EMBO/soda-nel huggingface dataset:
Npl3 stabilizes R-loops at telomeres to prevent accelerated replicative senescence (2020)
Figure 3

Figure 3 NPL3 stabilizes R-loops.

A-B) Npl3 associates to telomeres in early S. A) Cell cycle profiles of indicated strains. Cells were synchronized in G1 with alpha-factor for 2.5h and released into the cell cycle at 30°C in pre-warmed medium supplemented with the indicated HU concentrations. B) Npl3-TAP associates strongly to telomeres in early S (250 mM HU). Cross-linked samples from indicated strains were used in a TAP-ChIP. Enrichment at telomeres was determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to cells arrested in alpha factor n=3 (paired t-test two tailed *p<0.05, **p<0.01).

C) NPL3 over expression stabilizes telomeric R-loops. The indicated strains were grown on 1% raffinose 2% galactose to induce NPL3 over expression. Cross-linked samples were subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM relative to 250 mM HU eV n=3 (paired t-test two tailed *p<0.05, **p<0.01).

D) Deletion of NPL3 decreases telomeric R-loops. The indicated strains were crosslinked and subjected to DRIP. DNA-RNA hybrids were immunoprecipitated using the S9.6 antibody. R-loop levels were determined by quantitative PCR on indicated telomeres. Data represents mean % input +/- SEM n=3. Specificity of S9.6 antibody was determined treating a fraction of the samples with RNase H.

E-G) NPL3 stabilizes R-loops. E) R-loop dot blot. The indicated strains were grown on 1% raffinose 2% galactose for 72h and subsequently released into 1% raffinose 2% galactose or 2% glucose medium for 6 PDs. R-loop levels and dsDNA levels were determined using the S9.6 antibody and anti-dsDNA antibodies. Specificity of the S9.6 antibody was confirmed by treatment with RNase H. Loaded genomic DNA content is indicated at the bottom of the figure. F) R-loop dot blot quantification. Data represents R-loop signal as the S9.6 antibody signal relative to corresponding dsDNA signal n=3 (unpaired t-test two tailed *p<0.05). G) Serial dilutions of indicated strains were assayed on 1% raffinose 2% galactose media. Plates were imaged after 72h incubation at indicated temperatures.

Abbreviations: ns: not significant, eV: empty vector control, oE: over expression, ds: double-strand, HU: hydroxyurea. *p<0.05, **p<0.01
Wave patterns organize cellular protrusions and control cortical dynamics (2019)
Figure 1

Figure 1. Two distinct networks coordinately present as cortical excitable waves.

A Left, time lapse confocal images of PHcrac (red) and LimE (green) in waves at the basal surface of a giant cell. Right, intensity plot across the blue dotted arrow in image "30 sec".

B-E Various biosensors in excitable waves. B and D, snapshots of biosensors (top) and intensity plots (bottom) across the blue dotted arrows, with black plots showing the corresponding biosensors in images above, red (B) showing PHcrac in the same cell, and green (D) showing LimE. C and E, Scatter plots of biosensor peak locations relative to the peak of PHcrac (C) or the first peak of LimE (E) across propagating waves (blue solid lines indicate median). Positive values indicate that the peak precedes that of PHcrac (or LimE) along the direction of wave propagation. n=26, 26, 24, 21, 21, 32 cells for RalGDS, RBD, PKBA, RacGEF1, PAK1-GBD, and Coronin, respectively. Scale bars in images above represent 20 μm. Grey arrows in all plots represent the direction of wave propagation.

F Left, confocal images of PHcrac (red) and PAK1-GBD (green) in the same cells treated with LatA. Scale bars represent 10 μm. Right, temporal profiles of normalized mean cytosolic intensities of PHcrac (red) and PAK1-GBD (green) (mean±s.e.m., n=18 cells) in response to cAMP.

G Cartoon illustrating components and their intensity profiles during wave propagation.
Wave patterns organize cellular protrusions and control cortical dynamics (2019)
Figure 4

Figure 4. Perturbations to STEN reveal its molecular architecture.

A The STEN-CEN model architecture where Fs and Rs (Fc and Rc) denote the activator and inhibitor of STEN (CEN) respectively, showing the feedback connections between CEN and STEN. The positive feedback is indicated through the arrowhead while the bar at the end of the arrow refers to negative feedback.

B Time lapse images showing simulations of the coupled STEN-CEN model demonstrating CEN (top) and STEN (bottom) waves spreading in space. The threshold was lowered by increasing positive feedback of STEN (perturbation) at time 0. The white arrow (time 0, CEN) denotes a wave at a high threshold that disappears in the next image, while the red arrow (time 25, CEN) points to a lowered threshold wave that enlarges and spreads.

C A 3D representation of simulation waves under high (left) and low (right) threshold waves propagating in time.

D Quantification of simulated wave speed before and after threshold lowering (+perturb). Statistics are from 8 simulations. Error bars are from the student's t-test.

E Time lapse confocal images of LimE at the basal surface of a giant cell, which recruits Inp54p to membrane induced by rapamycin at time 0. The white arrow (-45 s) points to a nascent wave before Inp54p recruitment, while the red (60 s) arrow after.

F Color coded overlays of LimE before and after Inp54p recruitment. White arrows point to nascent waves at time 0.

G Kymographs of LimE in regions outlined by dotted line boxes in f.

H, I Box plots of mean wave speed (left) and fraction of fastest pixels (right) before and after Inp54p (H) or FRB (I) recruitment (red bars indicate median, n=25 cells each). Error bars are from the student's t-test.

J Time lapse confocal images of PHcrac at the bottom of giant cells under LatA treatment, where rapamycin is added at time 0 to induce Inp54p recruitment. White arrows point to wave patches right before rapamycin addition.

K Confocal image of PI5K (left) and LimE (right) at the bottom of the same cell. All scale bars in images represent 20 μm.

L Cartoon highlighting positive feedbacks in STEN.
Wave patterns organize cellular protrusions and control cortical dynamics (2019)
Figure 7

Figure 7. CEN exerts fast positive and slow negative feedbacks to STEN.

A Time lapse confocal images of PHcrac at the basal surface of a giant cell, which recruits RacGEF1ΔN to membrane at time 0 induced by rapamycin.

B Confocal images of PHcrac (left) and PHcrac merged with LimE (right, PHcrac as red, LimE as green) in a giant cell after RacGEF1ΔN recruitment.

C Kymograph of LimE (top, green) and PHcrac (bottom, red) across the dotted line in B.

D Confocal image of RalGDS (left) in a giant cell after RacGEF1ΔN recruitment and the corresponding kymograph (right).

E Time lapse confocal images of PHcrac in single cells, which recruit RacGEF1ΔN to membrane induced by rapamycin at time 0. White arrow points to PHcrac patches at cell cortex.

F Kymographs of PHcrac at cell cortex before and after RacGEF1ΔN recruitment.

G Temporal profiles of normalized PHcrac intensities at cell cortex before and after RacGEF1ΔN recruitment (mean±s.e.m., n=20 cells). Red dashed lines in F and G indicate rapamycin addition at time 0.

H Distribution of different sizes of PHcrac patches at cell cortex under LatA treatment, with (solid) or without (hollow) RacGEF1ΔN recruitment (~6000 cells from 3 independent experiments).

I Temporal profiles of normalized cytosolic intensities of PHcrac in response to 1 nM cAMP stimulation at time 0, with (red) or without (blue) RacGEF1ΔN recruitment (mean±s.e.m., n=25 cells each).

J Confocal images of GflB (top) and RapGAP1 (bottom) in waves at the basal surface of a giant cell.

K Intensity plots of GflB (blue) and RapGAP1 (orange) across the green dotted arrow in J.

L Scatter plots of first peak distances, of GflB relative to LimE, RapGAP1 relative to LimE, and RapGAP1 relative to GflB in the same giant cells (red lines indicate median, n=19 cells each).

M Confocal images of GflB (top) and RapGAP1 (bottom) at the basal surface of the same giant cell after RacGEF1ΔN recruitment.

N kymographs of GflB (top) and RapGAP1 (bottom) across the green dotted lines in m. All scale bars represent 20 μm. (o) Cartoon highlighting positive and negative feedback loops from CEN to STEN.
Wave patterns organize cellular protrusions and control cortical dynamics (2019)
Figure 8

Figure 8. Perturbations causing different cortical patterns also altered cellular protrusions correspondingly.

A, B Left, confocal images of LimE (top) and kymographs of cortical LimE (bottom) in single cells with PKBA (A) or RacGEF1ΔN (B) steadily recruited (scale bars, 10 μm). Right, color coded overlays of LimE in single cells.

C Box plots of roundness (left) and cell area (middle) comparing wt, cells with RacGEF1ΔN recruited, and spreading phases of oscillators after Inp54p recruitment (n=100 cells each). Right, box plots of coefficient of variance of cell area with the above conditions in a 10-minute time window (n=30 cells each). Red bars indicate median.

D Phalloidin staining of cells after perturbations. Maximal intensity projections are shown.

E Snapshots from level set simulations using the activity obtained from earlier simulations.
Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS (2022)
Figure 1

Figure 1: cGAS induces type I IFNs in response to VZV infection.

(A) Schematic detailing the experimental procedure for infection of PMA-differentiated THP1 cells with VZV by transwell assay. See text for details.

(B) Immunoblot of WT, cGAS KO, and MAVS KO THP1 cells used in (C-G).

(C) THP1 cells of the indicated genotypes were mock infected or infected with VZV for 24 and 48 hours using the transwell assay described in (A). Infected cells were quantified by surface staining for VZV-gE/gI and flow cytometry analysis. See Appendix Fig S3 for gating.

(D) RT-qPCR analysis of VZV ORF63 (immediate early (IE) gene), ORF54 (early (E) gene), and ORF40 (late (L) gene) transcripts in cells infected as in (C). Graphs show expression relative to GAPDH.

(E) RT-qPCR analysis of IFNB1, IFI44, and IFIT1 expression in cells infected as in (C). Graphs show expression relative to GAPDH.

(F) Activity of Lucia luciferase (secreted under an IRF3-dependent promotor) was determined in supernatants of cells infected as in (C) by QUANTI-Luc assay.

(G) Concentrations of CXCL10 in supernatants of cells infected as in (C) were determined by ELISA.

Data information: Panel (B) is representative of two independent biological experiments. Panels (C), (F), and (G) show pooled data from four repeats, where each data point represents an independent biological experiment (n=4 ± SEM). Panels (D) and (E) show pooled data from three repeats, where each data point represents an independent biological experiment (n=3 ± SEM). Statistical analysis in panels (C), (D), (F), and (G) was paired t-tests and in panel (E) paired ratio t-tests. hpi: hours post infection, WT: wild type, KO: knockout.

See also Fig EV1 and Appendix Fig S1, S2, and S3.
Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS (2022)
Figure 3

Figure 3. ORF9 interacts with cGAS.

(A) HEK293T cells were transfected with expression plasmids for ORF9-V5, human cGAS-FLAG, and human STING-HA. The next day, cells were lysed, and overexpressed proteins were immunoprecipitated with α-V5, α-FLAG, α-HA, or IgG isotype control antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies.

(B) THP1 monocytes stably transduced with either VZV FLAG-ORF9 or FLAG-GFP were PMA-differentiated overnight. The next day, cells were lysed and ectopically expressed proteins were immunoprecipitated using α-FLAG antibody. Input, unbound and IP fractions were subjected to immunoblotting using the indicated antibodies.

(C) HaCaT cells and PMA-differentiated THP1 cells were infected with WT VZV or VZVORF9-V5 through co-culture with infected MeWo cells for 48 hours. Cells were lysed and ORF9 was immunoprecipitated using α-V5 antibody. Input and IP fractions were subjected to immunoblotting using the indicated antibodies.

(D) HEK293T cells were seeded onto glass coverslips and were transfected with human cGAS-V5, FLAG-ORF9, or both together. The next day, cells were fixed, permeabilised and stained using α-V5-FITC, rabbit-α-FLAG, and goat-α-rabbit-AF647 antibodies, and DAPI. Mounted coverslips were imaged using confocal microscopy. Scale bars: 15 µm.

(E) Outline of the far western protocol.

(F) Far western analysis of cGAS-ORF9 interaction. Recombinant FLAG-ORF9 and FLAG-GFP protein were run on an SDS-PAGE gel and transferred to a membrane. After renaturation of proteins, the membrane was incubated with recombinant human cGAS as a probe, which was detected using α-cGAS antibody. After stripping, the proteins on the membrane were detected using α-FLAG antibody.

(G) Recombinant human cGAS-HA was mixed with recombinant FLAG-ORF9 or FLAG-GFP. Proteins were immunoprecipitated using α-HA and α-FLAG antibodies. Input, unbound and IP fractions were analysed by immunoblotting.

Data information: Recombinant proteins used in (F) and (G) are shown in Appendix Fig S4A. Panels (A) and (G) are representative of two independent experiments. Panels (B), (D), and (F) are representative of three independent experiments. Panel (C) is representative of two (HaCaT) and three (THP1) independent experiments.
Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS (2022)
Figure 4

Figure 4: VZV ORF9 binds DNA.

(A) Schematic detailing ORF9 truncation mutants used in (B).

(B) HEK293T cells were transfected with expression plasmids for human cGAS and ORF9 truncation mutants. The next day cells were lysed and ORF9 proteins were immunoprecipitated using α-FLAG antibody. IP fractions were subjected to immunoblotting using the indicated antibodies.

(C) Schematic detailing predicted structural features of VZV ORF9 in relation to predicted and crystal structure-derived features in HSV-1 VP22, KSHV ORF52, and MHV68 ORF52. See text for details.

(D) Recombinant cGAS, FLAG-ORF9, FLAG-GFP, and biotinylated VACV70mer dsDNA were incubated in the indicated combinations. Recombinant proteins were precipitated using streptavidin beads. Fractions were analysed by immunoblotting.

(E) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and fractions were subjected to immunoblotting using the indicated antibodies.

(F) VACV70mer dsDNA was incubated with indicated proteins and analysed by agarose gel EMSA. Triangles indicate concentrations of ORF9 (0.7, 1.4, 2.9, 5.7, 8.6 µM), cGAS (0.6, 1.1, 2.2, 4.5, 6.7 µM), and ORF9 (as before) in the presence of 4.5 µM cGAS.

Data information: Recombinant proteins used in (D) and (F) are described in Appendix Fig S4A and S4B, respectively. Results shown in panels (B) and (D-F) are representative of two independent experiments.
Varicella-Zoster Virus ORF9 Is an Antagonist of the DNA Sensor cGAS (2022)
Figure 6

Figure 6: ORF9 inhibits cGAS' catalytic activity.

(A) Protein sequence alignment of VZV ORF9, HSV-1 VP22, KSHV ORF52, and MHV68 ORF52. Residues selected for mutagenesis in ORF9 are highlighted in red. Residue colouring represents physico-chemical properties (green: hydrophilic, blue: positive, coral: aliphatic/hydrophilic, orange: aromatic, yellow: cysteine, red: negative, purple: conformationally special). See text for details.

(B) Schematic detailing position and identity of residues mutated in C-G.

(C) HEK293T cells were transfected with expression plasmids for FLAG-GFP or FLAG-ORF9 as indicated. The next day cells were lysed, and the lysate was spiked with biotinylated VACV70mer dsDNA. DNA was precipitated using streptavidin beads and proteins were analysed by immunoblotting using the indicated antibodies.

(D) HEK293T cells were transfected with expression plasmids for human cGAS and FLAG-GFP or FLAG-ORF9. The next day cells were lysed, and proteins were immunoprecipitated using α-FLAG antibody and analysed by immunoblotting using the indicated antibodies.

(E) THP1 cells stably transduced with GFP, ORF9, or ORF9-DM were transfected with the indicated amounts of E.coli dsDNA. The next day, IFNB1 expression was assessed by RT-qPCR.

(F) In vitro cGAS activity assay. Recombinant hcGAS was incubated with ATP, GTP, and radioactive α32P-ATP at 37°C with addition of dsDNA and other proteins as indicated (triangles represent concentrations of 0.5, 1, 2, 4, 6 µM). ORF9-DM was tested only at 0.5, 1, 2 µM. Reactions were treated with calf-intestinal phosphatase and products were analysed by thin-layer chromatography and phosphorimaging.

(G) Signal intensities from (F) were determined by densitometry analysis and normalized to the average BSA signal.

Data information: Recombinant proteins used in (F) and (G) are described in Appendix Fig S4B. Data shown in panels (C) and (D) are representative of two independent experiments. Panel (E) shows pooled data from two repeats, where each data point represents an independent biological experiment (n=2 +/- range). Panel (F) is representative of three independent experiments. Panel (G) shows pooled data from three repeats, where each data point represents an independent biological experiment (n=3 +/- SD).

EXPANDED VIEW FIGURE LEGENDS
TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment (2022)
Figure 1

Figure 1. TERT expression is associated with immunosuppressive signatures and triggers interferon response.

A Scatterplot representing differences in ssGSEA immune signatures in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA. -log10(padj) for enrichment of ssGSEA immune signatures in TERThigh versus TERTlow tumours is shown on the y-axis. Signatures more highly represented in TERThigh tumours are shown on the right, while those in TERTlow tumours are shown on the left.

B Heat map for ssGSEA of immune signatures in TERThigh and TERTlow tumours (n = 4,632 tumours in each group) across TCGA. Th1, Th2, Th17: T helper 1, 2, 17 cells, Tcm: central memory T cells, Tem: effector memory T cells, Tgd: gamma delta T cells, TFH: follicular helper T cells, Treg: regulatory T cells, MDSC: myeloid-derived suppressor cells, DC: dendritic cells, aDC: activated DCs, iDC: immature DCs, pDC: plasmacytoid DCs, NK cells: natural killer cells.

C Heat map of interferon-related genes in U2OS cells with TERT-WT or TERT-K626A ectopic expression.

D-F RT-qPCR analysis of interferon-related genes (D), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (E), and ELISA of IFNβ and CXCL10 in culture supernatant (F) in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A.

G-I RT-qPCR analysis of interferon-related genes (G), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (H), and ELISA analysis of CXCL10 in culture supernatant (I) in HeLa and HCT116 cells with TERT knockdown.

Data information: Data represent mean ± SEM of three biological replicates (D, F, G and I). *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. One-way ANOVA with Fisher's LSD tests were used for D and F, unpaired two-tailed Student's t-tests were used for G and I. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (E and H).
TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment (2022)
Figure 2

Figure 2. TERT triggers interferon response via induction of dsRNA transcribed from ERVs.

A Confocal microscopy of U2OS cells ectopically expressing TERT-WT or TERT-K626A. Total dsRNA is stained green, and nuclei are stained blue (DAPI). Scale bar = 20 µm.

B Genome-wide upregulation of ERVs in U2OS cells ectopically expressing TERT-WT or TERT-K626A. RNA-seq data for TERT-WT/TERT-K626A and empty vector control were used to calculate the log2 fold change values. Red dots indicate elements with increased expression, while blue dots indicate the elements with decreased expression.

C Representative TA-ERVs activated by TERT-WT or TERT-K626A.

D RT-qPCR analysis of TA-ERVs in U2OS and HeLa cells ectopically expressing TERT-WT or TERT-K626A.

E RT-qPCR analysis of TA-ERVs in HeLa and HCT116 cells with TERT knockdown.

F-H RT-qPCR analysis of interferon-related genes and TA-ERVs (F), western blot analysis of TERT, TBK1, pTBK1, IRF3, pIRF3, and GAPDH levels (G), and ELISA of IFNβ and CXCL10 in culture supernatant (H) in U2OS cells ectopically expressing TERT-WT or TERT-K626A using Dox-inducible system. GFP was used as negative control. Relative levels of pTBK1 and pIRF3 were quantified with ImageJ software and normalized to GAPDH, as indicated at the bottom of the blot (G).

Data information: Data represent mean ± SEM of three biological replicates (D, E, F and H). *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. One-way ANOVA with Fisher's LSD tests were used for D, F and H, unpaired two-tailed Student's t-tests were used for E.
TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment (2022)
Figure 3

Figure 3. TERT activates ERVs by interacting with Sp1.

A Identification of dsRNAs activated by TERT-WT or TERT-K626A using dsRIP-seq in U2OS cells. AluYb9 and tRNA-Thr-ACG as positive and negative controls, respectively.

B Quantification of antisense/sense transcripts of ERVs activated by TERT-WT or TERT-K626A in U2OS and HeLa cells using strand-specific RT-qPCR.

C Regression analysis of the correlation between TERT and TA-ERVs expression in 103 tumour samples across 11 cancer types. BRCA (breast invasive carcinoma), n = 17. cHL (classical Hodgkin lymphoma), n = 5. COADREAD (colon adenocarcinoma/rectum adenocarcinoma oesophageal carcinoma), n = 6. HGSC (high-grade serous ovarian cancer), n = 6. KIRC (kidney renal clear cell carcinoma), n = 12. LIHC (liver hepatocellular carcinoma), n = 9. LUAD (lung adenocarcinoma), n = 6. OSCC (oral squamous cell carcinoma), n = 8. PAAD (pancreatic adenocarcinoma), n = 10. PRAD (prostate adenocarcinoma), n = 6. THCA (thyroid carcinoma), n = 18.

D MeDIP-qPCR analysis of TA-ERVs sites in HeLa cells ectopically expressing TERT-WT or TERT-K626A. Primer sets recognizing exon 8 of the human ZC3H13 gene and a gene desert on chromosome 12 were used for positive and negative controls, respectively.

E ChIP-qPCR for TERT and Sp1 targets on TA-ERVs in U2OS cells with TERT ectopic expression and in HeLa cells. GAPDH promoter was used as a negative control, and VEGF promoter was used as a positive control.

Data information: Data represent mean ± SEM of three biological replicates (B, D and E). */#p < 0.05, **/##p < 0.01, ***/###p < 0.001, ns, not significant. Unpaired two-tailed Student's t-tests were used, and asterisks indicating significance for each ERV versus β-actin in TERT-WT while hashtags indicating significance for each ERV versus β-actin in TERT-K626A in B and E, one-way ANOVA with Fisher's LSD tests were used for D.