RHEV-CHIKV 90 dpv, *P =0.029 EV-CHIKV 90 dpv).


( B ) Half maximum inhibitory ( IC50 ) of pooled sera collected on 90 dpv were characterized in HEK293T infected with WT - CHIKV ( MOI 5 ) . Sera were serially diluted at 1 : 625000 , 1 : 125000 , 1 : 25000 , 1 : 16000 , 1 : 8000 , 1 : 5000 , 1 : 4000 , 1 : 2000 , 1 : 1000 , 1 : 500 , 1 : 250 . Sera dilution to obtain IC50 are shown in parenthesis .

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(B) Half maximum inhibitory concentration (IC50) of pooled sera collected on 90 dpv were characterized in HEK293T cells infected with WT-CHIKV (MOI 5). Sera were serially diluted at 1:625000, 1:125000, 1:25000, 1:16000, 1:8000, 1:5000, 1:4000, 1:2000, 1:1000, 1:500, 1:250. Sera dilution to obtain IC50 are shown in parenthesis.


Viremia were monitored over 2 . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

Viremia were monitored over 2 weeks. Viremia is detected with CHIKV nsP1 probe via qRT-PCR.


( E ) severity of joint inflammation were monitored over 2 .

(E) severity of joint inflammation were monitored over 2 weeks.


( A ) Representative hematoxylin eosin ( H & E ) of inflamed joint footpad at 6 dpc . Ed , region of edema ; red arrow , synovitis ; black arrow , normal synovial ; blue arrow , mild synovial hyperplasia ; D , degeneration of muscle ; N , necrosis of muscle ; R , regeneration of muscle .

(A) Representative hematoxylin and eosin (H&E) images of inflamed joint footpad at 6 dpc. Ed, region of edema; red arrow, synovitis; black arrow, normal synovial membrane; blue arrow, mild synovial hyperplasia; D, degeneration of muscle; N, necrosis of muscle; R, regeneration of muscle.


( B ) Histopathological scoring of edema , inflammation in different regions of joint footpad muscle pathology of WT - CHIKV challenged ( n = 5 ) at 6 dpc , performed blind . Three sections from each joint footpad were scored . data are expressed as means ± SD . Data were analyzed by one - way ANOVA with Tukey post - ; Edema ( * * * P = 0 . 000002 WT - CHIKV versus PBS , * * * P = 0 . 000004 RH - CHIKV versus PBS ) , Muscle necrosis ( * * P = 0 . 005521 WT - CHIKV versus PBS , * P = 0 . 02628 RH - CHIKV versus PBS ) , Muscle regeneration ( * * * P < 0 . 000001 WT - CHIKV versus PBS , * * * P < 0 . 000001 WT - CHIKV versus RH - CHIKV ) , Muscle inflammation ( * P = 0 . 040258 WT - CHIKV versus PBS , * P = 0 . 040258 RH - CHIKV versus PBS ) , Synovial inflammation ( * P = 0 . 014457 WT - CHIKV versus PBS , * P = 0 . 014457 RH - CHIKV versus PBS ) , Subcutaneous inflammation ( * * * P = 0 . 00004 WT - CHIKV versus PBS , * * * P = 0 . 000074 RH - CHIKV versus PBS ) .

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(B) Histopathological scoring of edema, inflammation in different regions of the joint footpad and muscle pathology of WT-CHIKV challenged animals (n=5) at 6 dpc, performed blind. Three sections from each joint footpad were scored. The data are expressed as the means ± SD. Data were analyzed by one-way ANOVA with Tukey post-test; Edema (***P =0.000002 WT-CHIKV versus PBS, ***P =0.000004 RH-CHIKV versus PBS), Muscle necrosis (**P =0.005521 WT-CHIKV versus PBS, *P =0.02628 RH-CHIKV versus PBS), Muscle regeneration (***P <0.000001 WT-CHIKV versus PBS, ***P <0.000001 WT-CHIKV versus RH-CHIKV), Muscle inflammation (*P =0.040258 WT-CHIKV versus PBS, *P =0.040258 RH-CHIKV versus PBS), Synovial membrane inflammation (*P =0.014457 WT-CHIKV versus PBS, *P =0.014457 RH-CHIKV versus PBS), Subcutaneous area inflammation (***P =0.00004 WT-CHIKV versus PBS, ***P =0.000074 RH-CHIKV versus PBS).


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at metatarsal region of footpad . ( A ) Viremia progression in ONNV - infected mice were monitored over 2 ( n = 5 ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

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Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (A) Viremia progression in ONNV-infected mice were monitored over 2 weeks (n=5). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


Three - week - old WT C57BL / 6 mice were infected with 1E6 PFU of WT - ONNV at metatarsal region of footpad . ( B ) joint inflammation in ONNV - infected mice were monitored over 2 ( n = 5 ) .

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Three-week-old WT C57BL/6 mice were infected with 1E6 PFU of WT-ONNV at the metatarsal region of the footpad. (B) joint inflammation in ONNV-infected mice were monitored over 2 weeks (n=5).


Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at metatarsal region of footpad . Challenge with 1E6 PFU of WT - ONNV was performed via same route at 3 months post - vaccination . Viremia progression of mice were monitored over 2 ( n = 5 per group ) . Viremia is detected with ONNV nsP1 probe via qRT - PCR .

Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. Viremia progression of the mice were monitored over 2 weeks (n=5 per group). Viremia is detected with ONNV nsP1 probe via qRT-PCR.


D ) Three - week - old WT C57BL / 6 mice were vaccinated subcutaneously with 1E6 PFU of WT - CHIKV or attenuated CHIKV ( RH ) at metatarsal region of footpad . Challenge with 1E6 PFU of WT - ONNV was performed via same route at 3 months post - vaccination . ( D ) joint inflammation of mice were monitored over 2 ( n = 5 per group ) .

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D) Three-week-old WT C57BL/6 mice were vaccinated subcutaneously with 1E6 PFU of WT-CHIKV or attenuated CHIKV (RH) at the metatarsal region of the footpad. Challenge with 1E6 PFU of WT-ONNV was performed via the same route at 3 months post-vaccination. (D) joint inflammation of the mice were monitored over 2 weeks (n=5 per group).


( A ) C - circle for samples . presence of C - circles was tested in 22 osteosarcomas by Rolling Circle Amplification ( RCA ) using Φ29 polymerase . Φ29 polymerase negative controls Φ29 polymerase - reactions starting respectively with 75 , 37 . 5 , 18 . 75 ng of were applied to dot blots C - circles were detected by hybridization with a 32P - ( CCCTAA ) 4 telomeric probe . U2OS HeLa correspond to positive negative control samples , respectively .

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(A) C-circle assay for tumor samples. The presence of C-circles was tested in 22 osteosarcomas by Rolling Circle Amplification (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA were applied to dot blots and C-circles were detected by hybridization with a 32P-(CCCTAA)4 telomeric probe. U2OS and HeLa cells correspond to positive and negative control samples, respectively.


( B ) Representative of telomere FISH ( red ) PML IF ( green ) colocalizations ( APBs ) in osteosarcoma samples . is counterstained blue with DAPI . APBs are indicated by arrows . Scale bars are 5 μm .

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(B) Representative images of telomere FISH (red) and PML IF (green) colocalizations (APBs) in osteosarcoma tumor samples. DNA is counterstained blue with DAPI. APBs are indicated by white arrows. Scale bars are 5 μm.


( C ) Representative image of TeSLA Southern Blot of two representative samples of ALT - positive ( left ) telomerase - positive ( right ) osteosarcomas . Nine TeSLA PCRs ( 30 pg each reaction ) were done for each sample . DIG - labelled MW ladder has been added a posteriori . ( D ) Plot of cumulative TeSLA fragment sizes ( in kb ) in ALT - positive ( n = 16 ) ALT - negative ( n = 6 ) osteosarcomas . Points error bars ( ± ) represent cumulative of TeSLA fragments from TeSLA PCRs ( n = 9 per sample ) from in both groups ( n = 16 in ALT - positive group , n = 6 in ALT - negative group ) . Kolmogorov - Smirnov was applied to identify statistical differences in TeSLA fragments distributions ( p = 0 . 0016 ) . ( E ) TeSLa fragment lengths in ALT - positive ( n = 16 ) ALT - negative ( n = 6 ) osteosarcomas . Each dot represents a TeSLA fragment , bars represent median value for ' s samples .

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(C) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs (30 pg each reaction) were done for each DNA sample. DIG-labelled MW ladder has been added a posteriori. (D) Plot of cumulative TeSLA fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs (n=9 per tumor sample) from all the patients in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA fragments distributions (p=0.0016). (E) Individual TeSLa fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA fragment, bars represent the median value for individual patient's samples.


( A ) Oncoprint graph showing distribution of mutations ( green rectangles ) copy number variations ( red rectangle , amplification ; blue rectangle , copy loss ) in samples . Tumors are distributed according to ALT - positivity ( ALT + ) or - negativity ( ALT - ) ATRX status ( ATRX - mutated or ATRX - wt ) .

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(A) Oncoprint graph showing the distribution of mutations (green rectangles) and copy number variations (red rectangle, amplification; blue rectangle, copy loss) in all samples. Tumors are distributed according to ALT-positivity (ALT+) or -negativity (ALT-) and ATRX status (ATRX-mutated or ATRX-wt).


( B ) Tissue sections of two representative high - grade osteosarcomas with HES ( hematoxylin eosin saffron ) ( left panel ) , anti - ATRX ( HPA064684 ) immunochemistry ( middle panel ) , anti - DAXX ( HPA008736 ) immunochemistry ( right panel ) , showing ATRX expression in two ALT - positive samples . In top panel , intratumoral ATRX expression is negative ( positive control osteoclasts are shown ) . In bottom panel , intratumoral ATRX expression is high . Scale bars are 250 μm .

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(B) Tissue sections of two representative high-grade osteosarcomas with HES (hematoxylin eosin saffron) staining (left panel), anti-ATRX (HPA064684) immunochemistry (middle panel), and anti-DAXX (HPA008736) immunochemistry (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX expression is negative (positive control osteoclasts are shown). In the bottom panel, intratumoral ATRX expression is high. Scale bars are 250 μm.


( A ) Copy - number - alteration ( CNA ) profiles of osteosarcomas . Representation of GISTIC with false discovery rate ( FDR ( q - value ) ) for cases ( n = 22 ; left panel ) , ALT - positive / ATRX - wt cases ( n = 11 ; middle panel ) , ALT - positive / ATRX - mutated cases ( n = 5 ; right panel ) .

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(A) Copy-number-alteration (CNA) profiles of osteosarcomas. Representation of the GISTIC analysis with false discovery rate (FDR (q-value)) for all cases (n=22; left panel), ALT-positive/ATRX-wt cases (n=11; middle panel), and ALT-positive/ATRX-mutated cases (n=5; right panel).


( B ) Gain / amplification of TOP3A gene region ( 17p11 ) is a genomic signature of ATRX - wt osteosarcomas . For each ATRX - mutated ( top part ) ATRX - wt ( bottom part ) case , from left to right , chromosome 17 regional genomic profiles were established , with CGH Analytics software . A focus was made within genomic interval ( 17 . 25 - 19 . 11 Mb ) of short arm of chromosome 17 ( hg38 human genome mapping ; Build 38 from NCBI , December 2013 version ) including TOP3 . Color profiles corresponding to different tumors are defined at top of each group . Most ATRX - wt cases show gain or amplification of this region ( bottom part ) , whereas no ATRX - mutated cases display this alteration ( top part ) .

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(B) Gain/amplification of TOP3A gene region (17p11) is a genomic signature of ATRX-wt osteosarcomas. For each ATRX-mutated (top part) and ATRX-wt (bottom part) case, from the left to the right, the chromosome 17 and regional genomic profiles were established, with CGH Analytics software. A focus was made within the genomic interval (17.25-19.11 Mb) of the short arm of chromosome 17 (hg38 human genome mapping; Build 38 from NCBI, December 2013 version) including TOP3. Color profiles corresponding to the different tumors are defined at the top of each group. Most ATRX-wt cases show gain or amplification of this region (bottom part), whereas no ATRX-mutated cases display this alteration (top part).


( C ) Chromosome 17 copy number variation showing amplification / gain of TOP3A region that do not extend into TP53 gene in ALT ATRX - wt tumors ( left panel ) , amplification of TOP3A region for which TP53 region is at edge of amplification / gain in ALT - positive ATRX - wt tumors ( right panel ) .

(C) Chromosome 17 copy number variation analysis showing amplification/gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX-wt tumors (left panel), and amplification of the TOP3A region for which TP53 region is at the edge of the amplification/gain in ALT-positive ATRX-wt tumors (right panel).


( A ) Volcano plot of supervised of mRNA expression profiles of osteosarcomas according to ALT - positive vs . ALT - negative samples , showing upregulated ( in red ) downregulated ( in green ) genes . Twelve tumors had sufficient quality to be analyzed : seven ALT - positive ATRX - wt , two ALT - positive / ATRX - mutated three ALT - negative .

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(A) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT-positive vs. ALT-negative samples, showing upregulated (in red) and downregulated (in green) genes. Twelve tumors had sufficient RNA quality to be analyzed: seven ALT-positive ATRX-wt, two ALT-positive/ATRX-mutated and three ALT-negative.


( B ) TOP3A gene expression according to ALT ATRX status : ALT + / ATRX - wt ( n = 7 ) , ALT + / ATRX - mut ( n = 2 ) ALT - ( n = 3 ) . Box - - whisker plot were defined with default parameters by median value ( central band at 50th percentile ) , interquartile ranges ( IQR , box limited by 25th 75th percentile ) whisker boundaries defined by minimum maximum value . An ANOVA statistical , Tukey ' s range were used to compare modalities .

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(B) TOP3A gene expression according to ALT and ATRX status: ALT+/ATRX-wt (n=7), ALT+/ATRX-mut (n=2) and ALT- (n=3). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined by minimum and maximum value. An ANOVA statistical test, and Tukey's range test were used to compare modalities.


( A ) Western blot showing ATRX expression in nine osteosarcoma lines HTC116 as positive control .

(A) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control.


( B ) Western blot showing TOP3A expression in nine osteosarcoma lines NY siTOP3A as control .

(B) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control.


( D ) C - circle showing inverse effect of ATRX ectopic expression on C - circle in ATRX - mutated ATRX - wt lines . Error bars represent mean ± from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney .

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(D) C-circle assay showing inverse effect of ATRX ectopic expression on C-circle levels in ATRX-mutated and ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test.


( E ) C - circle showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma Error bars represent mean ± from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney . Western blot of TOP3A expression is shown for U2OS overexpressing TOP3A .

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(E) C-circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test. Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A.


( B ) Representative of telomeric ( green ) PML ( red ) colocalizations ( APBs ) in osteosarcoma in vitro - immortalized ATRX - mutated or ATRX - wt lines according to TOP3A KD . Scale bars are 5 μm . ( C ) of APB frequency in osteosarcoma in vitro - immortalized ATRX - mutated or ATRX - wt lines according to TOP3A KD ; Error bars represent mean ± from n = 3 experiments , n = 150 scored per treatment , n . s . , non - significant ; Mann - Whitney . ( D ) Effect of TOP3A KD on Telomere Dysfunction - Induced Foci ( TIFs ) in osteosarcoma in vitro - immortalized lines ; Error bars represent mean ± from n = 3 experiments , n = 150 scored per treatment , * p < 0 . 05 , * * p < 0 . 005 ; Mann - Whitney .

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(B) Representative images of telomeric DNA (green) and PML protein (red) colocalizations (APBs) in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD. Scale bars are 5 μm. (C) Quantification of APB frequency in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s., non-significant; Mann-Whitney test. (D) Effect of TOP3A KD on Telomere Dysfunction-Induced Foci (TIFs) in osteosarcoma and in vitro-immortalized cell lines; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, *p < 0.05, **p < 0.005; Mann-Whitney test.


( E ) Distributions of TeSLA fragments in osteosarcoma in vitro - immortalized ATRX - mutated or ATRX - wt lines according to TOP3A KD ( left panel ; each dot represents a TeSLA fragment ) , representative TeSLA Southern Blot image for NY ( right panel ) .

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(E) Distributions of TeSLA fragments in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD (left panel; each dot represents a TeSLA fragment), and representative TeSLA Southern Blot image for NY (right panel).


( A ) FISH - IF of telomeres ( in red ) BLM ( in green ) in TOP3A - depleted BLM foci at telomeres are indicated by arrows . Error bars represent mean ± from n = 3 experiments , n = 150 scored per treatment , n . s . = non - significant , * * p < 0 . 01 , Mann - Whitney . Scale bars are 5 μm .

(A) FISH-IF of telomeres (in red) and BLM (in green) in TOP3A-depleted cells BLM foci at telomeres are indicated by white arrows. Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s. = non-significant, **p < 0.01, Mann-Whitney test. Scale bars are 5 μm.


( B ) ATSA assessing ALT - mediated telomeric synthesis ( telomere in red , EdU in purple ) in PML ( in green ) according to TOP3A KD . EdU signal at APB ( telomere / PML ) are indicated by arrows . Approximately 150 were divided into five groups ( 0 , 1 - 2 , 3 - 4 , 5 - 6n ≥ 7 ) on number of EdU + APBs . Scale bars are 5 μm .

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(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere in red, EdU in purple) in PML (in green) according to TOP3A KD. EdU signal at APB (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.


Representative low ( left ) high ( right ) power of H & E - stained lung sections from KrasG12D , KrasG12D : Adam17ex / + KrasG12D : Adam17ex / ex mice at 6 post Ad - Cre inhalation . Scale bars , 3mm ( left ) 300μm ( right ) . of lung parenchyma occupied by lesions ( B ) , incidence ( C ) , per whole mouse lung in indicated genotypes ( n = 6 per genotype ) .

Representative low (left) and high (right) power images of H&E-stained lung sections from KrasG12D, KrasG12D:Adam17ex/+ and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre inhalation. Scale bars, 3mm (left) and 300μm (right). Quantification of lung parenchyma area occupied by tumor lesions (B), and tumor incidence (C), per whole mouse lung in the indicated genotypes (n = 6 per genotype).


Representative of TTF - 1 - stained lung sections from indicated genotypes . Scale bar , 100μm . of TTF - 1 - positive / high power field ( HPF ) in lungs of indicated genotypes ( n = 6 per genotype ) .

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Representative images of TTF-1-stained lung sections from the indicated genotypes. Scale bar, 100μm. Quantification of TTF-1-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).


Representative of PCNA - stained lung sections from KrasWT , KrasG12D KrasG12D : Adam17ex / ex mice at 6 post Ad - Cre ( for KrasG12D ) or vehicle ( for KrasWT ) inhalation . Scale bar , 100μm . of PCNA - positive / high power field ( HPF ) in indicated mouse lungs ( n = 6 per genotype ) .

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Representative images of PCNA-stained lung sections from KrasWT, KrasG12D and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre (for KrasG12D) or vehicle (for KrasWT) inhalation. Scale bar, 100μm. Quantification of PCNA-positive cells/high power field (HPF) in the indicated mouse lungs (n = 6 per genotype).


qPCR expression analyses of cycle regulation genes ( normalized against 18SrRNA ) in lungs from indicated mice ( n = 6 per genotype ) . *

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qPCR expression analyses of cell cycle regulation genes (normalized against 18SrRNA) in lungs from the indicated mice (n = 6 per genotype). *


Representative immunoblots of KrasG12D KrasG12D : Adam17ex / ex lung lysates with indicated antibodies . Each lane is an mouse sample . Semi - quantitative densitometry of Myc ( relative to Actin ) in lung lysates from ( D ) . n = 6 per genotype .

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Representative immunoblots of KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies. Each lane is an individual mouse sample. Semi-quantitative densitometry of Myc protein levels (relative to Actin) in lung lysates from (D). n = 6 per genotype.


Representative ( F ) of CD45 - positive / HPF ( G ) in CD45 - stained lung sections from indicated genotypes at 6 post inhalations . In ( F ) , scale bar , 100μm . In ( G ) , n = 6 per genotype .

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Representative images (F) and quantification of CD45-positive cells/HPF (G) in CD45-stained lung sections from the indicated genotypes at 6 weeks post inhalations. In (F), scale bar, 100μm. In (G), n = 6 per genotype.


Representative immunofluorescence of lung sections of KrasG12D mice ( 6 post Ad - Cre inhalation ) co - stained for ADAM17 markers for club ( CC10 ) ( A ) , alveolar type II ( surfactant - C , SP - C ) ( B ) type I ( Podoplanin , Pdpn ) ( C ) , total immune ( CD45 ) ( D ) endothelial ( CD31 ) ( E ) . DAPI is blue . Scale bars , 100μm . Arrowheads in merged indicate dual - positive ADAM17 - expressing . of immunofluorescence from A - E ( n = 5 mice per stain ) .

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Representative immunofluorescence images of lung sections of KrasG12D mice (6 weeks post Ad-Cre inhalation) co-stained for ADAM17 and markers for club cells (CC10) (A), alveolar type II (surfactant protein-C, SP-C) (B) and type I (Podoplanin, Pdpn) (C) cells, total immune cells (CD45) (D) and endothelial cells (CD31) (E). DAPI nuclear staining is blue. Scale bars, 100μm. Arrowheads in merged images indicate dual-positive ADAM17-expressing cells. Quantification of immunofluorescence staining from A-E (n = 5 mice per stain).


Representative of H & E - stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D ( KrasKras ) or KrasG12D : Adam17ex / ex bone marrow ( KrasKras : ex / ex ) . Scale bar , 3mm . of lung parenchyma occupied by lesions in indicated bone marrow chimeras ( n = 6 mice per group ) .

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Representative images of H&E-stained lung sections from KrasG12D recipient mice reconstituted with KrasG12D (KrasKras) or KrasG12D:Adam17ex/ex bone marrow (KrasKras:ex/ex). Scale bar, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated bone marrow chimeras (n = 6 mice per group).


Representative of pADAM17 - stained lung sections from KrasWT KrasG12D ( 6 post inhalation ) mice . Scale bar , 100μm . of pADAM17 - positive / high power field ( HPF ) in lungs of KrasWT KrasG12D mice ( n = 6 per genotype ) .

Representative images of pADAM17-stained lung sections from KrasWT and KrasG12D (6 weeks post inhalation) mice. Scale bar, 100μm. Quantification of pADAM17-positive cells/high power field (HPF) in lungs of KrasWT and KrasG12D mice (n = 6 per genotype).


Representative immunoblots of KrasWT KrasG12D mouse lung lysates with indicated antibodies . Semi - quantitative densitometry of pADAM17 in lung lysates ( n = 6 per genotype ) .

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Representative immunoblots of individual KrasWT and KrasG12D mouse lung lysates with the indicated antibodies. Semi-quantitative densitometry of pADAM17 in lung lysates (n = 6 per genotype).


Representative immunoblots of KrasWT KrasG12D lung lysates with indicated antibodies . Semi - quantitative densitometry of pp38 pERK1 / 2 MAPK in KrasWT KrasG12D lung lysates ( n = 6 per genotype ) .

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Representative immunoblots of individual KrasWT and KrasG12D lung lysates with the indicated antibodies. Semi-quantitative densitometry of pp38 and pERK1/2 MAPK protein levels in KrasWT and KrasG12D lung lysates (n = 6 per genotype).


Representative of pADAM17 - stained lung sections from KrasG12D mice injected with a single dose of vehicle ( control ) , SB203580 p38 MAPK or U0126 ERK1 / 2 inhibitors . Scale bar , 100μm .

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Representative images of pADAM17-stained lung sections from KrasG12D mice injected with a single dose of vehicle (control), SB203580 p38 MAPK or U0126 ERK1/2 inhibitors. Scale bar, 100μm.


of pADAM17 - positive / HPF in lungs of indicated groups ( n = 5 per group ) .

Quantification of pADAM17-positive cells/HPF in lungs of the indicated groups (n = 5 per group).


Immunoblots with indicated antibodies on lysates from A549 NIH - H23 LAC lines treated with SB203580 for 24 hours .

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Immunoblots with the indicated antibodies on lysates from A549 and NIH-H23 LAC cell lines treated with SB203580 for 24 hours.


MTT viability assays of A549 NCI - H23 LAC treated with SB203580 .

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MTT viability assays of A549 and NCI-H23 LAC cells treated with SB203580.


ATP proliferation ( K ) assays of A549 NCI - H23 LAC treated with SB203580 .

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ATP proliferation (K) assays of A549 and NCI-H23 LAC cells treated with SB203580.


Representative immunoblots of KrasG12D KrasG12D : Adam17ex / ex lung lysates with indicated antibodies .

Representative immunoblots of individual KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies.


Representative of pERK1 / 2 - stained lung sections from indicated genotypes . Scale bars , 100μm . of pERK1 / 2 - positive / high power field ( HPF ) in lungs of indicated genotypes ( n = 6 per genotype ) .

Representative images of pERK1/2-stained lung sections from the indicated genotypes. Scale bars, 100μm. Quantification of pERK1/2-positive cells/high power field (HPF) in lungs of the indicated genotypes (n = 6 per genotype).


ELISA of sIL - 6R TNFα in serum from KrasG12D KrasG12D : Adam17ex / ex mice at 6 post Ad - Cre inhalation , along with control KrasWT mice at 6 post vehicle inhalation ( n = 6 per genotype ) .

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ELISA of sIL-6R and TNFα protein levels in serum from KrasG12D and KrasG12D:Adam17ex/ex mice at 6 weeks post Ad-Cre inhalation, along with control KrasWT mice at 6 weeks post vehicle inhalation (n = 6 per genotype).


Immunoblots of representative KrasWT , KrasG12D KrasG12D : Adam17ex / ex lung lysates with indicated antibodies .

Immunoblots of representative individual KrasWT, KrasG12D and KrasG12D:Adam17ex/ex lung lysates with the indicated antibodies.


Representative immunofluorescence of KrasG12D mouse lung sections ( n = 5 mice ) for ADAM17 either ( panels from left to right ) IL - 6R , TGFα , Notch1 , Nrg1 EGF . Sections were counterstained with DAPI ( blue ) . Scale bars , 100μm .

Representative immunofluorescence staining of KrasG12D mouse lung sections (n = 5 mice) for ADAM17 and either (panels from left to right) IL-6R, TGFα, Notch1, Nrg1 and EGF. Sections were counterstained with DAPI (blue). Scale bars, 100μm.


Representative of pERK1 / 2 - stained lung sections from KrasG12D mice treated with either vehicle ( control , Ctl ) or anti - IL - 6R antibodies 1F7 or 25F10 over 6 post Ad - Cre inhalation . Scale bar , 100μm . of pERK1 / 2 - positive / high power field ( HPF ) in lungs of indicated groups ( n = 5 per group ) .

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Representative images of pERK1/2-stained lung sections from KrasG12D mice treated with either vehicle (control, Ctl) or anti-IL-6R antibodies 1F7 or 25F10 over 6 weeks post Ad-Cre inhalation. Scale bar, 100μm. Quantification of pERK1/2-positive cells/high power field (HPF) in lungs of the indicated groups (n = 5 per group).


Representative immunoblots with indicated antibodies on whole lysates from immune epithelial isolated from lungs of mice ( n = 3 ) harbouring oncogenic KrasG12D allele that were stimulated with either PBS ( - ) or 100ng / ml Hyper - IL - 6 ( + ) . Graphs depict semi - quantitative densitometry of pERK1 / 2 MAPK in stimulated immune epithelial ( n = 2 experiments ) .

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Representative immunoblots with the indicated antibodies on whole cell lysates from primary immune and epithelial cells isolated from the lungs of mice (n = 3) harbouring the oncogenic KrasG12D allele that were stimulated with either PBS (-) or 100ng/ml Hyper-IL-6 (+). Graphs depict semi-quantitative densitometry of pERK1/2 MAPK protein levels in stimulated immune and epithelial cells (n = 2 experiments).


Representative of pADAM17 - stained lung sections from normal ( cancer - free ) control LAC . Scale bar , 100μm . of pADAM17 - positive in ( B ) normal ( cancer - free ) control individuals ( n = 8 ) LAC ( n = 18 )

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Representative images of pADAM17-stained lung sections from normal (cancer-free) control and LAC patients. Scale bar, 100μm. Quantification of pADAM17-positive cells in (B) normal (cancer-free) control individuals (n = 8) and LAC patients (n = 18)


of pADAM17 - positive pERK1 / 2 MAPK - positive in LAC either KRAS wild - type ( WT , n = 13 ) or mutant ( MUT , n = 5 ) .

Quantification of pADAM17-positive cells and pERK1/2 MAPK-positive cells in LAC patients either KRAS wild-type (WT, n = 13) or mutant (MUT, n = 5).


Linear regression analyses of pADAM17 with pERK1 / 2 ( D ) pp38 ( E ) MAPK in serial lung sections from LAC KRAS wild - type mutant .

Linear regression analyses of pADAM17 with pERK1/2 (D) and pp38 (E) MAPK staining in serial lung sections from LAC KRAS wild-type and mutant patients.


Immunoblots of lysates from normal human bronchial epithelial ( HBEC ) LAC lines A549 , NIH - H23 NIH - H2228 with indicated antibodies . Shown are lysates from 2 separate passages per . Pro , proADAM17 ; Mat , mature ADAM17 . Densitometric semi - of immunoblots showing relative expression of pADAM17 ( relative to Actin ) , pro mature forms of ADAM17 ( relative to Actin ) , pERK1 / 2 pp38 MAPK ( relative to their total counterparts ) .

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Immunoblots of lysates from normal human bronchial epithelial cells (HBEC) and LAC cell lines A549, NIH-H23 and NIH-H2228 with the indicated antibodies. Shown are lysates from 2 separate passages per cell line. Pro, proADAM17; Mat, mature ADAM17. Densitometric semi-quantification of immunoblots showing the relative expression levels of pADAM17 (relative to Actin), pro and mature forms of ADAM17 (relative to Actin), and pERK1/2 and pp38 MAPK (relative to their total counterparts) protein levels.


ELISA of sIL - 6R in culture supernatants from matching in ( F ) cultured over 24 hours . Data are from 4 independent experiments .

ELISA of sIL-6R protein levels in culture supernatants from matching cells in (F) cultured over 24 hours. Data are from 4 independent experiments.


MTT viability assays of A549 transduced with nontargeted control sgRNA ( Ctl ) ADAM17 sgRNA ( knockout , KO ) .

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MTT viability assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).


ATP proliferation ( J ) assays of A549 transduced with nontargeted control sgRNA ( Ctl ) ADAM17 sgRNA ( knockout , KO ) .

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ATP proliferation (J) assays of A549 cells transduced with nontargeted control sgRNA (Ctl) and ADAM17 sgRNA (knockout, KO).


ELISA of sIL - 6R in culture supernatants from matching A549 ( I J ) cultured over 24 hours . Data are from 3 independent experiments .

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ELISA of sIL-6R protein levels in culture supernatants from matching A549 cells (I and J) cultured over 24 hours. Data are from 3 independent experiments.


volume ( mm3 ) of KO Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 into NSG mice ( n = 5 per group ) .

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Tumor volume (mm3) of KO and Ctl A549 xenografts assessed every 3 days starting at one week post injection of 106 cells into NSG mice (n = 5 per group).


Weight (grams) (M) of tumor xenografts


representative images (N) of tumor xenografts


ELISA of human sIL - 6R in serum of indicated mice ( n = 5 per group ) . of sIL - 6R are below detection in serum from KO xenograft mice . ND , not detected .

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ELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). Levels of sIL-6R are below detection in serum from KO xenograft mice. ND, not detected.


Representative of pADAM17 pERK1 / 2 MAPK immunohistochemical on KO Ctl A549 xenograft tumors . Scale bars , 100μm .

Representative images of pADAM17 and pERK1/2 MAPK immunohistochemical staining on KO and Ctl A549 xenograft tumors. Scale bars, 100μm.


Immunoblots of lysates from A549 KO Ctl xenograft tumors with indicated antibodies .

Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies.


Representative of H & E - stained lung sections from KrasG12D mice treated with either vehicle ( control ) or ADAM17 prodomain inhibitor , A17pro ( 1mg / kg , 3 times a week ) , for 6 starting on day after Cre inhalation . Scale bars , 3mm . of lung parenchyma occupied by lesions in indicated groups ( n = 6 per group )

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Representative images of H&E-stained lung sections from KrasG12D mice treated with either vehicle (control) or the ADAM17 prodomain inhibitor, A17pro (1mg/kg, 3 times a week), for 6 weeks starting on the day after Cre inhalation. Scale bars, 3mm. Quantification of lung parenchyma area occupied by tumor lesions in the indicated groups (n = 6 per group)


Representative of lung sections from indicated mice from ( A ) stained with antibodies against TTF - 1 ( C ) , PCNA ( E ) pERK1 / 2 ( G ) . Scale bars , 100μm . of positive / high power field ( HPF ) in lungs of mice from ( C ) , ( E ) ( G ) stained for TTF - 1 ( D ) , PCNA ( F ) pERK1 / 2 ( H ) . n = 6 per group .

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Representative images of lung sections from the indicated mice from (A) stained with antibodies against TTF-1 (C), PCNA (E) and pERK1/2 (G). Scale bars, 100μm. Quantification of positive cells/high power field (HPF) in the lungs of mice from (C), (E) and (G) stained for TTF-1 (D), PCNA (F) and pERK1/2 (H). n = 6 per group.


ELISA of serum sIL - 6R in indicated mice ( n = 6 per group )

ELISA of serum sIL-6R protein levels in the indicated mice (n = 6 per group)


volume ( mm3 ) of a KRAS mutant PDX treated with vehicle control ( Ctl ) or A17pro ( 1mg / kg ) every second day . Treatment started when tumors reached a volume of 100 - 150mm3 after injection of 2 × 106 in NSG mice . n = 5 per group .

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Tumor volume (mm3) of a KRAS mutant PDX treated with vehicle control (Ctl) or A17pro (1mg/kg) every second day. Treatment started when tumors reached a volume of 100-150mm3 after injection of 2 × 106 cells in NSG mice. n = 5 per group.


representative images (L) of tumor PDXs


ELISA of human sIL - 6R in serum of indicated mice ( n = 5 per group ) . *

ELISA of human sIL-6R protein levels in serum of the indicated mice (n = 5 per group). *


Immunoblots with indicated antibodies of representative lysates from PDX tumors treated with vehicle control or A17pro .

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Immunoblots with the indicated antibodies of representative lysates from individual PDX tumors treated with vehicle control or A17pro.


Representative image of pADAM17 immunohistochemical on an untreated KRAS mutant PDX . Scale bar , 100μm .

Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor. Scale bar, 100μm.


Signal tracks showing cleavages generated by Med8 - MNase , Med17 - MNase , PMED8 - MNase in YPD at three highly expressed SAGA - dependent ( A ) three highly expressed TFIID - dependent ( B ) loci . time points for a given factor were scaled to same data range , PMED8 - MNase tracks were scaled to lower Mediator - MNase fusion range . Med14ChIP - seq Med15ChIP - chip data are shown for comparison . TSSs are indicated by arrows .

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Signal tracks showing cleavages generated by Med8-MNase, Med17-MNase, and PMED8-MNase in YPD at three highly expressed SAGA-dependent (A) and three highly expressed TFIID-dependent (B) loci. All time points for a given factor were scaled to the same data range, and PMED8-MNase tracks were scaled to the lower Mediator-MNase fusion range. Med14ChIP-seq and Med15ChIP-chip data are shown for comparison. TSSs are indicated by arrows.


( A ) Average plots of Mediator cleavages around TSSs of SAGA - TFIID - dependent genes . of cleavage maximum to TSS is indicated by dotted lines corresponding numbers .

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(A) Average plots of Mediator cleavages around the TSSs of SAGA- and TFIID-dependent genes. The distance of cleavage maximum to TSS is indicated by the dotted lines and corresponding numbers.


( B ) Signal tracks of Med8 - MNase free MNase cleavages at previously characterized UASs of CLB2 RPS5 genes . TSSs are indicated by arrows .

(B) Signal tracks of Med8-MNase and free MNase cleavages at the previously characterized UASs of the CLB2 and RPS5 genes. TSSs are indicated by arrows.


( A ) Average plots of Med8 cleavages around TSSs of SAGA - TFIID - dependent genes in Kin28AS treated with DMSO ( - NA - PP1 ) or 6 μM NA - PP1 ( + NA - PP1 ) .

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(A) Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes in Kin28AS cells treated with DMSO (-NA-PP1) or 6 μM NA-PP1 (+NA-PP1).


( B ) Signal tracks of Med8 - MNase cleavages at upstream regions of several genes previously shown to have increased promoter association of Mediator by ChIP - qPCR following NA - PP1 treatment of a Kin28AS ( Wong et al . , 2014 ) . time points for a given treatment were concatenated to generate a combined track . TSSs are indicated by arrows .

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(B) Signal tracks of Med8-MNase cleavages at the upstream regions of several genes previously shown to have increased core promoter association of Mediator by ChIP-qPCR following NA-PP1 treatment of a Kin28AS strain (Wong et al., 2014). All time points for a given treatment were concatenated to generate a combined track. TSSs are indicated by arrows.


( A ) Average plots of Med8 cleavages around SAGA - TFIID - dependent gene TSSs stratified into quintiles by level of NET - seq signal in a 200 bp window downstream of TSS .

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(A) Average plots of Med8 cleavages around SAGA- and TFIID-dependent gene TSSs stratified into quintiles by the level of NET-seq signal in a 200 bp window downstream of the TSS.


( B ) Average plots of Med8 cleavage around TSSs of 87 most highly transcribed SAGA - TFIID - dependent genes .

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(B) Average plots of Med8 cleavage around the TSSs of the 87 most highly transcribed SAGA- and TFIID-dependent genes.


( C ) Boxplots of transcription of 87 most highly transcribed SAGA - TFIID - dependent genes as determined by NET - seq . Significance was assessed by t - .

(C) Boxplots of the transcription levels of the 87 most highly transcribed SAGA- and TFIID-dependent genes as determined by NET-seq. Significance was assessed by t-test.


( D ) Scatterplots of average NET - seq in a 200 bp window downstream of TSS versus average Med8 cleavages in a 1 kb window upstream of TSS for SAGA - TFIID - dependent genes . Correspondence between datasets was assessed by Spearman correlation .

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(D) Scatterplots of average NET-seq counts in a 200 bp window downstream of the TSS versus average Med8 cleavages in a 1 kb window upstream of the TSS for SAGA- and TFIID-dependent genes. Correspondence between the datasets was assessed by Spearman correlation.


( A ) Average plot of Med8 cleavages around 193 Gcn4ChIP - chip peak midpoints ( MacIsaac et al . , 2006 ) in WT med15Δ .

(A) Average plot of Med8 cleavages around 193 Gcn4ChIP-chip peak midpoints (MacIsaac et al., 2006) in WT and med15Δ.


( B ) Average plots of Med8 cleavages around TSSs of genes upregulated downregulated ≥ 2 fold in SM ( Saint et al . , 2014 ) in WT med15Δ . Control cleavages were subtracted from SM cleavages at each base position .

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(B) Average plots of Med8 cleavages around the TSSs of genes upregulated and downregulated ≥2 fold in SM (Saint et al., 2014) in WT and med15Δ. Control cleavages were subtracted from SM cleavages at each base position.


( C ) qRT - PCR of SAGA - TFIID - dependent genes in WT med15Δ . Bars represent mean + for two biological replicates performed in triplicate . * , p < 0 . 05 ; † , p < 0 . 01 ; ‡ , p < 0 . 005 by unpaired Student ' s t - .

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(C) qRT-PCR analysis of SAGA- and TFIID-dependent genes in WT and med15Δ. Bars represent mean + SEM for two biological replicates performed in triplicate. *, p < 0.05; †, p < 0.01; ‡, p < 0.005 by unpaired Student's t-test.


( A ) Average plot of Taf1 cleavages around TSSs of SAGA - TFIID - dependent genes . A nucleosome occupancy profile as determined by MNase - seq is also shown for comparison . Dotted lines represent Taf1 cleavage maxima in TFIID - dependent gene bodies . NDR , taken to be - 1 to + 1 nucleosome midpoint , is indicated by a black rectangle .

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(A) Average plot of Taf1 cleavages around the TSSs of SAGA- and TFIID-dependent genes. A nucleosome occupancy profile as determined by MNase-seq is also shown for comparison. Dotted lines represent Taf1 cleavage maxima in TFIID-dependent gene bodies. The NDR, taken to be the -1 to +1 nucleosome midpoint distance, is indicated by a black rectangle.


( B ) Average plots of Taf1 cleavages around TSSs of SAGA - TFIID - dependent genes ± rapamycin to deplete Med14 - FRB .

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(B) Average plots of Taf1 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Med14-FRB.


Average plots of Med8 cleavages around TSSs of SAGA - TFIID - dependent genes ± rapamycin to deplete Taf1 - FRB .

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Average plots of Med8 cleavages around the TSSs of SAGA- and TFIID-dependent genes ± rapamycin to deplete Taf1-FRB.