EMBO/soda-nel huggingface dataset:
CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2 (2022)
Figure 5

Figure 5. CBX4 SUMOylates EZH2.

A. CBX4-SUMOylated candidates formed a large STRING network, which were further clustered into 12 sub-clusters utilizing k-means clustering. These 12 interconnected sub-clusters were shown in different colors.

B. SUMOylated proteins were proceeded to Gene Ontology (GO) analysis utilizing PANTHER classification system, which were classified into 21 functional classes.

C-F. Four highly interconnected STRING networks including mRNA splicing complex, PcG protein complex, NuRD complex and activation of SUMOylation.

G. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO1, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO1-EZH2 within each group were showed below the figure.

H. HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 or Flag-tagged CBX4, and siNC. In the last group, HA-tagged EZH2 was co-overexpressed with Flag-tagged SUMO4, Flag-tagged UBC9 and siCBX4. EZH2 was IP with anti-HA beads. Both total and IP samples were IB with antibodies against HA, Flag and GAPDH. The expression ratios of SUMO4-EZH2 within each group were showed below the figure.
CBX4 contributes to HIV-1 latency by forming phase-separated nuclear bodies and SUMOylating EZH2 (2022)
Figure 7

Figure 7. CBX4 depletion reactivates latent HIV-1 in cells from HIV-1-infected individuals.

A. PHA-activated primary CD4+ T cells were infected with wildtype HIV-1 viruses which harbored a GFP ORF after Nef gene, followed by infecting with shluc and shCBX4 lentiviruses respectively. The percentages of GFP-positive cells, which were HIV-1-expressing cells, were monitored every three days.

B, C. Primary CD4+ T cells were treated as in (A). On Day 6 post infection with HIV-1 viruses and shRNA lentiviruses, ChIP assays with antibodies against H3K27me3 and H2AK119Ub were conducted in both shluc and shCBX4 groups.

D. PHA-activated CD4+ T cells were infected with wildtype HIV-1 viruses as in (A). One group of HIV-1-infected CD4+ T cells were left untreated. One group of HIV-1-infected cells were infected with shluc. Another group of HIV-1-infected cells were infected with shCBX4. Two weeks later, one part of HIV-1-infected CD4+ T cells were reactivated by αCD3/αCD28/IL-2. One part of shluc- and shCBX4-infected cells were co-stimulated with LRA JQ-1. GFP-positive cells in each group were measured by flow cytometry to indicate reactivated HIV-1-infected cells.

E-G. Primary CD4+ T cells, which contained latent HIV-1-infected cells, were isolated from three HIV-1-infected individuals. One group of cells were untreated. One group of cells were activated with αCD3/αCD28/IL-2. Another two groups of cells were transfected with siNC and siCBX4 utilizing 4D-Nucleofector System respectively. Three days post transfection, the amounts of intracellular HIV-1 RNAs within each group were quantitated by RT-qPCR and represented as 103 copies per million CD4+ T cells.

H. The schematic of CBX4 body-mediated HIV-1 latency. Detailed information for the schematic was described in discussion.

Data information: Data represented mean ± SEM in biological triplicate. p-Values in (A-C) were calculated by two-way ANOVA with Sidak's multiple comparisons test. p-Values in (D-G) were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.

Expanded View Figure Legends
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress (2021)
Figure 1

Figure 1. Transcriptional modulation of glycolytic enzymes in productively or latently HIV-1 infected cells.

(A-C) Primary CD4+ T-cells were activated with α-CD3-CD28 beads and infected with HIV-1pNL4-3 or mock-infected. Cells were cultured for two weeks post-infection (p.i.) to model different infection stages (days 3-9 p.i., i.e. productive infection; day 14 p.i., i.e. latent infection) and subjected to microarray (A, B; n=2) or RNA-Seq (C; n=3) analysis. (A) Gene set enrichment analysis (GSEA) of the expression of the glycolytic pathway (HUMAN-GLYCOLYSIS) in mock-infected or HIV-1 infected cells. (B,C) Heatmaps of the relative expression of glycolytic enzymes upon HIV-1 infection. Data are expressed as Log2 fold change in HIV-1 infected vs mock-infected cells. For microarray data (B) expression values of infected and mock-infected cells at different time points were pooled. For RNA-Seq data (C), expression values in infected cells were normalized using the corresponding time point in mock-infected cells. Adjusted p values (q values) to account for multiple testing were calculated by Significance Analysis of Microarrays [SAM (Tusher et al, 2001)] and Deseq2 for RNA-Seq data (Love et al, 2014).

(D-F) scRNA-Seq of the expression of the entire glycolytic pathway or of glucose phosphate isomerase only (GPI) in primary CD4+ T-cells infected in vitro (D,E) or CD4+ T-cells of PLWH (F). In panels (D, E), cells were infected with VSVG-HIV-1-GFP and sorted for viral expression as detailed in (Golumbeanu et al, 2018). Following latency establishment, cells were left untreated or HIV-1 expression was reactivated through suberoyl anilide hydroxamic acid (SAHA) or α-CD3-CD28 engagement. Clusters 1 and 2 were identified by principal component analysis as described in (Golumbeanu et al, 2018). In panel (F), CD4+ T-cells were isolated from total blood of PLWH under ART as described in (Cohn et al, 2018). Viral expression was reactivated by treatment with phytohemagglutinin (PHA) and cells were sorted using antibodies against Env and Gag. Sorted cells were then subjected to scRNA-Seq analysis. The expression level of the HUMAN-GLYCOLYSIS pathway in (D) was calculated as the average expression of genes comprising the gene list; expression levels in cluster 1 and 2 were compared using Wilcoxon rank sum test. For panels (E, F), significance of GPI differential expression level between clusters (E) or between control and Env+ Gag+ conditions (F) was assessed by the Wilcoxon rank sum test encoded in FindMarkers Seurat R function. ** p< 0.01, *** p< 0.001; *** p< 0.0001. For panels (D,E) n = 1 donor, 43 cells (untreated),

90 cells (SAHA), 91 cells (TCR), for panel (F) n = 3 donors, 109 cells (control) and 85 cells (Gag+ Env+).
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress (2021)
Figure 4

Figure 4. Relative expression of HIV-1 gag and genes regulating the pentose phosphate pathway or antioxidant responses in CD4+ T-cells infected with wild type and Tat-deficient HIV-1.

Primary CD4+ T-cells were isolated from total blood of healthy donors and activated with α-CD3-CD28 beads for 72 h. Cells were then mock-infected or infected with wild type HIV-1pNL4-3 or with Tat-deficient HIV-1pNL4-3 (Bejarano et al, 2019). Cells were cultured for one week post-infection and gene expression was measured by qPCR.

(A) Relative expression of HIV-1 gag in cells infected with Tat-deficient HIV-1pNL4-3 as compared to cells infected with wild type HIV-1 pNL4-3.

(B-D) relative expression of the limiting rate enzyme of the pentose phosphate pathway, i.e. G6PD (B), and of genes regulating the thioredoxin and glutathione antioxidant pathways, i.e. TrxR1 (C) and GCLC (D), in HIV-1 infected as compared to mock infected cells.

Data information: Data were first normalized using 18S as housekeeping control and then expressed as Log2 fold mRNA expression in wild type vs Tat-deficient infection (panel A) or in infected vs mock infected cells (panels B-D), which were calculated using the 2-ΔΔCT method (Livak & Schmittgen, 2001).

GCLC= Glutamate-cysteine ligase; TrxR1= thioredoxin reductase 1. Data are expressed as mean ± SD of two replicates and were analyzed by Two-Way ANOVA followed by Sidak´s post-test. * p< 0.05
Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress (2021)
Figure 5

Figure 5. "Shock and kill" effect of combined thioredoxin (Trx) and glutathione (GSH) inhibition in primary lymphoid models of HIV-1 infection.

(A, B) Reactivation from HIV-1 latency (A) and relative cell viability (B) in primary Th17 cells following 24 h treatment with the Trx inhibitor auranofin (AF; 500 nM), the GSH inhibitor buthionine sulfoximine (BSO; 250 μM), or a combination of the two. GFP-HIV-1 expression was determined by FACS. Data are expressed as mean ± SD of three replicates and were analyzed by One-Way ANOVA followed by Tukey's post-test (A) or Two-Way ANOVA followed by Sidak´s post-test (B). Solid lines represent the means.

(C) Levels of integrated HIV-1 DNA following treatment for 48 h with AF and/or BSO in CD4+ T-cells derived from PLWH under suppressive antiretroviral therapy. Live cells were sorted after treatment, and integrated DNA was measured by Alu-PCR. The latency reactivating agent SAHA was used as a reference compound (Archin et al, 2012). Data were analyzed by non-parametric Friedman´s test followed by Dunn's post-test.

Data information: * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.

.
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 1

Figure 1: Monoclonal and polyclonal anti-AdV antibodies trigger inflammasome activation in human macrophages

(A) THP-1 cells were stimulated with (50 000 particles/cell) AdV-GFP and infection after 24 h measured by flow cytometry.

(B,C) Cells were stimulated as in (A) with AdV-GFP or AdV-GFP complexed with 20 μg/ml h9C12 or 20 mg/ml human IVIg. Cell supernatants were harvested at indicated time-points and cytokines measured by ELISA (B) or pro- and cleaved IL-1β was measured in whole cell lysates or in supernatants by western blot (C). Data is representative of two independent experiments. (D) HMDM were primed with 10 μg/ml pI:C for 2h, then stimulated for 16h with virus +/- antibody. Cytokines in the supernatants were measured by ELISA. (n = 7, mean ± s.e.m. *P-value ≤ 0.05, **P-value ≤ 0.005, paired, two-tailed t-test)
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 2

Figure 2: Opsonized AdV- dependent IL-1β and TNF release can occur without concurrent cell death.

IL-1 β, TNF (left axis) and LDH release (right axis) in the cell supernatants were measured 16 h after stimulation.

(A) AdV-GFP was incubated with 20 ug/ml h9C12 antibody or 20 mg/ml IVIg in PBS. These AdV-Ab complexes were then diluted 1:2 to give final doses of viral particles being added to WT THP-1 cells (with antibody complexed) as indicated. A single experiment representative of 3 independent experiments is shown.

(B) THP-1s were stimulated with a constant dose of AdV-GFP (50 000 pp/cell), and complexed with decreasing antibody concentrations as indicated. A single experiment representative of 2 independent experiments is shown.

(C,D) HMDM were stimulated directly (TNF) or primed (IL-1β, LDH) with AdV-Ab complexes as in (B). Data for each individual donor (2 total) is shown.

(E,F) THP-1 were stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release (E) or cell viability measured by PrestoBlue assay (F) (n=3, mean ± s.e.m. *P-value ≤ 0.05, **P-value ≤ 0.005, ***P-value ≤ 0.001 unpaired, two-tailed t-test)

(G, H) HMDM were primed, or not, and stimulated with AdV (50 000 pp/cell) complexed with h9C12 (20 ug/ml) or IVIg (20 or 0.8 mg/ml) for 16h and cell death was measured by LDH release (G) or cell viability measured by PrestoBlue assay (H). (n = 4, mean ± s.e.m. *P-value ≤ 0.05, **P-value ≤ 0.005, paired, two-tailed t-test)
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 4

Figure 4: IL-1β and TNF release in response to AdV and h9C12 monoclonal antibody is TRIM21 dependent

(A) Schematic showing the h9C12 antibody mutants that no longer engage Trim21 (H433A) or FC receptors (LLAA).

B, C (B) Primed or (C) un- primed HMDM were stimulated with AdV (50 000 pp/cell) +/- h9C12 (20 μg/ml) for 16h. (n = 4, mean ± s.e.m. *P-value ≤ 0.05, paired, two-tailed t-test)

(D) Ctrl or Trim21- deficient THP-1s were stimulated for 4 h with 1000 U/ml IFNα and TRIM21 expression measured by immunoblot.

(E,F) WT or TRIM21- deficient THP-1s were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 or 20 mg/ml IVIg or with 200 ng/well HT-DNA or 10 μM Nigericin for 16h, and IL-1β (E) and TNF (F) measured in the supernatant by ELISA. (n = 4 (E) or n = 3 (F), mean ± s.e.m. *P-value ≤ 0.05, **P-value ≤ 0.005, ***P-value ≤ 0.001 unpaired, two-tailed t-test)

(G) WT mice were injected i.v. with 2.5ug WT or H433A h9C12 antibody, then the next day injected i.v. with 2.5 x 1011 pp AdV-GFP. 4h later spleens were harvested and neutrophil influx measured by flow cytometry. (n = 6 mean ± s.e.m. ***P-value ≤ 0.001 unpaired, two-tailed t-test)

(H) HMDM were stimulated with AdV-GFP (250 pp/cell) in the presence of h9C12 antibodies at indicated doses. Infection was measured after 24h by flow cytometry. Infection relative to AdV alone is shown graphed on the left (n = 4 mean ± s.e.m). Representative plots and a comparison of the different h9C12 mutants at 0.2 ug/ml are also shown in the graph on the right(n = 4,mean ± s.e.m.).
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 5

Figure 5: TRIM21 is involved in inflammasome activation rather than enhancing pro-IL-1β expression.

(A) HMDM were stimulated with AdV (50 000 pp/cell) and antibody (h9C12; 20 ug/ml, IVIg: 20 mg/ml) or 10 μg/ml pI:C for 3h and gene expression was measured by qPCR (n = 4 mean ± s.e.m).

(B) HMDM were stimulated as in (A), or with 10 ng/ml LPS for 6 h and pro-IL-1β levels in the cytosol measured by western blot. Blot is representative of three independent donors.

(C) HMDM were stimulated as in (A), or with 10 ng/ml LPS for 3 h and NLRP3 mRNA expression measured by qPCR. Data shows average ± s.d. of 2 independent donors

(D) HMDM were stimulated as in (B) and NLRP3 levels in the cytosol measured by western blot. Blot is representative of two independent donors.

(E) TLR9 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve monocytes or HMDM derived from CD14+ve monocytes were assessed by qPCR and copy number was determined relative to actin copy number (n = 5 mean ± s.e.m).

(F) THP-1s expressing ASC-GFP were stimulated for 6 h with virus and antibody or 200 ng/well HT-DNA or 10 μM Nigericin, in the presence of the pan-caspase inhibitor zVAD-fmk. A representative image (scale bar 100 μm) and quantification of number of cells with ASC specks from three independent experiments (mean ± s.e.m, *P-value ≤ 0.05, paired, two-tailed t-test) is shown.
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 6

Figure 6: NLRP3 is required for inflammasome activation downstream of TRIM21

(A,B,C) Primed HMDM were treated with MCC950 (1 μM) for 30 mins before stimulation overnight with AdV (50 000 pp/cell) and h9C12 (20 μg/ml) or IVIg (20 mg/ml) or 10 μM Nigericin. IL-1β (A) or TNF (B) or LDH release (C) were measured in cell supernatants. Data shows average ± s.d. of two independent donors and is representative of three independent experiments.

(D) WT, NLRP3, ASC or Caspase-1 - deficient THP-1s were stimulated as in (A) and IL-1β in the supernatant measured by ELISA. Data is representative of two independent experiments.

(E) HMDM were primed with 10 ug/ml pI:C for 2h, treated with 100 μM VX-765 for 30 mins before stimulation for 16 h as in (A). IL-1β or LDH release was measured in cell supernatants. Data shows average ± s.d. of two independent donors

(F) Primed HMDM were treated with KCl as indicated for 1h, before being stimulated for a further 6h as in (A). IL-1β and TNF were measured in cell supernatants by ELISA. Data is representative of three independent donors.

(G) Primed HMDM were stimulated with AdV and 20 μg/ml h9C12 or 10 μM Nigericin for 3h, and then immunostained for ASC or intracellular antibody. Co-localisation was assessed by confocal microscopy. Data is representative of two independent donors.
Antibody and DNA sensing pathways activate the inflammasome during human macrophage infection. (2019)
Figure 7

Figure 7: Trim21 exposes AdV genomes to cGAS and STING to trigger NLRP3- dependent inflammasome activation

(A) AIM2 mRNA levels from PBMCs, CD19+ve B cells, CD14+ve Monocytes or HMDM derived from CD14+ve monocytes. were assessed by qPCR and copy number was determined relative to Actin copy number (n = 5 mean ± s.e.m).

(B) HMDM were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12 for 1h, washed 2x with SFM, then whole cell lysates harvested after a further 5h. Viral hexon and transgene (GFP) expression in the cytosol was assessed by western blot. Data is representative of two independent experiments.

(C) THP-1s expressing either a control guide RNA or targeting cGAS and STING were generated and stimulated with 1000 U/ml IFNα for 4 h and protein levels assessed by western blot.

(D) THP-1s deficient in cGAS and STING were stimulated with AdV (50 000 pp/cell) and 20 μg/ml h9C12, 200 ng/ well HT-DNA, 10 μM Nigericin or 10 ng/ml LPS for 16 h. Data shows combined data (mean ± s.e.m) of 3 experiments with absolute protein values (upper panel) or as % cytokine output of KO cells relative to Ctrl treated cells (lower panel), *P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).

(E) WT THP-1s were treated with 5 μM H151 for 30 mins before stimulation as in (D). Data in upper panel is representative of 3 independent experiments. Data in lower panel shows combined data of these 3 experiments (mean ± s.e.m) showing H151 treated cells relative to media treated cells (*P-value ≤ 0.05, ***P-value ≤ 0.001 unpaired, two-tailed t-test).

(F) ASC-GFP THP-1s were treated with 5 μM H151 for 30 mins before stimulation with AdV-mCherry (50 000 pp/cell) + 20 μg/ml h9C12 or 20 mg/ml IVIg or 200 ng/ well HT-DNA for 8h. A representative image (scale bar 50 μm) and quantification of number of cells with ASC specks from one representative experiment of three is shown.
Actin filaments regulate microtubule growth at the centrosome. (2019)
Figure 1

Figure 1: Cytoskeleton remodelling in B lymphocytes upon antigen stimulation.

IIA1.6 B lymphoma cells were stimulated with BCR-ligand- (IgM) or BCR-ligand+ (IgG) beads for 60 min, fixed and stained for F-actin (top) and α-tubulin (bottom). Scale bar: 3 µm.

Histograms show the quantifications of the polymerized tubulin and filamentous F-actin at the centrosome (dashed outline on the image, values correspond to the fraction of fluorescence in a 2-micron-wide area around the centrosome relative to the total fluorescence in the cell) and the total amount of polymerized fluorescent tubulin (bottom right, values were normalized with respect to the mean of control condition). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with Mann-Whitney test. Scale bar: 2 µm.

Percentage differences of centrosomal F-actin and centrosomal microtubule fluorescence intensities in cells stimulated with BCR-ligand+ beads with respect to cells stimulated with BCR-ligand- beads. The data set is identical to panel (B). Measurements were pooled from 3 independent experiments; IgM (BCR-ligand-): n= 88; IgG (BCR-ligand+): n= 93. Error bars correspond to standard deviations. P values were calculated with one-sample t-test (i.e. comparison to a theoretical mean of "0").

The graph shows the variations of the total amount of tubulin per cell with respect to the content of actin at the centrosome in an XY representation of individual measurements. The two lines correspond to linear regressions of the two sets of data relative to cells stimulated with BCR-ligand+ (activated cells) or BCR-ligand- (resting cells) beads.

E- - IIA1.6 B lymphoma cells were fixed and immuno-stained for F-actin (red) and microtubules (green). Images show the projection of maximal intensity of three confocal slices spaced by 0.5 µm apart from the centrosome. Scale bars: 2 µm (0.5 µm in the zoomed insets).
Actin filaments regulate microtubule growth at the centrosome. (2019)
Figure 3

Figure 3: Assembly of microtubules and actin filaments on isolated centrosomes.

Two sets of representative images showing fluorescent microtubules and actin filaments assembled from isolated centrosomes. Centrosomes were isolated from Jurkat cells expressing centrin1-GFP. Upper and lower lines show actin filaments and microtubules radiating from two distinct centrosomes with low (top) and high (bottom) densities of actin filaments. Scale bars: 10 μm.

The graph shows the number of microtubules per centrosome relative to the density of actin filaments. Inset shows actin filaments at the centrosome with a FIRE look-up table and a 20 µm-wide circle in which actin fluorescence intensity is measured. Measurements were pooled from 5 independent experiments; n= 50.

Microtubules (top line) and actin filaments (bottom line) assembly from isolated centrosomes in the presence of increasing concentration of monomeric actin (from left to right). Scale bar: 20 μm.

The graph shows the number of microtubules per centrosome in response to increasing concentrations of monomeric actin. Data were pooled from 2 independent experiments; 0 µM: n=21; 0.3 µM: n=17; 0.5 µM: n=17; 1.0 µM: n=17. ****:P<0.001 Mann-Whitney test.

The image shows the density of actin filaments (in the presence of 1µM of actin monomers) at the centrosome color-coded with the FIRE look-up table and the definition of central, peripheral and distal regions corresponding to decreasing concentrations of actin filaments. Scale bar: 20 μm. The graph shows the measurements of microtubule growth rate in each region. Data were pooled from 3 independent experiments: central zone: n=58, peripheral: n=104, outside: n=61; n. s. means no statistical difference between the data set according to Mann-Whitney test.

The graphs show the various intensities of centrosome immuno-staining with antibodies against gamma-tubulin on the same coverslip depending on the presence/absence of actin filaments (left) or on the amount of actin filaments (right). Data were pooled from 3 independent experiments. Left graph: without actin n=69, with actin n=26, right graph n=26. N.s. means no statistical difference between the data set according to Mann-Whitney test.