EMBO/soda-nel huggingface dataset:
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 2

Figure 2. G2/M DNA damage checkpoint abrogation is required for interferon signaling induced by ATR inhibition + ionizing radiation (IR).

A, MCF10A WT cells were pretreated with AZD6738(250 nM) and RO-3306 (9 μM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies.

B, Same condition as described in A, except that RO-3306 is replaced by PD-0332991 (1µM).

C, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cell cycle distribution was measured by flow cytometry. EdU (10 μM) was added into medium 2 hours before harvesting for flow cytometry.

D, MCF10A WT cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, and cells were harvested at indicated time points for immunoblotting.

E, MCF10A control cells and CDC25C-WT/constitutively active mutant CDC25C-S216A over-expressed cells were pretreated with DMSO or AZD6738(250 nM) for 2 hours, and then irradiated with 20 Gy, 24 hours later, cells were harvested for immunoblotting with indicated antibodies.

F, Same condition as indicated in E, except that cells were harvested 72 hours later.
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 3

Figure 3. The cGAS/STING-dependent cytosolic dsDNA sensing pathway is dispensable for interferon (IFN) signaling induced by ATR inhibition + ionizing radiation (IR).

A, MCF10A Wild-type (WT), cGAS knockout (KO), and STING KO cells were treated with cGAMP at indicated concentrations for 4 hours, and cells were harvested for immunoblotting.

B, MCF10A WT, cGAS KO, and STING KO cells were transfected with herring testis DNA (HT-DNA, 2 µg/ml) for 6 hours with lipofectamine 3000. Immunoblotting was performed with indicated antibodies.

C-F (C, D, and F) MCF10A WT or two independent clones for cGAS, STING, and IRF3 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. cGAS, STING, and IRF3 KO were verified by immunoblotting and Sanger DNA sequencing (Appendix Figure S7). (E) Same condition as described in C, except that cells were harvested for real-time quantitative PCR. IFNB1 mRNA level was measured and statistical analyzed by two-way ANOVA (*** means p value < 0.001). Data are presented as mean + standard error of the mean of three biological replicates.
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 4

Figure 4. The MAVS-dependent cytosolic RNA sensing pathway is indispensable for interferon (IFN) signaling induced by ATR inhibition + ionizing radiation (IR).

A-F (A, C, D, and F) MCF10A WT or two independent clones for MAVS, RIG-I, MDA5, and IRF7 KO cells were pretreated with DMSO or AZD6738 (250 nM) for 2 hours, and then irradiated with 20 Gy, 3 days later, cells were harvested for immunoblotting with indicated antibodies. MAVS, RIG-I, MDA5, and IRF7 KO were verified by immunoblotting and Sanger DNA sequencing (Appendix Figure S7). (B and E), Same condition as described in A, except that cells were harvested for real-time quantitative PCR instead of immunoblotting. IFNB1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 5

Figure 5. Cytosolic nucleic acid-sensing pathways involved in ionizing radiation (IR)-induced type I interferon (IFN) signaling differ in murine and human cells.

A, 4T1 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 10 Gy. 4 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.

B, Same condition as described in A. Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO 4T1 cells. Data are presented as mean + standard error of the mean of three biological replicates. All KO cells were verified by immunoblotting and Sanger DNA sequencing (Appendix Figure S7).

C, MC38 cells were pretreated with AZD6738 at indicated concentrations for 2 hours, and then irradiated with 20 Gy. 3 days later, Ifnb1 mRNA level was measured by real-time quantitative PCR. Data are presented as mean + standard error of the mean of three biological replicates.

D, Same condition as described in C. Ifnb1 mRNA levels were measured by real-time quantitative PCR in WT, Sting KO, Mavs KO, and Irf3 KO MC38 cells. Data are presented as mean + standard error of the mean of three biological replicates. All KO cells were verified by immunoblotting and Sanger DNA sequencing (Appendix Figure S7).
ATR inhibition potentiates ionizing radiation-induced interferon response via cytosolic nucleic acid-sensing pathways (2020)
Figure 6

Figure 6. DNA damage-elicited AT-rich DNA mediates type I interferon (IFN) signaling in human cells.

A, MCF10A WT, cGAS KO, STING KO, and MAVS KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.

B, 4T1 WT, Sting KO, Mavs KO, and Irf3 KO cells were treated with poly(dA-dT)/LyoVec at indicated concentrations for 24 hours. Immunoblotting was performed with indicated antibodies.

C, END-seq was used to map double-strand breaks (DSBs) associated with TA dinucleotide repeats in MCF10A cells with the indicated treatments. ATR inhibitor (ATRi) AZD6738 ( 250nM) was used with ionizing radiation (IR, 10 Gy) for 6 hours. NT, not treated.

D, MCF10A WT, MDA5 KO, RIG-I KO, and MAVS KO cells were transfected with AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.

E, MCF10A WT and STING KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with lipofectamine 3000 for 4 hours. Immunoblotting was performed with indicated antibodies.

F, 4T1 WT, Sting KO, and Irf3 KO cells were transfected with AT-rich and non-AT-rich oligos (2 μg/ml) with LyoVec for 24 hours. Immunoblotting was performed with indicated antibodies.

G, A proposed potential mechanism for the divergent cytosolic nucleic acid-sensing pathway activated in response to radiation alone or with ATRi among different cell lines.
A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotic cells (2023)
Figure 2

Figure 2. EGF activates Arf1 (mG*) and triggers the recruitment of GIV-GEM on Golgi.

A. Schematic showing the specific step being interrogated in panels (B-C), i.e., Arf1 activation under EGF stimulation.

B. Immunoblot shows GST-GGA-GAT domain bound Arf1 (active; top) and total Arf1 (input lysates; bottom) from equal aliquots of lysates of HeLa cells that were stimulated with EGF for the indicated time points prior to lysis.

C. Graphs display the model fitting for Arf1 activation dynamics. The experimentally determined Arf1 activation (in B) dynamics are displayed as black dots with error bars, representing mean ± S.E.M (n=3 biological replicates), and numerical simulation is shown by the blue continuous line.

D. Schematic showing the specific step being interrogated in panels (E-F), i.e., recruitment of GIV-GEM on Golgi.

E. HeLa cells expressing Arf1-HA were serum starved overnight (E, top) and subsequently stimulated with EGF for 5 min (E, bottom) prior to fixation with PFA. Fixed cells were stained for Arf1 (HA; green) and GIV (red) and nuclei (DAPI; blue). Panels on the left show overlay of all 3 stains and representative RGB plots of sections through the Arf1-stained pixels. Panels on the right display the magnified 3D surface plots of the boxed regions in the left panels. Scale bar = 10 µm.

F. Scatter plot shows the Mandler's overlap coefficient (MOC) for Arf1-HA and GIV colocalization in E that was calculated on 13-15 cells/experiment, n = 3 independent experiments. p values were determined using Mann-Whitney t-test: ***, 0.0002.
A Circuit for Secretion-coupled Cellular Autonomy in Multicellular Eukaryotic cells (2023)
Figure 5

Figure 5. Coupled GTPases are predicted to support secrete-and-sense autonomy and maintenance of cell number.

A. Schematic of the key features of the auto/paracrine loop that we hypothesize is regulated by the coupled GTPase circuit (left) and the corresponding phenomenological models to capture these key effects (right).

B-C. Model prediction for secretion as a function of stimulus in cells with coupled and uncoupled GTPases. Noise is introduced into the system in a similar way as described in Figure EV 4D-G. r2>0.99 in B; r2>0.94 in C.

D-E. The secretion (D) or the cell number (E) as a function of stimulus in coupled and uncoupled switches. The stimulus =0, 0.00046, 0.0046, 0.046, 0.115, 0.23 and 0.46 correspond to varying doses of EGF in simulations, ranging from 0, 0.1 nM, 1 nM, 10 nM, 25 nM, 50 nM, and 100 nM, respectively. The error bar denotes the S.D based on 1000 repeated independent simulations of ODEs when noise is in the stimulus and connections.

F. The bar plot depicts cell numbers achieved by cells with either coupled or uncoupled switches, at different levels of stimulus. For the first two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0.046 in E, respectively. For the last two bars, the height and error bars are the mean and S.D. of cell number when stimulus =0 in E, respectively.

G. Relation between cell number and EGF in the presence of noise, which was introduced in a similar way as described in Figure EV4 D-G. r2>0.95.
Characterization of histone inheritance patterns in the Drosophila female germline (2021)
Figure 2

Figure 2: Mitotic GSCs and CBs exhibit non-overlapping old versus new histone H3 patterns, but overall symmetric old and new segregation patterns.

(A-C) Telophase GSCs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 show non-overlapping patterns (A). Telophase GSCs expressing old (green) and new (red) histone H4 show moderate levels of overlap (B). GSCs expressing old (green) and new (red) histone H2A show high levels of overlap (C).

(D) A control where the old and new fluorescent tags have been switched shows the non-overlapping patterns of old (red) versus new (green) H3Rev histone in mitotic telophase GSCs marked by H3S10p (gray).

(E) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the GSC division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev GSCs, H2A GSCs, and H4 GSCs. Data gathered over 16 separate experiments for H3 GSCs. Values are mean + 95% CI.

(F-H) Telophase CBs marked by H3S10p (gray) expressing old (green) and new (red) histone H3 also show non-overlapping patterns. (F) Telophase CBs expressing old (green) and new (red) histone H4 (G) and H2A (H), which depict more overlap like GSCs.

(I) A control with switched old and new fluorescent tags shows the non-overlapping old (red) versus new (green) H3Rev histone patterns in mitotic telophase CBs marked by H3S10p (gray).

(J) Quantification of log2 ratios of total old and new histone inherited by each future daughter cell of the CB division, where a value of 0 is equal inheritance at exactly a 1:1 ratio. Data gathered over 9 separate experiments for H3Rev CBs and H2A GSCs. Data gathered over 12 separate experiments for H4 CBs and H3 CBs. Values are mean + 95% CI.

Data information: Asterisk: niche. Scale bars: 2μm. Dashed box (cyan): biased spec inheritance. Zoomed-in images of biased spectrosome inheritance (gray) are displayed to the right of panels (F-I). Scale bars: 0.5μm. See Dataset EV2 for individual data points for Fig 2E and Fig 2J.
Characterization of histone inheritance patterns in the Drosophila female germline (2021)
Figure 4

Figure 4: Oligopaint IF-FISH reveals differential old versus new H3 inheritance at key genes for maintaining stem cell fate or promoting differentiation in WT female GSCs.

(A-B) Old (red) versus new (green) histone H3 distribution patterns and percentage of signal strength with a single genomic locus labeled with fluorescent probes (cyan) for dad (A) and bam (B) in WT telophase female GSCs, marked by anti-H3S10p (gray).

(C) Quantification of the probe association ratios with old versus new H3 for each candidate gene in WT female GSCs. Data gathered over 8 separate experiments.

Data information: Asterisk: niche. Arrowheads: probe signal. Scale bars: 1μm. Zoomed-in images of each probe are displayed to the right of each figure panel. Scale bars: 0.1μm. See Dataset EV4 for individual data points for Fig 4C.

Expanded View Figure Legends:
Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling (2016)
Figure 4

Figure 4. Proliferation in the IFE is hair cycle dependent and occurs mainly during early anagen phase.

(a-b) Ki67 immunohistochemistry of back skin sections at different phases of the hair follicle cycle determined using a bioluminescent reporter mice of the canonical Wnt signaling pathway (a) and corresponding scatter plots quantification. (Scale bar 100µm) (b). Analysis focused on the basal layer of the IFE. Each dot represents one image. Proliferation in the IFE is significantly increased during the anagen phase except in mid-anagen. Unpaired t test, ****p<0.0001, **p=0.0020. EA n = 32, EMA n = 13, MA n = 15, C n = 21, T n = 19 from 6 mice. Black arrows: positive Ki67 cells. (c) Photomicrographs represent 2D optical sections from Z stack acquisition of whole mounted back skin stained in anagen and telogen with anti-Ki67 antibody or upon BrdU incorporation. Ki67+ cells are labelled with Alexa 647 (red). The density of Ki67+ cells increased in the anagen phase (iii and iv) compared to telogen (i and ii). White arrows: ki67+ cells. (Scale bar = 20µm). Similarly, short term BrdU pulse (2h) resulted in significant staining of anagen epidermal areas (vi, red nuclear stain) reinforcing hair follicle openings as compared to telogen areas.
Bimodal behaviour of interfollicular epidermal progenitors regulated by hair follicle position and cycling (2016)
Figure 5

Figure 5. Small clones not attached to hair follicles harbor epidermal populations that are quiescent and can be mobilized upon stimulation.

a-b) Skinbow mice were induced for 5 consecutive days and pulsed with BrdU for 3 consecutive days with 2 injections per day. Skin was collected either immediately after the last BrdU injection at 72hours or after periods of 1 to 5 weeks of chase. a) Photomicrographs show optical sections from a confocal image stack acquired on a whole mounted skin preparation immunostained with anti-BrdU antibody (red). White dots represent hair follicles at different chase time points. b) At 5 weeks of chase, BrdU staining (green) in skinbow skin revealed single cells distant from hair follicles. White dashed circles: hair follicles. White line: small clones. White arrows: BrdU+ cells. (scale bar = 20 µm). c-d) In Skinbow mice induced at P42-46, TPA was applied for one day, 3 weeks post-induction and animals were sacrificed at 48hours following a short pulse of BrdU (2h). c) Photomicrographs show optical sections from a confocal image stack acquired on a 0.5 mm back skin section immunostained with anti-ki67 antibody. Ki67+ cells are labelled with Alexa 647 (red). Back skin from treated and untreated areas was collected 48hrs after TPA topical application. d) Multiple optical section from a Z stack acquisition of whole mount preparation of skin treated with TPA and stained with anti-BrdU (green). Small clones not attached to HF (2 examples figured by dashed lines) can be tracked from the corneocyte layer down to the basement membrane where BrdU staining demonstrates their proliferation. e) (i to iv) Representative individual sections from Z stacks, show small clones (lines) identified in backskin treated (i and ii) or not with TPA (iii and iv). (scale bar = 100 µm). (v to viii) Maximal intensity projection from stack acquired on whole mounted skin. A contribution of the infundibulum and the bulge to the IFE is observed in back skin treated with TPA (v to vi) compared to skin treated with vehicle (vii and viii). (scale bars = 100 µm). f) Reduction in small clone frequency in the areas treated with TPA. Histogram chart showing the clone size distribution and relative frequency of small clones not attached to HF in areas treated with TPA and not treated with TPA in a same mouse. Mann Whitney test, ** p=0.0079. Data pooled from 3 mice represented as median with interquartile range. The number of clones analyzed is indicated on the graph. * p=0.0272. Data are represented as mean ± s.e.m.
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 1

Figure 1:

FLNA editing in cardiac patients from GTEX donors.

(A) ADAR2 is most strongly expressed in vascular tissues. Graph showing ADAR2 gene expression data derived from GTEx for few representative human tissues. Note the highest expression of ADAR2 in the tibial artery followed by colon and esophagus. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.

(B) FLNA gene expression in few representative human tissues from >500 different donors using GTEx data. Tibial artery, colon and esophagus show highest gene expression similar to ADAR2 expression. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.

(C) Bar graph shows FLNA RNA editing (%) among few representative human tissues. Note very high editing levels in the arterial system (red bars).

(D) Scatter plot shows the log fold change in editing levels of several candidates between ventricles of healthy donors and heart samples of cardiomyopathy patients. FLNA marked in the graph is one significant discriminator. Y axis is the -log 10 of the p value for the difference between healthy and sick. The colors (red vs green) reflect the threshold cutoffs randomly assigned to distinguish the sites, which demonstrate large, highly significant differences between the two groups. Fold change cutoff of 0.9 was used.

(E) Scatter plot shows the FLNA RNA editing in control and diseased human tibial arteries and aorta. At least 4 control and 7 diseased human donors were used in each case. P value <0.05 measured by t test was considered significant.
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 2

Figure 2:

Generating mice deficient in Filamin A editing.

(A) Scheme showing the FLNA wild-type (wt) allele, the targeting vector and the FLNAΔECS neo-allele. The loxP flanked PGK-neo cassette in the targeting vector replaced the editing complementary sequence (ECS) using homologous recombination, which was then deleted using Cre recombinase. Right, southern blotting analysis screened for positive clones shown. The positions of loxP sites, restriction enzyme (EcoRI) and Southern blotting probe are also indicated.

(B) Sequencing electropherograms show the average FLNA editing levels in wt and FLNAΔECS tissues (Stomach, colon, heart, lung and tibial artery). Editing levels were checked in tissues from 3 independent mice and the value below the chromatogram depict the average value of 3 replicates.

(C) FLNA mRNA expression levels measured by qPCR in wt and FLNAΔECS colon tissue showed no difference.

(D) FLNA protein levels measured by western blotting are identical in wt and FLNAΔECS stomach tissue. Y-axis represents FLNA expression normalized to tubulin levels.

(E) FLNA editing levels (%) in wt whole dorsal aorta, tunica media and tunica adventitia. Tunica media consisting of smooth muscle cells show the highest FLNA RNA editing.

Data information: Arrows indicate the FLNA editing site. For (C,D,E) data shown as mean ± SD from three independent experiments. ** P<0.05
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 3

Figure 3:

FLNAΔECS aortae and primary vSMCs show hypercontraction in response to U46619.

A,B) Treatment of aortic rings with thromboxane A2 receptor agonist U46619 leads to (A) hypercontraction and (B) increased contraction force in FLNAΔECS aortae in myograph chambers. Emax was higher in FLNAΔECS aortae without any difference in EC50 as compared to wt aortae. For each condition, 10-12 aortic rings from at least 4 wild-type (wt) and 4 FLNAΔECS mice were used. Data shown as mean ± SEM. * P<0.05, ** P<0.01, *** P<0.001 (Student's t test)

(C) Graph shows the normalized cell contraction measured by Xcelligence Real time cell analyzer in wt (red) and FLNAΔECS (green) primary vascular smooth muscle cells (vSMC) indicating hypercontraction in FLNAΔECS cells in response to 10μM U46619. Data shown as mean ± SD from three independent experiments.

(D) Quantification of cell index measurements plotted as percentage of contraction following different concentrations of U46619. Data shown as mean ± SD from three independent experiments. P value <0.05 (Student's t test) was considered significant. The difference between wt and FLNAΔECS was significant at every concentration of U46619.
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 4

Figure 4:

FLNAΔECS vSMCs have increased Rho/ROCK activity and myosin phosphorylation.

(A) Myosin light chain phosphorylation detected by a phospho-specific antibody (pMLC) and normalized to total myosin light chain (MLC). Tubulin served as a loading control. Quantification shows a significant increase in pMLC in FLNAΔECS vSMCs.

(B) Rhotekin Pull down activation assay was performed on wt and FLNAΔECS vSMC lysates and activated (GTP) bound RhoA was compared between the 2 genotypes. FLNAΔECS vSMCs show more GTP-loaded RhoA than wt vSMCs. Tubulin was used as a loading control.

(C) Thromoboxane A2 stimulation with U46619 increased phosphorylation of myosin light chain phosphatase (pMYPT1) in FLNAΔECS vSMCs as shown by western blotting on vSMC lysates with phosphor-specific MYPT1 antibody.

(D) CPI17 phosphorylation levels were measured in wt and FLNAΔECS vSMCs in untreated cells and in the presence of ROCK and PKC inhibitor using pCPI17 antibody.

Data information: For (A,B,C,D) data shown as mean ± SD from at least three independent experiments. ** P<0.05 (Student's t test)
RNA editing of filamin A pre-mRNA regulates vascular contraction and diastolic blood pressure (2018)
Figure 5

Figure 5:

FLNAΔECS mice are hypertensive during resting periods.

A-C) Telemetry measurements were done on mice continuously for 72 hrs and reading was recorded every minute to analyse blood pressure in wt and FLNAΔECS mice. Scatter plots show diastolic blood pressure over 72 hrs (A), diastolic blood pressure during inactive phase (B) and diastolic pressure during active phase (C). Note: Significant increase in diastolic blood pressure in FLNAΔECS mice during the inactive phase. 9 mice (5 males marked as square + 4 females marked as round) were analyzed for each genotype. Data shown as mean ± SEM. P value <0.05 (Student's t test) was considered significant. Boxes represent the 25th and the 75th percentile with median represented by the black line in the box. The whiskers depict the minimum and the maximum value.