EMBO/soda-nel huggingface dataset:
Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity (2021)
Figure 4

Figure 4. CCT complex depletion leads to decreased cell viability when A3A is expressed

(A) Knockdown of one CCT complex member results in depletion of other CCT subunits. Following siCCT4 or siCCT7 transfection, cell lysates were evaluated by immunoblot for expression of additional CCT subunits by endogenous antibodies for CCT1, CCT4, CCT5, and CCT7. GAPDH was used as a loading control.

(B) Decreased cell viability upon CCT depletion and A3A expression. CCT4 (left) and CCT7 (right) subunits were depleted by siRNA transfection in U2OS cells with dox-inducible A3A transgenes. Immunoblot analysis of cell lysates showed decreased expression of targeted CCT subunit, but no alteration in A3A expression. Scr indicates non-targeted siRNA, used as control. Ku-86 is a loading control. Following siRNA transfection to deplete CCT subunits, U2OS-A3A cells were treated with dox. Viability was determined by colorimetric change after addition of a water-soluble tetrazolium salt. Percent viability was normalized to untreated controls. Statistical analysis was performed using a paired two-tailed t-test, n=3 biological replicates; error bars, SEM.

(C) Increased cell death resulting from A3A expression along with depletion of CCT. U2OS-A3A cells were depleted of CCT4 by siRNA, treated with dox, or combination. Cell death was measured by staining cells with fluorescent labeled calcein AM (live) and DNA (dead) stains. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t test, n=3; error bars, SEM.

(D) Viability of cells with catalytically inactive A3A. U2OS-A3A-C106S cells were induced with dox to express the catalytically inactive A3A mutant (C106S), depleted of CCT4 by siRNA, or combination. Cells were subsequently evaluated by live-dead staining assessed by FACS. Bar chart shows quantitation of FACS results averaged over three biological replicates. Statistical analysis was performed using a paired two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT4 after siRNA targeting. Ku-86 is a loading control.

(E) Increased DNA damage signaling upon CCT depletion and A3A expression. Depletion of CCT7 in K562-A3A cells was achieved by siRNA targeting. Expression of A3A was induced by dox treatment. Intracellular staining with anti-γH2AX antibody was analyzed by flow cytometry. Bar chart shows quantification of FACS results obtained over three biological replicates. Statistical analysis was performed using a two-tailed t-test, n=3; error bars, SEM. Immunoblot analysis of cell lysates showed decreased expression of CCT7 after siRNA targeting and A3A expression upon dox treatment. Ku-86 is a loading control. **p-value <0.01, ***p-value <0.01, ****p-value <0.0001, n.s. non-significant.
Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity (2021)
Figure 5

Figure 5. A3A mutational signatures are enriched in cancers with CCT mutations

(A) Tumors with CCT gene mutations have an increased mutational burden. All cancer genome sequences in TCGA were evaluated for mutation burden and specific base substitutions. Cancers with CCT gene mutations predicted to cause a negative impact on protein function were classified as deleterious and were compared to those with no mutations in any CCT gene. Wilcoxon rank sum test applied to all paired comparisons between tumors with and without CCT gene mutations yielded p<0.001.

(B) The APOBEC3 mutational signature is enriched in tumors with CCT gene mutations. Single base substitution (SBS) patterns, as defined by COSMIC Mutational Signatures v2, were evaluated in CCT-mutated and CCT non-mutated cancer genome sequences. Depicted are the mutational signatures attributable to APOBEC3 activity (SBS 2 and SBS 13). Denoted p-values determined by Wilcoxon rank sum test.

(C) Quantification of APOBEC3 mutational signatures among three tumor types that have previously been associated with high APOBEC3 activity. Breast, bladder, and cervical cancer samples from the TCGA database were divided into CCT-mutated and non-mutated genomes, as above. The number of mutations contributing to SBS 2 and SBS 13 in each tumor type was quantified and displayed as an average. Comparison by Wilcoxon rank sum test yielded p-values denoted.

(D) The contribution of APOBEC3 mutational signatures (SBS 2 and SBS 13) and all other SBS signatures defined by COSMIC v2 was evaluated in all tumors and independently in breast, bladder, and cervical cancer genomes within the TCGA database. A comparison of mutational signature contributions between CCT-mutated and non-mutated tumors is shown. A z test of proportions was used to compare whether the percentage of 'other' signatures is significantly different between CCT-mutated and non-mutated tumors, p-values show statistical significance in breast and cervical cancers but not in bladder cancer.

Data information: Box and whisker plots depict median and interquartile range (25-75%).

EXPANDED VIEW FIGURE LEGENDS
Determinants of epigenetic resistance to HDAC inhibitors 
in dystrophic fibro-adipogenic progenitors (2022)
Figure 2

Figure 2. Integrative analysis of ChIP- and RNA-seq data reveals pathological features reminiscent of cellular senescence in old mdx FAPs. YC: young mdx control; OC: old mdx control.

A) Acetylation tracks for H3K9/14ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in YC and OC FAPs;

B) Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR;

C) Graphs showing the H3K9/14 acetylation levels for the representative genes shown in (A) measured by ChIP-qPCR.

D) Acetylation tracks for H3K27ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in young and old mdx FAPs;

E) Graphs showing RNA levels for the representative genes shown in (D)) measured by qPCR;

F) Graphs showing the H3K27 acetylation levels for the representative genes shown in (D) measured by ChIP-qPCR.

G) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days (EdU in red/DAPI in blue). Scale bar = 25 μm.

H) Graph showing the percentage of EdU positive cells for the staining shown in (G).

I) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs (LMNB1 in green/DAPI in blue). Scale bar = 25 μm.

J) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number,in the staining shown in (I).

K) Graph showing the relative expression of Lmnb1 in YC and OC FAPs measured by qPCR.

L) Representative images of the γH2AX and KI-67 staining in freshly isolated YC and OC FAPs (γH2AX in green/KI-67 in red/DAPI in blue). Scale bar = 10 μm. Magnification in the insert (scale bar = 3 μm) shows γH2AX positive CCF in OC FAPs.

M) Graph showing the percentage of cells positive for γH2AX positive CCF, for the staining shown in (L).

N) Graph showing the percentage of cells positive for KI-67 for the staining shown in (L).

O) Graph showing the relative expression of Ki67 in YC and OC FAPs measured by qPCR.

Data information: All data are presented as average ± SEM (n=3, biological replicates); (*) indicates statistical analysis by Student's t-test, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001.
Determinants of epigenetic resistance to HDAC inhibitors 
in dystrophic fibro-adipogenic progenitors (2022)
Figure 4

Figure 4. HDACi reduce the activation of SASP-related signalling pathways in old mdx FAPs. YC: young mdx control; YT: young mdx in vivo treated with TSA for 15 days; OC: old mdx control; OT: old mdx in vivo treated with TSA for 15 days.

A) Acetylation tracks for H3K9/14ac and H3K27ac ChIP-seq (on the left) and corresponding FPKM values by RNA-seq (on the right) for representative genes in YC, YT, OC and OT FAPs.

B) Graphs showing RNA levels for the representative genes shown in (A) measured by qPCR.

C) Graphs showing the H3K9/14 acetylation levels for the representative genes shown in (A) measured by ChIP-qPCR.

D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm;

E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D)

F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm;

G) Box plot showing the percentage of pP38 positive FAPs in the staining shown in

(F).

H) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm;

I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).

Data information: In (B, C) data are presented as average ± SEM, in (E, G, I) the central band of the boxplot shows the median, the box estends from the 25th to 75th percentiles, whiskers are down to the minimum and up to the maximum value (n=3, biological replicates); (*) indicates statistical analysis by Student's t-test in the comparison against YC, *P≤0.05, **P≤0.01, ***P≤0.001, ****P≤0.0001; (†) indicates statistical analysis by Student's t-test in the comparison against OC, †P≤0.05, ††P≤0.01, †††P≤0.001, ††††P≤0.0001; (#) indicates statistical analysis by one-way ANOVA, #P≤0.05, ##P≤0.01, ###P≤0.001 ####P≤0.0001.…
Prolonged contact with dendritic cells turns lymph node-resident NK cells into antitumor effectors (2016)
Figure 2

Figure 2. Effect of LPS on tumor vascularization.

(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel. (B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.
Prolonged contact with dendritic cells turns lymph node-resident NK cells into antitumor effectors (2016)
Figure 3

Figure 3. Anti-tumor effector NK cells are generated in the lymph node.

(A,B) Absolute numbers of IFN-+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics. (C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme. (D) Percent of IFN-+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.
Prolonged contact with dendritic cells turns lymph node-resident NK cells into antitumor effectors (2016)
Figure 4

Figure 4. Blocking NK cell recruitment at the draining lymph node does not interfere with the generation of effector NK cells.

(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cell ingress in the lymph node from blood. Where indicated mice were challenged with LPS. (B) Absolute numbers of IFN-+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment. (C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration. (D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN- antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown. (E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment. (F) Percent of IFN-+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody. (A, B, C, E, F) n (number of animals per group)=4. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.
Prolonged contact with dendritic cells turns lymph node-resident NK cells into antitumor effectors (2016)
Figure 7

Figure 7. IL-18 is the contact-dependent signal required in DC-NK cell interactions.

(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m. (B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures. (C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS–activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 1

Figure 1. TG2 transamidase activity increases during C. trachomatis infection along with its expression.

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).

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.

F - 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.
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 2

Figure 2. TG2 activity is needed for optimal C. trachomatis developmental and enhances hydrosalpinx upon C. muridarum infection in mice.

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.

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.
Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells (2020)
Figure 3

Figure 3. TG2 controls glucose import.

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.

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 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 > 0.05.

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.