EMBO/soda-nel huggingface dataset:
Aurora B-dependent polarization of the cortical actomyosin network during mitotic exit (2021)
Figure 5

Figure 5 - Aurora B inhibition affects DNA-dependent actin clearance during anaphase

(A) Stills from time-lapse sequence of representative Hela cells expressing Lifeact-GFP and H2B-mCherry, exiting mitosis under different conditions i.e. Control siRNA, knock-down of centralspindlin protein RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). Scale bar - 10 µm

(B) High magnification view of boxed regions in (A) pseudo-colored showing failure to reduce actin during anaphase in cells treated with Aurora B inhibitor and even greater effect with Aurora B inhibitor upon RACGAP1 depletion. Scale bar - 1 µm.

(C) Quantification of actin intensity at the poles of cells at 6 mins post anaphase onset, before furrow formation under different conditions as in (A). Actin fails to be cleared from the poles upon Aurora B kinase inhibition during mitotic exit and is stronger when both centralspindlin and Aurora B kinase activity are affected, while it is cleared upon RACGAP1 depletion. Data represented as mean±SD for treatments - siControl- 0.98±0.05, siRACGAP1 - 0.99±0.04, ZM - 1±0.039, siRACGAP1+ZM - 1±0.037. Ordinary one-way ANOVA with multiple comparison- siControl vs siRACGAP1 - n.s., p=0.896, siControl vs ZM - **p=0.0018, siControl vs siRACGAP1+ZM - ****p<0.0001 and ZM vs siRACGAP1+ZM - p=0.1528.

(D) Schematic representation for quantifying actin intensity profile along the cortex as in E (E) Quantification of average actin intensity profile of siControl, siRACGAP1, ZM and siRACGAP1+ZM treated cells shown in (A). While actin levels reduced from the polar regions in both siControl and siRACGAP1 treated cells seen by levels lower than 1 (dashed red line) in segments 2-8, they failed to be cleared from ZM and siRACGAP1+ZM treatment conditions. Date represented as mean±SD .

(F) Stills of representative Hela cells expressing Lifeact-GFP and H2B-mCherry captured at early anaphase under different conditions i.e. Control siRNA, knock-down of RACGAP1, in the presence and absence of Aurora B kinase inhibitor (2µM ZM). The masks below were generated for each cell and the shortest distance between the centroid of DNA (+) and the masked cortical actomyosin network (*) was quantified in (G) with Matlab. Scale bar - 10 µm

(G) Quantification of shortest distance between centroid of DNA and cortical actomyosin network. While chromatin is able to reach close to the cortex in both siControl and siRACGAP1 treated cells, the distance of chromatin from cortex increases upon ZM treatment and is further enhanced in the double treatment condition. Data represented as mean±SD. siControl - 4.7±0.87, siRACGAP1 - 4.4±0.64, ZM - 5.47±0.87, siRACGAP1+ZM - 6.35±1.14 microns. Ordinary one-way ANOVA with multiple comparison, siControl vs ZM, ***p=0.002, siControl vs siRACGAP1, p=0.2823, siControl vs siRACGAP1+ZM, ****p<0.0001, siRACGAP1 vs siRACGAP1+ZM, ****p <0.0001, ZM vs siRACGAP1, ****p<0.0001, ZM vs siRACGAP1+ZM, ***p=0.0004 .

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DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma (2022)
Figure 1

Figure 1. DNA-PK catalytic activity promotes nucleic acid-dependent type I IFN responses in cGAS-deficient cells.

A T98G cells were challenged or not with dsDNAs or with 2'3'cGAMP for 6 h prior to whole cell extraction and Western Blot (WB) analysis using indicated antibodies.

B IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR in samples treated as in A (n=3 independent experiments).

C Gli4 cells were treated as in A prior to WB analysis using indicated antibodies.

D IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR in samples treated as in C (n=3 independent experiments).

E Phorbol 12-myristate 13-acetate (PMA)-treated THP1 cells were incubated for 24 h with conditioned media derived from T98G cells treated or not with dsDNA. IFIT1, IFIT2, MXA, OAS1 mRNA levels were analysed by RT-qPCR. Graphs present a representative biological triplicate (n=3 independent experiments).

F T98G cells were challenged or not with dsDNA for 6 h, in the presence or not of the NU7441 DNA-PKcs inhibitor, prior WB analysis using indicated antibodies.

G IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR in samples treated as in F (n=4 independent experiments).

H T98G cells were treated with non-targeting (CTRL) or DNA-PKcs-targeting siRNAs for 72 h prior to 6 h challenge with dsDNA. IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR. Graphs present a representative biological triplicate (n=3 independent experiments).

I T98G cells were treated or not with 0.16 µM camptothecin (CPT) for 48 h prior to immunofluorescence analysis using dsDNA- and 53BP1-specific antibodies, and DAPI nuclear staining (n=3 independent experiments). Scale bar, 20 µm.

J Whole cell extracts from T98G cells treated or not for 72 h with 0.16 µM CPT were analyzed by WB using indicated antibodies.

K IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR in samples treated as in J (n=3 independent experiments).

L T98G cells were treated or not with 0.16 µM CPT for 72 h, in presence or not of NU7441, prior to assessment of IFNB and CXCL10 mRNA levels by RT-qPCR. Graphs present a representative biological triplicate (n=3 independent experiments).

Data information: All immunoblots are representative experiments (n=3 independent experiments). All graphs present means ± standard error from the mean (SEM). P-values were determined by Student's t-test. ns: not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma (2022)
Figure 2

Figure 2. DNA-PK-dependent detection of cytosolic dsDNA drives STING-independent IRF3-dependent type I IFN responses in cancer cells lacking cGAS.

A T98G cells were treated or not with dsDNA for 6 h prior to IF analysis using DNA-PKcs- and pDNA-PKcs-specific antibodies, and DAPI nuclear staining (n=3 independent experiments). Scale bar, 20 µm.

B Whole cell extracts from T98G cells were incubated with 80nt-long biotinylated ssDNA or dsDNA prior to pull-down using streptavidin-affinity beads. Input and eluates were analyzed by WB using indicated antibodies.

C T98G cells were treated with non-targeting (CTRL) or KU70-targeting siRNAs prior to whole cell extract preparation, and pull-down as in B. Inputs and eluates were analyzed by WB using indicated antibodies.

D T98G cells were treated with CTRL or KU70-targeting siRNAs prior to challenge with dsDNA for 6 h. IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR. Graphs present a representative biological triplicate (n=3 independent experiments).

E CTRL or STING-/- T98G cells were transfected or not with dsDNA for 8 h prior to analysis of IFNB and CXCL10 mRNA levels (n=3 independent experiments).

F As in E, except that CTRL or IRF3-/- T98G cells were transfected or not with dsDNA for 6 h prior to analysis (n=3 independent experiments).

G CTRL or STING-/- T98G cells were treated or not with CPT for 72 h prior to analyses of IFNB and CXCL10 mRNA levels (n=3 independent experiments).

H As in G, except that CTRL and IRF3-/- T98G cells were used. Graphs present a representative biological triplicate (n=3 independent experiments).

I Schematic representation of cytosolic dsDNA-dependent type I IFN induction in T98G cells.

Data information: All immunoblots are representative experiments (n=3 independent experiments). All graphs present means ± SEM. P-values were determined by Student's t-test. ns: not significant. ** p < 0.01, *** p < 0.001, **** p < 0.0001.
DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma (2022)
Figure 3

Figure 3. cGAS and DNA-PKcs cooperate for optimal dsDNA-induced type I Interferon responses.

A T98G cells stably expressing cGAS (T98GcGAS) or not (T98GEmpty) were transfected or not with dsDNA for 6 h prior to whole cell extraction and WB analysis using indicated antibodies.

B IFNB and CXCL10 mRNA levels were analyzed by RT-qPCR in samples treated as in A. Graphs present a representative biological triplicate (n=5 independent experiments).

C T98GEmpty and T98GcGAS were transfected or not with biotinylated dsDNA prior to whole cell extraction and pull-down using streptavidin-affinity beads. Inputs and eluates were analyzed by WB using indicated antibodies.

D T98GEmpty and T98GcGAS were transfected or not with dsDNA for 6 h in the presence or not of NU7441 prior to IFNB and CXCL10 expression analysis. Graphs present a representative biological triplicate (n=5 independent experiments).

E T98GEmpty and T98GcGAS were treated or not with CPT for 72 h in combination or not with NU7441 (48 h) prior to IFNB and CXCL10 expression analysis. Graphs represent a biological triplicate (n=4 independent experiments).

F THP-1CTRL and THP-1cGAS-/- were transfected or not with dsDNA for 6 h in presence or not of NU7441 prior to IFNB and CXCL10 expression analysis (n=3 independent experiments).

G Intracellular cGAMP levels were analyzed in samples treated as in E by ELISA (n=3 independent experiments).

H Intracellular cGAMP levels were analyzed in samples treated as in F by ELISA. Graphs present a representative biological triplicate (n=2 independent experiments).

I T98G expressing a catalytic dead cGAS allele (T98GcGAS-CD) and T98G were treated as in A prior to IFNB and CXCL10 levels analysis. Graphs present a representative biological triplicate (n=3 independent experiments).

J T98GcGAS were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.

K THP-1 were transfected or not with dsDNA in presence or not of NU7441, prior to WB analysis using indicated antibodies.

L Schematic representation of the molecular mechanisms involved in the cooperation between DNA-PKcs and cGAS for type I IFN induction.

Data information: All immunoblots are representative experiments (n=3 independent experiments). All graphs present means ± SEM. P-values were determined by Student's t-test. ns: not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma (2022)
Figure 4

Figure 4. cGAS re-expression in glioblastoma cancer cells impairs tumorigenesis

A The proliferation of T98GEmpty and T98GcGAS was monitored in 2D cultures over 4 days (n=3 independent experiments).

B The volume of spheroids formed by T98GEmpty and T98GcGAS was monitored over 8 days (n=3 independent experiments).

C Representative images of T98GEmpty and T98GcGAS spheroids measured in B, at day 1 and day 8. Scale bar, 250 µm.

D The volume of subcutaneous T98GEmpty and T98GcGAS tumors in nude mice was measure every 3-4 days by caliper (n=6 mice per group).

E Representative pictures of T98GEmpty and T98GcGAS tumors from D, at day 90 post subcutaneous engraftment.

F T98GEmpty and T98GcGAS stably expressing a GFP reporter (T98G-GFPEmpty and T98G-GFPcGAS, respectively) were xenotransplanted into the head of tg(mfap4:RFP) zebrafish line at 3 days post fertilization (dpf). Zebrafish embryos were imaged daily over 3 days. The graph represents the mean (±SEM) percentage of tumor growth normalized by the area on the day of transplantation (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos). Scale bar, 200 µm.

Data information: All graphs present means ± SEM. P-values were determined by Student's t-test. ns: not significant. * p < 0.05, ** p < 0.01, **** p < 0.0001. One-way Anova with Tukey's multiple comparisons test was used for mice analyses. Mann-Withney test was performed to analyze tumor growth in zebrafish.
DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma (2022)
Figure 5

Figure 5. cGAS re-expression in glioblastoma cancer cells promotes macrophage recruitment

A Graph represents the mean (± SEM) fold migration of THP-1 cells through a 3 µm transwell insert when conditioned media from T98GEmpty or T98GcGAS was applied to lower chamber for 6 h. CCL2 was used as positive control (n=8 biological replicates).

B CXCL10, CCL2 and CCL5 protein levels in conditioned media from T98GEmpty and T98GcGAS cells were assessed using proteome profiler. Proteins that were found to be the most upregulated in T98GcGAS are shown. Representative immunoblots (n=3 independent experiments).

C T98GEmpty and T98GcGAS were transfected or not with dsDNA prior to analyses of CCL2 and CCL5 by RT-qPCR (n=4 independent experiments).

D Zebrafish embryos injected with T98G-GFPEmpty, T98G-GFPcGAS, or PBS at 3 dpf (Figure 4E) were imaged at 24 h post transplantation. Representative images of macrophage recruitment (purple) in the head (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos). Scale bar, 200 µm.

E Graph presents the quantification of macrophages recruited at tumor site 24 h post xenotransplantation in D (n=21 T98G-GFPEmpty and n=29 T98G-GFPcGAS embryos).

F Heads of zebrafish treated as in D were isolated prior to RNA extraction and analysis of M1 (tnfa and cxcl11) or M2 (arg2 and il10) polarization markers. Each value in the graph is the mean of 25 embryos.

Data information: All graphs present means ± SEM. P-values were determined by Student's t-test. ns: not significant. * p < 0.05, ** p < 0.01, **** p < 0.0001. Mann-Withney test was performed to analyze macrophage recruitment in zebrafish.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 1

Figure 1 - Establishment of patient-derived organoids from solid HGSOC deposits.

A Summary of cancer patient data with TNM and FIGO classifications showing advanced stage of disease at the time of surgery.

B Graphic representation of the standard experimental procedure for tumor patient material. Samples were obtained at the time of primary debulking surgery from the high purity tumor deposits in peritoneum/omentum.

C In vitro niche dependency of HGSOC tumor cells. Phase-contrast pictures illustrate that isolated ovarian cancer cells rely on EGF supplementation for growth, while they do not grow at all in Wnt3a supplemented medium. Also, inhibition of BMP signaling through Noggin has strong negative effect on the initial growth. Scale bar: 500 µm. E - EGF, F - FGF10, N -Noggin, R - R-spondin1, B - Basic medium, P - Passage

D Cancer organoids express HGSOC markers Pax8 and EpCAM and have lost the cystic phenotype suggesting complete breakdown of epithelial polarity as seen on confocal images from two representative organoid lines. Scale bar: 20 µm.

E HE staining of organoids and respective tissue confirm high similarity in cellular structure and tissue organization. Scale bar: 100 µm.

F HGSOC organoids show differential response to carboplatin treatment, confirming patient-specific sensitivity of the cultures. Cell viability assay was performed after 5 days of treatment with different concentrations of carboplatin on mature organoids from three different donors. Data represent mean ±SD of technical triplicates.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 2

Figure 2 - HGSOC organoids match tumor tissue in mutational profile and gene expression biomarkers.

A Overview of mutations found in organoid cultures and matching tumor tissue obtained by targeted sequencing of 121 HGSOC related genes confirms almost identical profile between parental tissue and in vitro long-term organoid culture. Color code indicates type of mutations which were detected.

B Representative western blot showing loss or overexpression of p53 in individual organoid lines.

C Strong nuclear p53 signal in immunostainings of HGSOC organoids is indicative of a gain-of-function TP53 mutation. Scale bars: 100 µm (Tissue) and 50 µm (Organoid).

D Multi-Dimensional-Scaling (MDS) plot based on the gene expression profiles (microarrays) of 3 healthy FT and 8 tumor tissue samples and their respective organoid cultures shows 4 clusters: Normal FT tissue, normal FT organoids, cancer organoids and cancer tissue.

E Box plots depicting overall constant level in normalized expression of major HGSOC marker genes between organoids and parental tissue. Data represent the median, quartiles, maximum, and minimum of the normalized expression from 8 different donors.

F Heat map of differentially expressed genes between cancer and healthy tissue/organoids reveals upregulation of several HGSOC biomarkers and reduction of FT differentiation markers in the cancer samples. Differential expression determined by single-color microarray for 8 different patient samples was significant for all genes with p < 0.05 except for OVGP1 and TOP2A in organoids.

Source data are available online for this figure.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 3

Figure 3 - Stable triple knockdowns of tumor suppressors p53, PTEN and RB in healthy FT organoids.

A Experimental approach for genetic manipulation of FT epithelial cells (FTECs). FTECs were sequentially transduced in 2D culture with replication-deficient viruses containing specific shRNAs and different selection markers. After sorting cells were seeded in Matrigel for organoid formation. Scale bars: 2 cm (tissue), 200 µm (2D culture) and 500 µm (3D culture).

B Confirmation of robust knockdowns of p53, PTEN and RB in FT organoids on RNA and protein level. Relative mRNA levels were normalized to the WT mRNA level and are given as the mean ±SEM from 3 different donors. A representative western blot from one donor is depicted. RNA as well as protein samples were taken at passage 1 or 2 of each organoid line. KD - Knockdown, Vec Ctrl - Vector Control, WT- Wild type

C Cyclin E1 overexpression as a downstream effect of RB knockdown was confirmed by western blot analysis.

D Confocal images of triple KD organoids reveal increased DNA damage (yH2AX, marked by asterisks), atypic nuclei (DRAQ5) and loss of apicobasal polarity. Scale bar: 20µm.

E Presence of ciliated cells (arrowheads), as revealed by immunostaining against detyrosinated Tubulin (dtyrTub) in triple KD organoids, proves capacity for terminal differentiation. Scale bar: 50µm.

Source data are available online for this figure.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 4

Figure 4 - Triple KD organoids show improved growth in ovarian cancer medium.

A In long-term 3D culture triple KD organoids show loss of growth capacity and premature growth arrest compared to the controls as indicated by the representative growth curves of FT234. Scale bar: 500 µm.

B Growth curves and phase-contrast images of triple KD organoids grown in OCM compared to FTM reveal successful rescue of long-term growth capacity. Scale bar: 500 µm.

C Levels of DNA damage (yH2AX) and apoptosis (cleaved caspase-3, PARP1) do not differ among the different media as shown by representative western blots comparing triple KD organoids and their controls.

D Triple KD (p53/PTEN/RB) organoids are characterized by a HGSOC gene expression signature as revealed by microarray analysis comparing WT and KD organoids grown in either OCM or FTM. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.00005.

E Differential expression of stemness- and differentiation-related genes of triple KD organoids grown in OCM vs. FTM. Fold-changes were derived from microarray data and indicate and an increase in stemness (CD133, SOX2, MYCN) and a drop in differentiation (OVGP1, PGR, FOXJ1) under OCM conditions. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p < 0.05.

F FOXJ1 expression determined by qPCR was significantly diminished in triple KD organoids grown in OCM compared to FTM, suggesting a decrease in differentiation capacity. Data represent the mean ±SEM from technical triplicates for two independent knockdown cultures.

G Proportion of ciliated cells (detyrosinated Tubulin; dtyrTub) is significantly reduced in KD organoids under OCM growth conditions, confirming inhibition of differentiation as illustrated by confocal images (Scale bar: 50 µm). The quantification plot depicts the mean ±SEM from 3 independent biological replicates, based on quantification of the average number of ciliated cells per counted nuclei. 1000 cells were counted per individual experiment. *p < 0.05, two-sided Student's t-test.

Source data are available online for this figure.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 5

Figure 5 - Wnt depletion supports preservation of stemness in HGSOC organoids.

A The proportion of CD133+ cells was determined by FACS analysis after growing triple KD and control cells in FTM vs. OCM for two passages. The graph is representative of two biological replicates.

B Growth capacity increases when KD organoids are grown in OCM medium as shown by difference in total viable cell number (representative graph of 3 independent experiments). The luminescent cell viability assay was performed in triplicate and the bar plot depicts the mean ±SEM. * p < 0.05, two-sided Student`s t-test; KD - Knockdown, Vec Ctrl - Vector Control, WT - Wild type

C Phase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.

D The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.

E Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.
Stable expansion of high-grade serous ovarian cancer organoids requires a low Wnt environment (2019)
Figure 6

Figure 6 - Unifying induction of MYCN and Wnt inhibitors in HGSOC and KD samples.

A Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.

B Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.

C Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.

D FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.

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