EMBO/soda-nel huggingface dataset:
PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin (2021)
Figure 8

Figure 8. Leukocyte migration through endothelium generates tension on VE-cadherin required for dephosphorylation of VE-cadherin-Y731.

A-C Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL (A, B) or VE-cad FL-TS (C). HUVEC were stimulated 4h with TNFα prior to adding IL-8 for 4 min, followed by flow with buffer alone (A) or together with PMNs (B, C). The connected data points represent FRET efficiency of same junctions compared before and after addition of flow/PMNs.

D Quantification of FRET efficiency (percentage) in junctions of HUVEC expressing VE-cad FL or VE-cad FL-TS. Cells were either exposed to flow and PMNs or to flow alone, fixed using 4 % PFA and washed with PBS. FLIM measurements were performed at sites of transmigration (PMNs) or at junctions without PMNs (Flow).

E Representative images of HUVEC expressing VE-cad FL at a site of PMN transmigration (left micrographs) or without PMN (right micrographs). Maximum intensity projection of a Z-stack of YPet fluorescence (VE-cadherin in green) and CellTracker DeepRed (PMN in red) is shown for the upper micrographs. The part of the PMN above the HUVEC monolayer is encircled in yellow and the part underneath in blue. The amplitude averaged lifetime of YPet per pixel (FLIM measurement) of the same cells is shown in the bottom micrographs.

F HUVEC were grown on collagen-coated polyacrylamide gels of varying physiologic stiffness of 0.2 kPa and 20 kPa, and stimulated with TNFα for 17 h prior to adding HL60-derived neutrophils for 20 min. VE-cadherin was immunoprecipitated and precipitates as well as total cell lysates were analyzed by Western blotting for indicated antigens. Relative quantifications of pY731 blot signals adjusted to the amount of precipitated VE-cadherin are shown on the right (from seven independent experiments).

Data information: Graphs represent data from n = 50 (A, B) and 51 (C) measurements pooled from ten independent experiments. Graph in D represents measurements (n = 32, 30, 14, 14) of three independent experiments. Graph in F represents data of seven independent experiments. Bars and error bars indicate mean ± SEM. Statistical significance was tested with paired t-test (A, B, C), and unpaired t-test (D, F), ****P <0.0001, ***P<0.001, n.s., not significant.
RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 2

Figure 2: A type I IFN response is unleashed in ADAR1 knockout cells upon expression of LGP2.

A) HEK293 cells were genetically engineered to knockout ADAR1 using CRISPR/Cas9. Cells were treated for 24h with recombinant type I IFN to upregulate ADAR1 p150 and ISG60 to confirm correct gene editing and type I IFN responsiveness, respectively. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3).

B) ADAR1-knockout HEK293 cells (clone 1) were co-transfected with an empty vector (EV) or a FLAG-LGP2-encoding vector (LGP2) combined with a vector encoding GFP-tagged ADAR1 p110 or p150. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 expression, normalized to ACTB. Data are means ± s.d. from a representative of four biological replicate experiments.

C) ADAR1-knockout cells (clone 1) were transfected as in (B). Protein lysates were analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=4).
RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 3

Figure 3: RNA binding by LGP2 is required for receptor oligomerization and type I IFN induction in ADAR1 knockout cells.

A) ADAR1-knockout HEK293 cells (clone 1) were modified with a lentiviral-based inducible system to express FLAG-LGP2 WT or a FLAG-LGP2 RNA binding mutant (K138E/R490E/K634E, denoted as "KRK mutant") in a doxycycline-regulated manner. Cells were treated 72h with doxycycline (dox). Protein lysates were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies (n=3). iEV = inducible empty vector; iLGP2 = inducible LGP2.

B) Cells generated in (A) were treated with doxycycline for 72h to induce LGP2 WT or KRK mutant gene expression. The type I IFN response (IFN-β and IFIT1 transcripts) and LGP2 (DHX58) expression were monitored by RT-qPCR analysis. All transcripts were normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.

C) Cells generated in (A) were plated on coverslips and treated with or without doxycycline for 72h. Cells were fixed, permeabilized and stained with anti-FLAG (red) and anti-IRF3 (green) antibodies. Nuclei were stained with DAPI (blue). Scale bar is 50 μm. Total nuclei (>500 nuclei per experimental condition) and IRF3-positive nuclei were counted using semi-automated software analysis and plotted as percentage IRF3-positive nuclei of total nuclei per field of view (a representative of two biological replicate experiments is quantified). The boxplot indicates the interquartile range as a box, the median as a central line, and the whiskers extend from the minimum to the maximum value. Statistical analysis was performed using a Kruskal-Wallis test with a Dunn's post hoc test for multiple comparisons. ns, not significant; ****, p<0.0001.

D) Cells generated in (A) were treated with doxycycline for 72h. During the last 24h, recombinant type I IFN was added to upregulate endogenous MDA5 protein expression. Protein lysates were analyzed by SDD-AGE and SDS-PAGE using the indicated antibodies to determine protein oligomerization and total expression levels, respectively (n=3).

E) MDA5-knockout HEK293 cells, generated in Fig. 1D, were transfected with an ADAR1-targeting siRNA (siADAR1) or a control siRNA (siCtrl) and 8h later with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of MDA5. Cells were harvested 72h post-siRNA transfection and the type I IFN response was monitored by RT-qPCR analysis of IFIT1 and ISG15 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.
RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 4

Figure 4: The function of LGP2 in sensing unedited self RNA involves its canonical role as dsRNA sensor that requires RNA binding and ATP hydrolysis.

A) Schematic illustration of the domain structure of LGP2 and various point mutants and truncation mutants that are used in this study. The N-terminal domain (NTD) of LGP2 is composed of a conserved DExH/D helicase domain, subdivided into the helicase 1 (Hel1), helicase 2 (Hel2) and helicase insertion (Hel2i) domain, and a pincer motif (P). The NTD is followed by a C-terminal domain (CTD), involved in RNA binding.

B) HEK293 WT or ADAR1-knockout cells (clone 1) were transiently transfected with an empty vector (EV) or a vector encoding the indicated WT, truncation, or point mutant(s) of LGP2. Cells were harvested 72h post-transfection and the type I IFN response was monitored by RT-qPCR analysis of IFN-β and IFIT1 transcript expression, normalized to ACTB. Data are means ± s.d. from a representative of three biological replicate experiments.

C) Cells were treated as in (B). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies (n=3). The dotted line indicates the juxtaposition of two non-adjacent lanes.
RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 5

Figure 5: Reduced tumor cell growth upon loss of ADAR1 is dependent on LGP2.

A) Kaplan-Meier plots showing overall survival of ADARlow and ADARhigh patients stratified by DHX58 levels in bladder cancer (BLCA; n=407), breast cancer (BRCA; n=1099) and sarcoma (SARC; n=263) TCGA datasets. Median cut-offs were used for patient stratification and logrank test P values are shown.

B) CAL27 cells transduced with doxycycline-inducible shRNAs targeting ADAR1 or GFP (negative control) were treated with doxycycline and transfected with two independent siRNAs targeting LGP2 (siLGP2 #1 or #2) or a control siRNA (siCtrl). To determine cell confluency at 120h post-transfection, cells were fixed, stained with Crystal Violet, and imaged. Images of a representative experiment are shown (n=3).

C) Crystal Violet was extracted from stained cells (B) and the dye intensity was quantified using a colorimetric assay (OD590). OD590 values of doxycycline-treated cells were normalized to the OD­590 values of untreated cells. Quantification of data from three independent experiments is shown as mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.
RNA sensing via the RIG-I-like receptor LGP2 is essential for the induction of a type I IFN response in ADAR1 deficiency (2022)
Figure 6

Figure 6: Depletion of ADAR1 in combination with epigenetic repressors trigger type I IFN induction in an LGP2-dependent manner.

A) HT29 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed by RT-qPCR analysis of IFN-β, IFIT1, and ISG15 transcript expression, normalized to ACTB.

B) LIM1215 cells were treated with or without 300 nM 5-AZA-CdR for 2 days and subsequently washed and transfected with the indicated siRNAs. Cells were harvested 72h post-transfection and the type I IFN response was analyzed as in (A).

C) LIM1215 cells were treated with 250 nM palbociclib or a DMSO control for 7 days. Three days after treatment initiation, cells were transfected with the indicated siRNAs and cultured for an additional 72h. The type I IFN response was monitored as in (A).

Data information: Data from three biological independent experiments are shown with mean ± s.d.. Statistical analysis was performed using ordinary two-way ANOVA with Tukey's post hoc test. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Expanded View Figure Legends
Dynamic loading and redistribution of the Mcm2‐7 helicase complex through the cell cycle (2014)
Figure 1

Cyclin E/Cdk2 activity modulates Mcm2‐7 loading during G1

FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.
Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).
Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).
Dynamic loading and redistribution of the Mcm2‐7 helicase complex through the cell cycle (2014)
Figure 2

The full complement of Mcm2‐7 is loaded during an unperturbed G1/S transition

Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.
Dynamic loading and redistribution of the Mcm2‐7 helicase complex through the cell cycle (2014)
Figure 3

The cyclin E/Cdk2 kinase‐dependent phase of Mcm2‐7 loading requires the canonical pre‐RC assembly pathway

Schematic of the experiment.
FACS profiles of DNA content for each cell population assayed.
Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.
Dynamic loading and redistribution of the Mcm2‐7 helicase complex through the cell cycle (2014)
Figure 4

Mcm2‐7 loading is restricted to ORC binding sites in the absence of cyclin E

Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.
Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.
Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.
Dynamic loading and redistribution of the Mcm2‐7 helicase complex through the cell cycle (2014)
Figure 5

The Mcm2‐7 chromatin distribution in G1 is dependent on cyclin E/Cdk2 activity and the transcription machinery

Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.
Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.
“Meta”‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.
Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.
Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.
A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression (2015)
Figure 1

Figure 1. Public and private miRNA vulnerabilities among ovarian cancer cell lines.

(A) Two-way unsupervised hierarchical cluster of Z-score distributions by Euclidian distance. Any miRNA mimic with a z-score ≤ -2 in at least one cell line was included. Seed sequences of each miRNA mimic are on the right. Bars correspond to arbitrary cluster boundaries. Seeds in red correspond to miRNAs with decreased expression in serous, clear cell, or endometrioid ovarian cancer relative to normal tissue (Iorio et al, 2007).

(B) The histogram indicates the number of non-redundant hits binned according to the number of responsive cell lines.

(C) Expression of miRNAs miR-146a and miR-505 correlated with overall survival in ovarian cancer patients. The validation cohort (n=150 samples) is shown. See Figure EV1 for the training cohort.

(D) Intersection of miRNA sensitivities in the non-clonal short-term cultures and miRNAs with reduced expression in serous ovarian cancer (Iorio et al, 2007). p-value from hypergeometric distribution.
A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression (2015)
Figure 2

Figure 2. Tumor cells with epithelial features are selectively sensitive to miR-155 and miR-181.

(A) The consequence of miR-155 and miR-181b mimics on cell viability, 5 days post transfection, relative to a negative control miRNA mimic is shown across a panel of normal and ovarian cancer cell lines as indicated. Each data point is the mean of n=3/cell line. In the screen, miR-155 was found to reduce cell viability (Z-score < -2) in one cell line, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. miR-181 was found to reduce viability (Z-score < -2) in 2 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors. N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34), EOC, epithelial ovarian cancer cell lines. miR-155 data points for each cell line are shown in black while miR-181b data points are displayed in red.

(B) Immunoblots indicate suppression of serum-induced AKT phosphorylation at S473 and T308 in response to miR-155 and suppression of T308 phosphorylation in response to miR-181b.

(C) miR-155 and miR-181b reduced cell viability in GDC0941-sensitive breast cancer cell lines. Error bars indicate mean ± SD (n=3).

(D) Differential localization of E-cadherin in PEO1 and PEO4 cells 48 hours post transfection with the indicated oligos. Cells were counterstained with phalloidin and DAPI.

(E) Stimulation of PEO4 cells with 10 ng/mL TGFβ1 significantly sensitized the cells to AKT inhibition with AKT Inhibitor X. Each point represents the mean of 3 experiments ± SD and * denotes p-value < 0.05 by Student’s T-test.

(F) Cell viability upon expression of miR-155 or miR-181a mimics in 41 NSCLC cell lines and 5 human bronchial epithelial cell lines. Each data point is the mean of n=3/cell line. R2 from Pearson correlation.
A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression (2015)
Figure 3

Figure 3. miR-517a attacks a common molecular vulnerability in ovarian cancer cells.

(A) Consequence of miR-517a on EOC cell viability and 41 NSCLC cell lines (as in 2A). N, normal cell lines (IHH, HOSE, HBEC3, HBEC13, HBEC30, and HBEC34). miR-517a was found to significantly reduce cell viability (Z-score < -2) in 5 cell lines in the screen, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.

(B, C) In vivo delivery of neutral liposome incorporated miR-517a mimic reduced tumor burden by weight (B) and nodule number (C) in an orthotopic xenograft model using SKOV3 cells. Box-and-whisker plot from n=8. p-value from Student’s T-Test. LD, low-dose (200 μg/kg); HD, high-dose (400 μg/kg).

(D) Correlation of the consequence of ARCN1 depletion and miR-517a sensitivity in 12 NSCLC cell lines (plot as in 2D).

(E) Immunoblots indicate miR-517a-induced depletion of ARCN1 and USP1 in the indicated cell lines.

(F) Consequence of the indicated siRNAs and miR-517a on the viability of the indicated cell lines.

(G) Immunoblots indicate that miR-517a suppresses expression of ARCN1 in multiple miR-517a sensitive cell lines.
A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression (2015)
Figure 4

Figure 4. miR-124-induced reprograming of EOC gene expression profiles.

(A) Consequence of miR-124 on EOC cell viability (as in 2A). N, normal cell lines (IHH and HOSE). In the screen, miR-124 was found to significantly reduce cell viability (Z-score < -2) in 6 cell lines, and TCGA expression data revealed no significant change in expression of this miRNA in ovarian tumors.

(B) miR-124 induced expression of neuronal marker proteins Tuj1 (TUBB3) and MAP2 in ES2 cells 48 hours post transfection. Cells were counterstained with DAPI and phalloidin.

(C) miR-124 responsive genes in PEO1 cells were enriched for TargetScan predicted targets. p-value from hypergeometric distribution.
A genome-scale screen reveals context-dependent ovarian cancer sensitivity to miRNA overexpression (2015)
Figure 5

Figure 5. Selective addiction of EOC to the SIX4 homeobox transcription factor.

(A) Immunoblots indicate miR-124-induced SIX4 depletion. siSIX4 is shown as an antibody control.

(B) Consequence of SIX4 depletion on EOC cell viability (as in 2A, N, normal cell lines: HOSE, HBEC30).

(C) in vivo knockdown of SIX4 using neutral liposome incorporated siRNA reduced tumor burden and ascites volume in an orthotopic xenograft mouse model. Box-and-whisker plot from n=8. p-value from Student’s T-Test.

(D) Consequence of SIX4 depletion in PEO1 cells on E-cadherin plasma membrane accumulation 48 hours after transfection. Cells were counterstained with phalloidin and DAPI.

(E) Immunoblots indicate consequence of SIX4 depletion on LKB1 accumulation and AMPK pathway activation.
SATB2-LEMD2 interaction links nuclear shape plasticity to regulation of cognition-related genes (2020)
Figure 1

Figure 1 - SATB2 interacts with the INM protein LEMD2

Model of a hypothetical NL-chromatin tether containing SATB2. Schematic representation of the nuclear envelope consisting of two lipid bilayers, the inner nuclear membrane (INM) and outer nuclear membrane (ONM), and the lamin polymer underlying the INM (modified after Zuleger et al., 2011). Depicted are the INM protein LEMD2, lamins and BAF, identified as SATB2 interaction partners (Cera et al., 2019).

Immunoprecipitation of SATB2 from mouse neonatal cortical lysates. LEMD2 was detected by Western blotting in the SATB2-immunoprecipitate from control but not SATB2-deficient cortical lysate. The equal input of total protein from control and SATB2-deficient cortical lysates was controlled by ERK2 detection. Representative images of the immunoblots are shown; I (Input), F (Flow-through), B (Beads).

Reverse immunoprecipitation with LEMD2 antibody and control IgG antibody, followed by WB detection of LEMD2 and SATB2. Lysates from primary cortical culture lysates were used.

GST pull-down assays. Representative images of n = 3 independent experiments are shown. GST pull-down assay of transiently overexpressed V5-tagged LEMD2 (i), SATB2 (ii), and LEMD2ΔLEM (iii) from HeLa cell lysates using GST, GST-SATB2, and GST-LEMD2(413-503) hybrid proteins.

Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with expression plasmids encoding V5-tagged full-length SATB2 (Satb21-733) and EGFP as control. The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).

The CUT-like domain of SATB2 is required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from lysates of HeLa cells transfected with V5-tagged Satb21-247 and Satb21-157 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells transfected with the deletion mutant containing the CUT-like domain (Satb21-247). The equal input of total protein was controlled by GAPDH detection. Representative images of the immunoblots are shown; I (Input), F (Flow‐through), B (Beads), M (Molecular weight marker).

CUT1 and CUT2 domains are required for the interaction with LEMD2. Immunoprecipitations using anti-V5-tag antibody from HeLa cells transfected with V5-tagged Satb2346-733 and Satb2616-733 deletion mutants. LEMD2 and BAF were detected only in immunoprecipitates from HeLa cells expressing the CUT1 and CUT2 containing deletion mutant (Satb2346-733).
SATB2-LEMD2 interaction links nuclear shape plasticity to regulation of cognition-related genes (2020)
Figure 5

Figure 5 - NEE-activated cFos-positive neurons are more likely than non-active neurons to bear nuclear infoldings in Satb2 floxed but not in Satb2CamCre mice

Representative confocal images (Z-axis projection) of CA1 pyramidal neurons of: (top panel) Satb2 floxed mice (n = 3) maintained in their home cages (HC), (middle panel) Satb2 floxed mice (n = 4) subjected to NEE for 1.5 h, (bottom panel) Satb2CamkCre mice (n = 5) exposed to NE for 1.5 h. Brain sections were stained for cFos and Lamin B2. Scale bars: 10 μm.

Higher magnification views of boxed areas in A (middle panel). Arrows show examples of infolded, cFos-positive nuclei of the CA1 field of Satb2 floxed mice subjected to NEE. Scale bars: 5 μm.

2 x 2 contingency tables showing the number of analyzed CA1 pyramidal neuron nuclei grouped by category: cFos+/cFos-, infolded/non-infolded in Satb2 CKO mice (left) and Satb2 floxed mice (right). In floxed animals exposed to novel environment, cFos-positive nuclei were more likely than cFos-negative nuclei to bear infoldings, χ -square test of independence, χ2 (1, N = 3578) = 54.649, p < 0.00001. No significant relationship between cFos expression and nuclear infoldings was identified for the CA1 neurons of Satb2CamkCre mice (χ2 (1, N = 3003) = 0.80, p = 0.3693).