EMBO/SourceData huggingface dataset:
Figure 6(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).
Figure 7(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.
Figure 1Representative 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).
Figure 2Representative 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). *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.
Figure 3Representative 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).
Figure 4Representative 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).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.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.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.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).
Figure 5ELISA 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.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).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).
Figure 6Representative 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).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.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).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.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 xenograftsrepresentative images (N) of tumor xenograftsELISA 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.Immunoblots of lysates from individual A549 KO and Ctl xenograft tumors with the indicated antibodies.
Figure 7Representative 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.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.Weight (grams) of tumor PDXsrepresentative images (L) of tumor PDXsELISA 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.Representative image of pADAM17 immunohistochemical staining on an untreated KRAS mutant PDX tumor. Scale bar, 100μm.
Figure 2(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.
Figure 3(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.
Figure 4(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.(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.
Figure 5(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.(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.
Figure 6(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.
Figure 1A - 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).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.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.
Figure 2A - 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.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.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.
Figure 3A - 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.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 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.
Figure 4A - 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.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).
Figure 5B - 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.