EMBO/soda-nel huggingface dataset:
Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor (2020)
Figure 4

Figure 4: H3K27me3 amplification and SMARCB1 deletion drives mithramycin sensitivity in rhabdoid tumor.

A, B: Mithramycin displaces BRD9 and SMARCE1 SWI/SNF subunits from chromatin in a time-dependent manner in G401 (A) but only BRD9 with SMARCB1 re-expression in G401 (B) cells. Western blot analysis showing whole cell lysate (Total), cytoplasmic soluble (CS), nuclear soluble (NS), and chromatin bound (Chr) fractions collected after exposure to solvent (S) or 100nM mithramycin for 8 or 18h and probed for the SWI/SNF subunits (BRD9, SMARCB1 or SMARCE1) or H3 (chromatin fraction control) and GAPDH (soluble fraction control).

C: BT12 cells show a threshold of exposure that leads to irreversible growth inhibition. The cells were exposed to 100nM MMA for the indicated times followed by replacement with drug-free medium. Beyond 8h (red) of mithramycin exposure, the cells do not recover proliferative potential and exhibit a phenotype consistent with cell death. Data represents mean with standard deviation derived from three independent experiments.

D: Mithramycin leads to H3K27me3 amplification in a time-dependent manner that precedes the induction of apoptosis as measured by the cleavage of PARP. Western blot lysates collected at 2h, 4h, 6h, 8h, 12h, 18h, 24h of continuous 100nM mithramycin treatment.

E: Suppression of EZH2 expression and activity antagonizes mithramycin activity in BT12 rhabdoid tumor cells. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the PRC2 subunit EZH2 or treatment with the EZH2 inhibitor EPZ-6438 relative to mithramycin alone (media) or a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.

F: Suppression of SMARCB1 sensitizes U2OS osteosarcoma cells (wild type SWI/SNF) to mithramycin. Data represents dose response curves of mithramycin in BT12 cells following a 48h exposure in the presence of siRNA silencing of the SWI/SNF subunit SMARCB1 relative to a non-targeting siRNA (siNeg). Data represents mean with standard deviation derived from three independent experiments.

G. Western blot showing concentration-dependent increase in H3K27me3 following exposure to 100nM, 50nM, 25nM mithramycin for 18h in SMARCB1- silenced U2OS cells relative to loading control (H3). Knockdown of SMARCB1 following siRNA suppression triggers mithramycin-dependent H3K27me3 amplification, while siNegative (control) does not have an effect on H3K27me3 following mithramycin exposure.
Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor (2020)
Figure 5

Figure 5: Mithramycin does not lead to general transcription inhibition and instead favors pro-survival pathways.

A-C: Volcano plot showing gene expression trends at 8h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (A) or 1.5 log2FC (drug over control) (B) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (C). Labels indicate names of genes that meet the logFC and q-value threshold.

D-F: Volcano plot showing gene expression trends at 18h MMA treatment. Dashed lines represent a 2 log2FC (drug over control) (D) or 1.5 log2FC (drug over control) (E) and 10e-7 q-value threshold. Quantification of induced and repressed genes in (F). Labels indicate names of genes that meet the logFC and q-value threshold.

G: SP1 protein expression is reduced following mithramycin exposure. Western blot showing suppression of SP1 expression compared to loading control (GAPDH) after 100nM mithramycin exposure for 1h, 4h, 8h, 12h, 18h.

H, I: The loss of SP1 expression is associated with a decrease in SWI/SNF occupancy of the SP1 promoter and an increase in H3K27me3. Data represents ChIP-qPCR analysis following 8-hours or 18-hours of 100nM mithramycin exposure and immunoprecipitation of SMARCC1 (8-hour, P= 0.0006; 18-hour P=0.0001) (H) and H3K27me3 (8-hour, P= 0.009; 18-hour P=0.0001) (I) . ChIP quantitation is percent input (ng of immunoprecipitated DNA/input DNA *100) determined by absolute quantitation of sheared chromatin relative to a standard curve. Data represents mean with standard deviation derived from three independent experiments. P-values were determined by one-way ANOVA using Dunnet test for multiple comparisons.

J: fgsea analysis of gene ontology terms upregulated in primary AT/RT tumors as described in (Wang et al., 2017) following 8h of mithramycin exposure. Mithramycin downregulates the expression of these pathways. P-values derived from the fgsea analysis package.

K, L: fgsea analysis of hallmark pathways downregulated after 8-hours of MMA exposure (K) or upregulated after 18-hours MMA exposure (L). P-values derived from the fgsea analysis package.
Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor (2020)
Figure 6

Figure 6: Mithramycin reprograms rhabdoid tumor promoters to trigger a change in cellular state and favor a differentiation phenotype.

A: Heatmaps depicting ATAC-seq global chromatin structure following 8-hours (middle) and 18-hours (right) 100nM MMA treatment. A 2kb window is centered on the TSS. Chromatin accessibility clusters at the TSS and does not change globally relative to solvent. Quantification of genes that gain accessibility or reduce accessibility on the right.

B: CTCF is the top down-regulated motif from the ATAC-seq analysis (P = 1e-58).

C: Schematic for the 18 state chromHMM model built for MRTs and collapsed into 6 super states. Chromatin states were called if the state was present in at least 50% of samples. ATAC-seq and H3K27ac ChIP-seq peaks were queried against the 6 super states. P-values derived from the homer motif analysis package.

D: Donut plots representing the percentage of each chromatin super state across treatment time (from 8h to 18h) that increased 1.5-fold (left) or decreased 1.5-fold (right). Below, Motif analysis of the top up-regulated motifs from the H3K27ac ChIP-seq gene lists that pass a 1.5 logFC. P-values derived from the homer motif analysis package using a binomial algorithm.

E: IGV tracks of rhabdoid tumor genes previously identified to be occupied by non-canonical SWI/SNF (Michel et al., 2018). ID3 and JUND3 decrease in H3K27ac occupancy and chromatin accessibility following exposure to mithramycin for 8-hours or 18-hours.

F: IGV tracks of rhabdoid tumor genes previously identified to gain H3K27me3 upon SWI/SNF loss (Erkek et al., 2019). CDK6 and CDK2 decrease in H3K27ac occupancy and chromatin accessibility following exposure to 8-hours and 18-hours mithramycin treatment.

G: IGV tracks of genes identified to have an increase in accessibility, H3K27ac and gene expression following mithramycin exposure. BMP1 and ADIPOR1 play crucial roles in bone and adipogenic differentiation, respectively.
Mithramycin induces promoter reprogramming and differentiation of rhabdoid tumor (2020)
Figure 7

Figure 7: EC8042 leads to durable tumor regression in a rhabdoid tumor xenograft model.

A: Immunohistochemistry analysis recapitulates the biochemistry described in vitro for mithramycin. G401 tumor sections at 20X magnification stained with H&E, cleaved caspase 3 (CC3; apoptosis) or H3K27me3. A marked increase in CC3 that correlates with H3K27me3 staining is seen only in mice treated with the continuous infusion schedule but not vehicle. Scale bar (lower left): 50μm.

B: Prolonged durable response and cure of mice bearing G401 xenografts treated with 30mg/kg EC8042 administered continuously over 72-hours. Treatment duration indicated by gray shaded box. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).

C: Kaplan-Meier survival curves indicating extended survival for mice bearing established G401 xenografts treated with the 3-day continuous infusions of EC8042 in (B). The shaded box indicates the duration of treatment. Asterisk indicate an animal sacrificed due to unknown causes not related to tumor progression or drug toxicity (see text).

D: 20X image of section of G401 treated tumors stained for H3K27me3, cleaved caspase 3, and γH2AX. The sections compare vehicle to treatment started on day 1 with 30 mg/kg of EC8042 administered continuously for 72-hours (3-day pump). H3K27me3 increases and correlates with apoptosis (CC3), however induction of CC3 is modest. γH2AX staining does not increase with treatment indicating DNA damage is not responsible for these effects. Scale bar (lower left): 50μm.
Targeted stabilization of Munc18-1 function via pharmacological chaperones (2020)
Figure 1

Figure 1. Syntaxin-1 fragments rescue deficits in Munc18-1 levels and solubility.

A, Localization of disease-causing missense mutations of Munc18-1b in its tertiary structure (PDB code 4JEU) Burkhardt et al, 2008(), with annotation of residues and binding sites of syntaxin-1 Habc domain and N-terminus. Highlighted in blue are the residues 406 and 544 that we have analyzed in this study in detail.

B, Syntaxin-1 domain structure. The N-terminus (residues 1-28) binds to domain 1 of Munc18-1 while the Habc domain (residues 28-180) and the SNARE domain (residues 180-264) bind in Munc18-1's central cleft. The transmembrane domain (TMD; residues 264-288) does not participate in Munc18-1 binding.

C, Total protein levels of Munc18-1. HEK293T cells transfected with WT or mutant Munc18-1 variants and either GFP, syntaxin-11-264 or syntaxin-11-180 were lysed and lysates were analyzed by quantitative immunoblotting to indicated proteins, normalized to β-actin (Synt-1A = syntaxin-1A). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001 by Kruskal-Wallis test, followed by Dunn's multiple comparison test; n = 11 independent experiments; exact p values are shown in Appendix Table S1).

D, Solubility of Munc18-1. HEK293T cells transfected as in (C) were solubilized in 0.1% Triton X-100 (TX) and equal volumes of soluble and insoluble fractions were analyzed by quantitative immunoblotting. TX-soluble Munc18-1 was measured as percent of total Munc18-1 by quantitative immunoblotting. Data are means ± SEM (*p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by Kruskal-Wallis test followed by Dunn's multiple comparison test, or by one-way ANOVA followed by Bonferroni post-hoc test; n = 5-9 independent experiments; exact n and p values are shown in Appendix Table S1).
Targeted stabilization of Munc18-1 function via pharmacological chaperones (2020)
Figure 5

Figure 5. Rescue of spontaneous neurotransmitter release in cortical mouse neurons expressing Munc18-1 G544D.

A, B. Munc18-1 knockout neurons expressing G544D Munc18-1 were plated on a microelectrode array and subjected to analysis of mean firing rate before addition of compounds (0 h) or 48 hours after vehicle (DMSO) or compound addition. 16 electrodes per well were analyzed for neuronal firing (A; purple boxes indicate network activity). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test, or Kruskal-Wallis test followed by Dunn's post-hoc test; n = 11-16 independent experiments; exact n and p values are shown in Appendix Table S1).
Targeted stabilization of Munc18-1 function via pharmacological chaperones (2020)
Figure 6

Figure 6. Rescue of synaptic vesicle cycling in mouse cortical neurons expressing mutant Munc18-1.

A-D. Uptake of synaptotagmin-1 antibody during high K+ stimulation. Neurons expressing cre recombinase and/or WT, R406H or G544D Munc18-1b with or without compound were subjected 7 days after lentiviral infection to an antibody uptake assay. Endocytosed synaptotagmin-1 antibody was quantified by immunostaining (A-C; scale bar = 30 µm; 4-PBA = 4-phenylbutyrate), via counting the number of pixels > intensity of 15 (D). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-10 independent experiments; exact n and p values are shown in Appendix Table S1).
Targeted stabilization of Munc18-1 function via pharmacological chaperones (2020)
Figure 7

Figure 7. Rescue of evoked neurotransmitter release in mouse cortical neurons expressing mutant Munc18-1.

A-G. Representative images (A) or traces (B-D & E-G) of changes in fluorescence of synaptophysin-pHluorin upon stimulation of neurons (scale bar in panel (A) = 2 µm).

H, I. Plot of change in fluorescence from baseline to peak fluorescence for Munc18-1 G544D (H) or R406H (I). Data are means ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 by one-way one-way ANOVA and Dunnett's multiple comparison test; n = 7-23; exact n and p values are shown in Appendix Table S1). Note, that compound 10 is fluorescent at a similar wavelength than pHluorin, so experiments at concentrations higher than 1 µM were not possible.
Targeted stabilization of Munc18-1 function via pharmacological chaperones (2020)
Figure 8

Figure 8. Rescue of deficits in mutant C. elegans.

A-L (A, C, E, G, I, K) Heat-induced paralysis. Indicated worm strains maintained in compounds at indicated concentrations were exposed to 37 °C over a period of 120 min, and paralysis was scored at indicated time points. Data are means ± SEM (n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1). (B, D, F, H, J, L) Quantification of paralysis at 20 minutes. Data are means ± SEM (*,#p < 0.05, **,##p < 0.01, ***,###p < 0.001, # as compared to WT, * as compared to mutant by two-way ANOVA and Dunnett's multiple comparison test; n = 3-4 independent experiments on ten worms per experiment; exact n and p values are shown in Appendix Table S1).

M. Image of a worm, highlighting head and tail ganglia, as well as the ventral nerve cord and motor neurons.

N, O. Rescue of the subcellular localization of UNC-18 in worms expressing G544D UNC-18. C. elegans expressing WT::GFP or G544D::GFP at 1, 5, 20 or 100 µM compound were immobilized, and the ventral nerve cord was imaged. Solid arrowheads point to pairs of bigger puncta, broken arrowheads to single, smaller puncta (N, O). Scale bar in (N) and (O) = 10 µm. 4-PBA = 4-phenylbutyrate.

P. Quantification of soluble and aggregated Munc18-1. Data are means ± SEM (**p < 0.01, ***p < 0.001 by one-way ANOVA and Dunnett's multiple comparison test; n = 3-4 worms; exact n and p values are shown in Appendix Table S1).
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 1

BACE1 is modified with bisecting GlcNAc in vivo

Bisecting GlcNAc modification by GnT-III.

BACE1 or APP was immunoprecipitated from mouse brains and blotted with E4-PHA lectin (lower) or anti-BACE1 or APP antibodies (upper). hAPP indicates the APP23 transgenic mouse model for AD.

Proteins from mouse brain membrane fractions were treated with or without PNGase F and then immunoblotted for BACE1 or for syntaxin 6 (loading control).

LC–MS base peak chromatogram of desialo-alditol N-glycans derived from mouse brain BACE1. To simplify the results, N-glycans were chemically desialylated before LC–MS analysis. BACE1-specific glycans, judged by comparison with N-glycan structures from anti-BACE1 IgG (shown in Supplementary Fig S2C), are highlighted by red squares. Asterisks indicate glycans demonstrated by MS/MS analysis to contain a bisecting GlcNAc structure. Numbers in parentheses indicate the charge state.

Source data are available online for this figure.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 2

BACE1 is abnormally hyper-modified with bisecting GlcNAc in AD patients

Summary of clinical and histological data of non-AD control (NC), early-stage AD (eAD) or AD patients.

BACE1 from temporal lobe membrane fractions of NC, eAD, or AD patients was immunoprecipitated and blotted with E4-PHA (upper) or anti-BACE1 (lower). The signal intensity of E4-PHA relative to that of BACE1 was calculated (n = 10). The graph shows means ± SEM (*P < 0.05; one-way ANOVA with post hoc Tukey–Kramer test. P = 0.014 for NC versus eAD, P = 0.028 for NC versus AD).

Source data are available online for this figure.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 3

Impaired β-site cleavage of APP in Mgat3−/− brain

APP metabolites from 3-month-old mouse brain membrane (A) or soluble (B) fractions were immunoblotted. The signal intensity was quantified (n = 4–5). All graphs show means ± SEM (*P < 0.05; Student's t-test for (A) βCTF and (B) sAPPα, and Mann–Whitney U-test for the others. P = 0.386 for APP, P = 0.602 for αCTF, P = 0.045 for βCTF, P = 0.218 for sAPPα, P = 0.022 for sAPPβ).

Source data are available online for this figure.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 4

Reduced Aβ load and ameliorated cognitive function in Mgat3−/− mouse brain

Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from (A) 3-month-old or (B) 12-month-old hAPP/Mgat3+/+, hAPP/Mgat3+/−, or hAPP/Mgat3−/− brains (n = 5). P = 0.015 for TBS Aβ40, P = 0.034 for TBS Aβ42, P = 0.001 and 0.012 for Gn–HCl Aβ40, P = 0.024 for Gn–HCl Aβ42 in (A), P = 0.040 for Gn–HCl Aβ40, P = 0.047 for Gn–HCl Aβ42 in (B).

Amounts of Aβ40 or Aβ42 in the TBS-soluble or Gu-HCl-extractable fraction from 3- to 4-month-old Mgat3+/+ or Mgat3−/− brains (n = 7–8). An outlier value was rejected by the Smirnov–Grubbs' test (P < 0.05). P = 0.045 for Gn–HCl Aβ40.

Immunostaining of Aβ plaques in 12-month-old mouse brain (left). Scale bar, 300 μm. The number of FSB-stained Aβ plaques in brain sections prepared from 12-month-old male mice was quantified (right) (n = 6). P = 0.004 for hippocampus, P = 0.0003 for cortex.

The Y-maze test was performed using 12-month-old male Mgat3+/+, Mgat3−/−, hAPP/Mgat3+/+, or hAPP/Mgat3−/− mice (n = 8–10). **P = 0.004 for Mgat3+/+ versus hAPP/Mgat3+/+, *P = 0.022 for hAPP/Mgat3+/+ versus hAPP/Mgat3−/−.

Data information: All graphs show means ± SEM (*P < 0.05, **P < 0.01. For comparison between two groups, Student's t-test was used for (C) and (D) cortex, and Mann–Whitney U-test was performed for (D) hippocampus. In other cases, two-way ANOVA with a post hoc Tukey–Kramer test (A, B) or the Student–Newman–Keuls test (E) was used.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 5

BACE1 is directed to late endosomes/lysosomes in Mgat3−/− cells

BACE1 from 2-week-old mouse brains was immunoprecipitated and immunoblotted (left). Immunoprecipitated BACE1 activity was measured in vitro (right, n = 3). The values of Bace1−/− samples were subtracted as a background. N.S., P = 0.101.

MEF homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, APP, rab5, or rab7. EE, early endosome; LE, late endosome.

Brain homogenates were fractionated by sucrose density centrifugation and immunoblotted for BACE1, rab5, or rab9. EE, early endosome; LE, late endosome. Signal intensities were quantified and are shown in the right graphs.

Immunostaining of 12-month-old mouse cerebral cortex for BACE1 and APP. A typical image in the vicinity of plaque-forming area is shown for hAPP/Mgat3+/+ brain. The area of co-localization was quantified using random images of cerebral cortex (n = 10). *P = 0.015.

Immunoblot of BACE1 or GAPDH (loading control) from immortalized MEFs treated with a proteasome inhibitor (MG132) or a lysosome inhibitor (chloroquine; CQ).

Immunostaining of 12-month-old mouse cerebral cortex for BACE1 (F), or nicastrin (G), and Lamp1. LE, late endosome. Scale bar, 10 μm. The area in which co-localized staining was observed was quantified as a percentage of the total BACE1-positive area (right, n = 10). **P = 0.007.

Data information: All graphs show means ± SEM (Student's t-test).

Source data are available online for this figure.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 6

Down-regulation of BACE1 protein in aged Mgat3−/− mouse brain

Immunostaining of 12-month-old brain sections (cerebral cortex) with anti-BACE1. Scale bar, 100 μm.

Proteins from the membrane fractions of 12-month-old mouse brains (n = 4–5) were immunoblotted for BACE1, and BACE1 intensity was quantified. *P = 0.044.

Lysates from MEFs were immunoblotted for APP, BACE1, or GAPDH (loading control), and BACE1 intensity was quantified (n = 4).

Amount of Aβ40 in the culture medium of immortalized MEFs (n = 3). *P = 0.045.

MEFs were transfected with control siRNA or GGA3-siRNA and then immunoblotted for BACE1, GGA3, or GAPDH (loading control) (n = 6).

Data Information: All graphs show means ± SEM (*P < 0.05; Student's t-test for (B) and the Mann–Whitney U-test for (D)).

Source data are available online for this figure.
An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. (2015)
Figure 7

Limited impairment of BACE1 activity in Mgat3−/− brain

Membrane fractions from 3-week-old Mgat3+/+, Mgat3−/−, and Bace1−/− mice were immunoblotted for CHL1 (upper), contactin-2 (middle), or syntaxin 6 (lower) (n = 4–5). The graphs show means ± SEM (**P < 0.01; two-way ANOVA with post hoc Tukey–Kramer test, P = 0.006 for Mgat3+/+ versus Bace1−/−, P = 0.008 for Mgat3−/− versus Bace1−/− in CHL1/syn6, P = 0.001 for Mgat3+/+ versus Bace1−/−, P = 0.002 for Mgat3−/− versus Bace1−/− in Contactin-2/syn6).

Number of pups surviving at 4 weeks following a cross between heterozygous male and female mice.

Source data are available online for this figure.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 1

Figure 1. cGAS is constantly present in the cytosol and nucleus and is impacted by cell cycle.

A-C. Left: Immunofluorescence images of cGAS in the nucleus (DAPI) and cytosol (phalloidin) in BMDMos cultured at low/high density (A), with/without serum (B), or with/without Aphidicolin (Aphi) (C). Scale bar: 50 μm. Right: Corresponding quantification of the nuclear cGAS from 6 different fields with n > 50 cells.

D-F. Immunoblot estimation of cGAS in nuclear/cytosolic subcellular fraction of BMDMos cultured under indicated conditions. Lamin B and α-Tubulin are nuclear and cytosolic markers respectively.

G-I. Flow cytometric analysis of cell cycle of BMDMos depicted in D-F.

Data information: Data are presented as means ±SEM. Statistical significance was assessed using unpaired student's t test. **** P ≤ 0.0001.
Chromatin-bound cGAS inhibits DNA repair hence accelerates genome destabilization and cell death (2019)
Figure 2

Figure 2. cGAS promotes irradiation-induced micronuclei generation and cell death.

A, B. Micronuclei (indicated by arrow head) in GFP-NLS- or GFP-hcGAS expressing HEK293 cells before (0 h) or 24 h after γ-irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show Mean ± SEM (n=3 independent experiments) representing six different microscopic fields with over 200 cells.

C. IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n=3 independent experiments.

D. Experimental outline for micronuclei generation and cell death after γ-irradiation.

E. Micronuclei (indicated by arrow head) and cGAS staining in WT and cGAS-/- BMDMos exposed to γ-irradiation (10 Gy). Scale bar: 10 μm.

F. Average MNs/cell in BMDMos. MN graphs show Mean ± SEM (n=3 independent experiments) representing eight different microscopic fields with over 200 cells.

G. Cell death in WT and cGAS-/- BMDMos that were first synchronized at G2/M, then γ-irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ±SD, x biological triplicates (n=3) per treatment group are shown.

Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two-tailed Student's t-test. *** P ≤ 0.001; **** P ≤ 0.0001. Statistical significance in (G) was assessed using two-way ANOVA test, ****P < 0.0001.