( 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 .

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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 weeks . Viremia is detected with CHIKV nsP1 probe via qRT - PCR .

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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 weeks .

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


( 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 .

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(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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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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 the metatarsal region of the footpad . ( B ) joint inflammation in ONNV - infected mice were monitored over 2 weeks ( n = 5 ) .

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

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 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 .

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

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 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 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

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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 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 .

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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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 ) .

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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 ) 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 .

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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 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 ) .

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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 ) 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 ) .

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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 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 ) .

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(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 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 .

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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 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 .

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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 protein expression in nine osteosarcoma cell lines and HTC116 as positive control .

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


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

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


( 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 .

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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 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 .

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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 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 .

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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 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 ) .

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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 ) 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 .

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(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 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 .

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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 ) 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 ) .

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

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 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 ) .

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

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 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 ) .

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

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 cell cycle regulation genes ( normalized against 18SrRNA ) in lungs from the 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 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 .

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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 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 .

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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 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 ) .

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

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 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 ) .

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

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 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 ) .

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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 individual KrasWT and KrasG12D mouse lung lysates with the 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 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 ) .

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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 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 .

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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.


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

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

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


Immunoblots with the indicated antibodies on lysates from A549 and NIH - H23 LAC cell 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 and NCI - H23 LAC cells 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 and NCI - H23 LAC cells treated with SB203580 .

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


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

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

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


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 ) .

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

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 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 ) .

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

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


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 .

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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 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 ) .

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

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 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 ) .

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

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 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 )

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

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)


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 ) .

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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 ) and pp38 ( E ) MAPK staining in serial lung sections from LAC KRAS wild - type and mutant patients .

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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 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 .

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

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 protein levels in culture supernatants from matching cells in ( F ) cultured over 24 hours . Data are from 4 independent experiments .

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

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 cells transduced with nontargeted control sgRNA ( Ctl ) and ADAM17 sgRNA ( knockout , KO ) .

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

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


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

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

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


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 .

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

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.


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 ) .

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

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 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 .

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

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

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

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


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

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


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 )

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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 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 .

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

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 protein levels in the indicated mice ( n = 6 per group )

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

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


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 .

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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 protein levels in serum of the indicated mice ( n = 5 per group ) . *

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

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


Immunoblots with the indicated antibodies of representative lysates from individual 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 staining on an untreated KRAS mutant PDX tumor . 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 , 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 .

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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 the TSSs of SAGA - and TFIID - dependent genes . The distance of cleavage maximum to TSS is indicated by the dotted lines and 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 and free MNase cleavages at the previously characterized UASs of the CLB2 and RPS5 genes . TSSs are indicated by arrows .

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(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 the TSSs of SAGA - and TFIID - dependent genes in Kin28AS cells 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 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 .

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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 - and TFIID - dependent gene TSSs stratified into quintiles by the level of NET - seq signal in a 200 bp window downstream of the 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 the TSSs of the 87 most highly transcribed SAGA - and 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 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 .

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(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 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 .

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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 and med15Δ .

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(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 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 .

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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 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 .

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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 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 .

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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 the TSSs of SAGA - and 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 the TSSs of SAGA - and 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.


A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C . trachomatis L2 ( multiplicity of infection MOI = 1 ) in the presence or not of BP . In the indicated samples CP4d was added 2 h before infection . Cell lysates were run on SDS - PAGE , proteins were transferred to a membrane and BP incorporation was revealed with HRP - conjugated streptavidin . BP incorporation is enhanced in infected samples , and is inhibited by CP4d . The two main bands present in all samples correspond to naturally biotinylated host proteins ( Haneji & Koide , 1989 ) . After blotting the membrane was stained with Coomassie blue to control for equal loading . B - Same as in ( A ) , except that where indicated 250 μM doxycycline ( doxy , left ) or 7 μM cycloheximide ( CHX , right ) were added 24 h or 2 hpi , respectively .

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A - Whole cell lysates were prepared with HeLa cells infected or not for 48 h with C. trachomatis L2 (multiplicity of infection MOI=1) in the presence or not of BP. In the indicated samples CP4d was added 2 h before infection. Cell lysates were run on SDS-PAGE, proteins were transferred to a membrane and BP incorporation was revealed with HRP-conjugated streptavidin. BP incorporation is enhanced in infected samples, and is inhibited by CP4d. The two main bands present in all samples correspond to naturally biotinylated host proteins (Haneji & Koide, 1989). After blotting the membrane was stained with Coomassie blue to control for equal loading. B - Same as in (A), except that where indicated 250 μM doxycycline (doxy, left) or 7 μM cycloheximide (CHX, right) were added 24 h or 2 hpi, respectively.


C - Whole cell lysates were prepared with TG2 + / + and TG2 - / - MEFs infected or not for 48 h with C . trachomatis L2 in the presence or not of BP , and analyzed as in ( A ) .

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C - Whole cell lysates were prepared with TG2+/+ and TG2-/- MEFs infected or not for 48 h with C. trachomatis L2 in the presence or not of BP, and analyzed as in (A).


D - Western blot with anti - TG2 antibodies on total cell lysates infected or not with C . trachomatis L2 for the indicated time . The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments , with the results of the Student ' s ratio - paired t - test . NI : not infected .

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D -Western blot with anti-TG2 antibodies on total cell lysates infected or not with C. trachomatis L2 for the indicated time. The histogram displays the mean ± SD of TG2 expression relative to actin from four independent experiments, with the results of the Student's ratio-paired t-test. NI: not infected.


E - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . Where indicated , 40 μM CP4d was added 2 hpi . tgm2 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least four times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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E - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. Where indicated, 40 μM CP4d was added 2 hpi. tgm2 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least four times. P-values of Student's ratio-paired t-test <0.05 are shown.


F - Cells were treated for 18 h with the indicated concentration of human recombinant IL - 6 before measuring TG2 transcription relative to actin like in ( E ) . The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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F - Cells were treated for 18 h with the indicated concentration of human recombinant IL-6 before measuring TG2 transcription relative to actin like in (E). The data are presented as relative mRNA levels compared to untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


Cells were left uninfected or infected with C . trachomatis L2 in the presence of the indicated concentration of anti - IL - 6 receptor antibodies . Forty - eight h later TG2 transcription relative to actin was measured like in ( E ) . The data are presented as relative mRNA levels compared to uninfected / untreated cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated at least three times . P - values of Student ' s ratio - paired t - test < 0 . 05 are shown .

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Cells were left uninfected or infected with C. trachomatis L2 in the presence of the indicated concentration of anti-IL-6 receptor antibodies. Forty-eight h later TG2 transcription relative to actin was measured like in (E). The data are presented as relative mRNA levels compared to uninfected/untreated cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated at least three times. P-values of Student's ratio-paired t-test <0.05 are shown.


A - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were disrupted and bacterial titers ( IFU = inclusion forming unit ) were determined by re - infecting fresh HeLa cells as described in the methods . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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A - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were disrupted and bacterial titers (IFU=inclusion forming unit) were determined by re-infecting fresh HeLa cells as described in the methods. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


B - HeLa cells were transfected with control siRNA or two siRNAs against TG2 . Two days later , the efficiency of the silencing was assessed by western blot using anti - TG2 antibodies and anti - actin antibodies as loading control ( bottom ) . Duplicate wells were infected with L2incDGFP and progeny was analyzed as in ( A ) ( top ) . The mean ± SD of four independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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B - HeLa cells were transfected with control siRNA or two siRNAs against TG2. Two days later, the efficiency of the silencing was assessed by western blot using anti-TG2 antibodies and anti-actin antibodies as loading control (bottom). Duplicate wells were infected with L2incDGFP and progeny was analyzed as in (A) (top). The mean ± SD of four independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


C - Same as in ( A ) except that C . trachomatis serovar L2 ( left ) or D ( right ) were grown in primary cells isolated from fallopian tubes . For serovar D , IFU were determined 48 hpi . The mean ± SD of four to five independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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C - Same as in (A) except that C. trachomatis serovar L2 (left) or D (right) were grown in primary cells isolated from fallopian tubes. For serovar D, IFU were determined 48 hpi. The mean ± SD of four to five independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


D - HeLa cells were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with L2incDGFP at MOI = 0 . 15 . Thirty hours later the cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( left ) and the mean fluorescence of the infected population ( right ) ± SD are shown for three independent experiments . P - values of Student ' s paired t - test are indicated when < 0 . 05 . A representative field for each condition is shown , scale bar = 10 μm .

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D - HeLa cells were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with L2incDGFP at MOI=0.15. Thirty hours later the cells were fixed and analyzed by flow cytometry. The percentage of infected cells (left) and the mean fluorescence of the infected population (right) ± SD are shown for three independent experiments. P-values of Student's paired t-test are indicated when <0.05. A representative field for each condition is shown, scale bar = 10 μm.


E - Primary epithelial cells isolated from fallopian tubes were pre - treated with the indicated concentrations of CP4d ( or DMSO alone ) for 2 h before being infected with C . trachomatis serovar L2 ( left ) or D ( right ) . Twenty - four hours later the cells were fixed , bacteria were stained using FITC - labeled anti - Chlamydia - LPS antibodies , and the mean size of the inclusions manually determined using ImageJ , on twenty inclusions per experiment . The mean ± SD of three independent experiments are shown . P - values of Student ' s paired t - test are indicated when < 0 . 05 .

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E - Primary epithelial cells isolated from fallopian tubes were pre-treated with the indicated concentrations of CP4d (or DMSO alone) for 2 h before being infected with C. trachomatis serovar L2 (left) or D (right). Twenty-four hours later the cells were fixed, bacteria were stained using FITC-labeled anti-Chlamydia-LPS antibodies, and the mean size of the inclusions manually determined using ImageJ, on twenty inclusions per experiment. The mean ± SD of three independent experiments are shown. P-values of Student's paired t-test are indicated when <0.05.


F - Mice were infected intravaginally with 105 IFU of C . muridarum . Twenty - five days later the mice were sacrificed and the upper genital tract , from the uterine horn to the oviduct , was collected . The right part was used for bacterial burden assessment ( top left ) . The left part was rinsed with PBS and observed with a binocular magnifier ( right ) to determine the hydrosalpinx score ( bottom left ) . Each dot represents one mouse , the mean ± SD is shown . P - values of Mann - Whitney test are indicated when < 0 . 05 .

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F - Mice were infected intravaginally with 105 IFU of C. muridarum. Twenty-five days later the mice were sacrificed and the upper genital tract, from the uterine horn to the oviduct, was collected. The right part was used for bacterial burden assessment (top left). The left part was rinsed with PBS and observed with a binocular magnifier (right) to determine the hydrosalpinx score (bottom left). Each dot represents one mouse, the mean ± SD is shown. P-values of Mann-Whitney test are indicated when <0.05.


A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria ( MOI = 0 . 2 ) . Cells were disrupted 30 h later and the bacterial titer determined by re - infecting fresh wild type cells . The mean ± SD of three independent experiments are shown .

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A - MEFs were grown for 24 h culture medium complemented with the indicated concentration of glucose before being infected with L2 IncDGFP bacteria (MOI = 0.2). Cells were disrupted 30 h later and the bacterial titer determined by re-infecting fresh wild type cells. The mean ± SD of three independent experiments are shown.


B - Cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 48 h . Where indicated 40 μM CP4d was added 2 hpi . GLUT - 1 and GLUT - 3 transcripts were measured by real - time RT - PCR and normalized to actin transcripts following the ∆ ∆ Ct method . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated four times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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B - Cells were infected with C. trachomatis L2 (MOI=1) for 24 or 48 h. Where indicated 40 μM CP4d was added 2 hpi. GLUT-1 and GLUT-3 transcripts were measured by real-time RT-PCR and normalized to actin transcripts following the ∆∆Ct method. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated four times. P-values of Student's ratio-paired t-test are indicated when <0.05.


C - Relationship between TGM2 and GLUT - 1 ( top ) and GLUT - 3 ( bottom ) expression across 265 HGSOCs . Expression comparisons were performed using Spearman ' s rank correlation test .

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C -Relationship between TGM2 and GLUT-1 (top) and GLUT-3 (bottom) expression across 265 HGSOCs. Expression comparisons were performed using Spearman's rank correlation test.


D - HeLa cells were infected with C . trachomatis L2 ( MOI = 1 ) for 24 or 46 h . HIF - 1α transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative

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D -HeLa cells were infected with C. trachomatis L2 (MOI=1) for 24 or 46 h. HIF-1α transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative


E - HeLa cells were transfected with control siRNA or two siRNAs against HIF - 1α . Two days later , cells were infected with C . trachomatis L2 ( MOI = 1 ) for 48 h . The indicated transcripts were measured by real - time RT - PCR and normalized to actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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E - HeLa cells were transfected with control siRNA or two siRNAs against HIF-1α. Two days later, cells were infected with C. trachomatis L2 (MOI=1) for 48 h. The indicated transcripts were measured by real-time RT-PCR and normalized to actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


F - TG2 - / - MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C . trachomatis L2 ( MOI = 1 ) . Mouse GLUT - 1 transcripts were measured by real - time RT - PCR and normalized to mouse actin transcripts . The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD . Each experiment was performed in duplicate and repeated three times . P - values of Student ' s ratio - paired t - test are indicated when < 0 . 05 .

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F - TG2-/- MEFs stably transformed or not with the indicated TG2 construct were infected for two days with C. trachomatis L2 (MOI=1). Mouse GLUT-1 transcripts were measured by real-time RT-PCR and normalized to mouse actin transcripts. The data are presented as relative mRNA levels compared to uninfected cells and shown as the mean ± SD. Each experiment was performed in duplicate and repeated three times. P-values of Student's ratio-paired t-test are indicated when <0.05.


A - HeLa cells were infected with C . trachomatis ( MOI = 1 ) and 40 µM CP4d was added or not 2 hpi . After 24 h 0 . 5 mM BP was added and cells were lysed at 48 hpi . Lysates were precipitated with streptavidin - coated beads . After separation with SDS - PAGE , proteins were transferred to a membrane and blotted with anti - GFPT antibody followed with HRP - conjugated secondary antibody .

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A - HeLa cells were infected with C. trachomatis (MOI = 1) and 40 µM CP4d was added or not 2 hpi. After 24 h 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin-coated beads. After separation with SDS-PAGE, proteins were transferred to a membrane and blotted with anti-GFPT antibody followed with HRP-conjugated secondary antibody.


B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP . Samples were incubated for 3 h at 37 ° C before separation by SDS - PAGE . Proteins were transferred to a membrane and BP was revealed using HRP - conjugated streptavidin . rhGFPT1 is 77 . 5 kDa .

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B - In vitro assay testing the ability of purified TG2 to crosslink purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS-PAGE. Proteins were transferred to a membrane and BP was revealed using HRP-conjugated streptavidin. rhGFPT1 is 77.5 kDa.


D - In vitro assay was performed as described in B using wild type rhGFPT1 ( WT ) , rhGFPT1 Q58N , rhGFPT1 Q328N or rhGFPT1 Q555N as substrates . The reaction was performed at 37 ° C for 30 min . After probing with HRP - Streptavidin the membrane was washed and probed with anti - GFPT antibodies followed with HRP - conjugated secondary antibodies . The ratio of modified protein ( streptavidin signal ) to the total GFPT is shown , normalized to its value with WT rhGFPT1 . The mean ± SD of five independent experiments is shown , the p - value of the Student ' s ratio - paired t - test is indicated when < 0 . 05 .

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D - In vitro assay was performed as described in B using wild type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N or rhGFPT1 Q555N as substrates. The reaction was performed at 37 °C for 30 min. After probing with HRP-Streptavidin the membrane was washed and probed with anti-GFPT antibodies followed with HRP-conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown, normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, the p-value of the Student's ratio-paired t-test is indicated when <0.05.


E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 ° C for 45 min with fructose - 6 - P and glutamine . The production of glucosamine - 6 - P was measured using HPAEC - PAD . Results of three independent experiments are shown , with mean ± SD , and p - value of the Student ' s paired t - test is indicated ( * P < 0 . 05 ) .

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E - Lysates of cells treated or not for 6 h with ionomycin were incubated at 37 °C for 45 min with fructose-6-P and glutamine. The production of glucosamine-6-P was measured using HPAEC-PAD. Results of three independent experiments are shown, with mean ± SD, and p-value of the Student's paired t-test is indicated (*P < 0.05).


B - Endocervical epithelial cells were pre - treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin ( or an equivalent volume of DMSO ) and 0 . 5 µM BP . Six hours later whole cell lysates were analyzed by western blot . The membrane was first blotted with HRP - conjugated streptavidin to detect TG2 activity , then extensively washed and probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . Last the membrane was probed with anti - actin as a loading control .

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B - Endocervical epithelial cells were pre-treated or not with 40 μM CP4d for 2 h before addition of the indicated concentration of ionomycin (or an equivalent volume of DMSO) and 0.5 µM BP. Six hours later whole cell lysates were analyzed by western blot. The membrane was first blotted with HRP-conjugated streptavidin to detect TG2 activity, then extensively washed and probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. Last the membrane was probed with anti-actin as a loading control.


C - The same experimental procedure as described in ( B ) was applied to HeLa cells treated for 48 h prior to ionomycin treatment ( 8 µM ) with siRNA control or directed against TG2 .

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C - The same experimental procedure as described in (B) was applied to HeLa cells treated for 48 h prior to ionomycin treatment (8 µM) with siRNA control or directed against TG2.


A - HeLa cells were infected or not ( NI ) with C . trachomatis ( MOI = 1 ) , then lysed 24 or 48 hpi . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - O - GlcNAcylation antibody followed with HRP - conjugated secondary antibodies . After extensive washes the membrane was blotted again with anti - GFPT and anti - actin antibodies before revelation with HRP - conjugated secondary antibodies .

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A - HeLa cells were infected or not (NI) with C. trachomatis (MOI = 1), then lysed 24 or 48 hpi. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-O-GlcNAcylation antibody followed with HRP-conjugated secondary antibodies. After extensive washes the membrane was blotted again with anti-GFPT and anti-actin antibodies before revelation with HRP-conjugated secondary antibodies.


B - HeLa cells were infected or not with C . trachomatis ( MOI = 1 ) . Twenty - four hours later , 8 µM ionomycin ( or DMSO alone ) and 0 . 5 mM BP were added . After 6 h of treatment , cells were lysed and proteins revealed as in ( A ) .

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B - HeLa cells were infected or not with C. trachomatis (MOI = 1). Twenty-four hours later, 8 µM ionomycin (or DMSO alone) and 0.5 mM BP were added. After 6 h of treatment, cells were lysed and proteins revealed as in (A).


C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed . After separation with SDS - PAGE , proteins were transferred to a membrane , probed with anti - GFPT and anti - actin antibody before revelation with HRP - conjugated secondary antibodies .

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C - HeLa cells treated for 72 h with siRNA targeting GFPT1 or GFPT2 were lysed. After separation with SDS-PAGE, proteins were transferred to a membrane, probed with anti-GFPT and anti-actin antibody before revelation with HRP-conjugated secondary antibodies.


D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2 . Two days later the cells were infected for the indicated times ( MOI = 0 . 3 ) before fixation , rupture of the cells and measurement of bacterial diameter . The mean diameter ± SD and p - values of Student ' s paired t - test on 4 independent experiments are shown .

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D - HeLa cells were transfected with control siRNA or siRNAs against GFPT or TG2. Two days later the cells were infected for the indicated times (MOI=0.3) before fixation, rupture of the cells and measurement of bacterial diameter. The mean diameter ± SD and p-values of Student's paired t-test on 4 independent experiments are shown.


E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not ( siCTRL ) were infected with C . trachomatis ( MOI = 0 . 15 ) . Thirty hours later cells were fixed and analyzed by flow cytometry . The percentage of infected cells ( top ) and the mean fluorescence of the infected population ( middle ) ) ± SD are shown for at least four independent experiments . Duplicate wells were lysed and used to re - infect fresh HeLa cells to determine the bacterial titer ( bottom ) . P - values of Student ' s ratio - paired t - test are indicated .

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E - HeLa cells treated for 48 h with siRNA targeting GFPT1 or not (siCTRL) were infected with C. trachomatis (MOI = 0.15). Thirty hours later cells were fixed and analyzed by flow cytometry. The percentage of infected cells (top) and the mean fluorescence of the infected population (middle) ) ± SD are shown for at least four independent experiments. Duplicate wells were lysed and used to re-infect fresh HeLa cells to determine the bacterial titer (bottom). P-values of Student's ratio-paired t-test are indicated.


C - E KRAS and TP53 alterations in the cancer genome atlas ( TCGA ) pan - cancer MPM dataset ( n = 86 patients ) . Shown are clinical and molecular data plot with alteration frequencies ( C ) and patients reclassified as KRAS - or TP53 - altered ( asterisks ) , copy number variation data summary ( D ) , and segments of the KRAS and TP53 loci ( E ) .

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C-E KRAS and TP53 alterations in the cancer genome atlas (TCGA) pan-cancer MPM dataset (n = 86 patients). Shown are clinical and molecular data plot with alteration frequencies (C) and patients reclassified as KRAS- or TP53-altered (asterisks), copy number variation data summary (D), and segments of the KRAS and TP53 loci (E).


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients ( A ) and data summaries of mononucleotide change signatures ( B ) , of indices of genomic instability and mutation burden ( C ) , of clinical

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Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients (A) and data summaries of mononucleotide change signatures (B), of indices of genomic instability and mutation burden (C), of clinical features


Molecular and clinical features of the cancer genome atlas ( TCGA ) pan - cancer MPM patients ( n = 87 ) stratified by the presence of KRAS standalone ( n = 10 ) and combined KRAS / TP53 ( n = 7 ) alterations . Shown are unsupervised hierarchical clustering of n = 86 patients ( gene expression data were not available for one patient ) by 40 genes significantly overexpressed in KRAS / TP53 - altered over KRAS - altered over KRAS / TP53 - normal patients overall survival ( F ) .

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Molecular and clinical features of the cancer genome atlas (TCGA) pan-cancer MPM patients (n = 87) stratified by the presence of KRAS standalone (n = 10) and combined KRAS/TP53 (n = 7) alterations. Shown are unsupervised hierarchical clustering of n = 86 patients (gene expression data were not available for one patient) by 40 genes significantly overexpressed in KRAS/TP53-altered over KRAS-altered over KRAS/TP53-normal patients overall survival (F).


H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from ( A - G ) .

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H Data summary of mutant allelic frequency of KRAS compared with NF2 and BAP1 in all mutated samples from (A-G).


Pleural fluid cell pellets and supernatants from 10 patients ( called CRCINA # ) with pleural effusion and pleural tumor samples from 17 patients ( called TR # ) with MPM were subjected to digital droplet polymerase chain reaction ( ddPCR ) for the detection of mutant ( MUT ) copies of KRAS codon 12 / 13 ( KRASG12 / 13 ) and KRAS codon 61 ( KRASQ61 ) , as well as copies of TP53 and TERT . Diagnoses were lung adenocarcinoma ( LUAD ; n = 4 ) and MPM ( n = 23 ) . The assays were designed for detection of down to 1 : 20000 copies using EKVX ( KRASWTTP53G610T ) , A549 ( KRASG12STP53WT ) , NCI - H460 ( KRASQ61HTP53WT ) , NCI - H3122 ( KRASWTTP53E285V ) , and NCI - H3255 ( KRASWTTP53G560 - 1A ) human LUAD cells as controls . Shown are individual patient ( KRAS plot ) and individual sample ( TP53 plot ) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot ( A ) Any number of KRAS - mutant droplets detected in any sample ( KRAS plot in A ) and any patient that failed to achieve normal TP53 ploidy by any sample ( TP53 plot in A ) was deemed altered .

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Pleural fluid cell pellets and supernatants from 10 patients (called CRCINA #) with pleural effusion and pleural tumor samples from 17 patients (called TR#) with MPM were subjected to digital droplet polymerase chain reaction (ddPCR) for the detection of mutant (MUT) copies of KRAS codon 12/13 (KRASG12/13) and KRAS codon 61 (KRASQ61), as well as copies of TP53 and TERT. Diagnoses were lung adenocarcinoma (LUAD; n = 4) and MPM (n = 23). The assays were designed for detection of down to 1:20000 copies using EKVX (KRASWTTP53G610T), A549 (KRASG12STP53WT), NCI-H460 (KRASQ61HTP53WT), NCI-H3122 (KRASWTTP53E285V), and NCI-H3255 (KRASWTTP53G560-1A) human LUAD cells as controls. Shown are individual patient (KRAS plot) and individual sample (TP53 plot) allelic frequencies with color code and limits of normal TP53 allelic frequency as vertical dashed lines in the TP53 plot (A) Any number of KRAS-mutant droplets detected in any sample (KRAS plot in A) and any patient that failed to achieve normal TP53 ploidy by any sample (TP53 plot in A) was deemed altered.


F Representative May - Gruenwald - Giemsa - stained pleural fluid cytocentrifugal specimen from a KRASG12D ; Trp53f / f mouse showing macrophages ( MΦ , black arrow ) , lymphocytes ( LΦ , purple arrow ) , and neutrophils ( NΦ , green arrow ) and summary of cellular and biochemical features of effusions of KRASG12D ; Trp53f / f mice ( n = 10 ) .

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F Representative May-Gruenwald-Giemsa-stained pleural fluid cytocentrifugal specimen from a KRASG12D;Trp53f/f mouse showing macrophages (MΦ, black arrow), lymphocytes (LΦ, purple arrow), and neutrophils (NΦ, green arrow) and summary of cellular and biochemical features of effusions of KRASG12D;Trp53f/f mice (n = 10).


G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [ n is given in table in ( E ) ] .

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G Gross macroscopic and microscopic images of visceral and parietal tumors stained with hematoxylin and eosin or PCNA [n is given in table in (E)].