Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: A Pt(IV)-conjugated brain penetrant macrocyclic peptide shows pre-clinical efficacy in glioblastoma

doi: 10.1016/j.jconrel.2022.10.051

Figure Lengend Snippet: Dose-regime and efficacy studies of Pt(IV)-M13 reveal DNA-damage effects on GBM tumors. a) Percentage of weight variation of naïve nude mice administered after platinum compound tail-vein injection at the indicated concentrations. Black arrows indicate days of injection (d1, d4, d9, d13, d17, d19). Mean and standard deviation are shown, n = 5 animals per group. b) Average clinical score from study shown in a). Mice were monitored every 2 days for symptomatic changes. Symptoms were classified from no symptoms (0), visible (2), to fatal (4). Eye redness, eye twitching and weight loss were the most visible effects. c) Efficacy study of Pt(IV)-M13 (5 mg/kg, 0.3 mM) compared to cisplatin MTD (5 mg/kg, 3 mM). d) Immunostaining of ϒH2AX (Ser139) using anti-rabbit Alexa Fluor 647, and nuclei with Hoechst 33342. Representative image of independent tissue triplicates from the efficacy study c). Scale bar, 10 μm. e) Area percentage of positive cells with ϒH2AX foci within the tumor core from study in d). Statistical significance for c) using log-rank test, *p = 0.020. Median survival for each group is shown. For e), mean and standard deviation are shown, n = 3. Two-way ANOVA test **p < 0.005, ***p = 0.0004.

Article Snippet: For DNA damage staining, tissue slides were incubated with rabbit or mouse anti-phospho-ser139 histone γH2AX antibody (Cell Signaling Technologies) at a dilution of 1:100.

Techniques: Injection, Standard Deviation, Immunostaining

Journal: Aging (Albany NY)

Article Title: Caspase-3 knockout attenuates radiation-induced tumor repopulation via impairing the ATM/p53/Cox-2/PGE 2 pathway in non-small cell lung cancer

doi: 10.18632/aging.103984

Figure Lengend Snippet: Radiations induce DNA damage, caspase-3 activation, and tumor repopulation in NSCLC cells. ( A ) Confocal images of immunostained A549 and H460 cells showing γH2AX foci following 8 Gy irradiation at 48 h. Scale bars: 25 μm. ( B , C ) The left panel shows flow cytometry analysis of A549 ( B ) and H460 ( C ) cell death after 0 Gy or 8 Gy irradiation on day 3. Apoptosis was monitored by Annexin V/propidium iodide (PI) double staining. The right panel shows quantitative analysis of early apoptosis and total cell death in 0 Gy- or 8 Gy-irradiated A549 ( B ) and H460 ( C ) cells (*** p <0.001, Student’s t test, n = 3). ( D ) Cleaved caspase-3 induced by 8 Gy radiations was assayed by western blotting, and β-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) served as loading controls. ( E ) Representative confocal images of immunostained A549 and H460 cells showing cleaved caspase-3 following exposure to 8 Gy radiations on day 3. Scale bars: 25 μm. ( F ) The 8 Gy-irradiated NSCLC cells promoted the growth of living NSCLC reporter cells. The upper panel depicts luciferase activities showing the growth of A549 Fluc and H460 Fluc cells that were seeded alone or with 0 Gy- or 8 Gy-irradiated NSCLC cells. The lower panel shows the representative bioluminescence images (** p <0.01, *** p <0.001, one-way analysis of variance [ANOVA], n = 4).

Article Snippet: Cells were incubated with antibodies against γH2AX (#80312, Cell Signaling Technology), caspase-3 (#9662, Cell Signaling Technology), or EndoG (#ab9647, Abcam) overnight at 4°C, followed by incubation with an Alexa Fluor 488- or 594-conjugated secondary antibody (Proteintech) for 1 h at room temperature.

Techniques: Activation Assay, Irradiation, Flow Cytometry, Double Staining, Western Blot, Luciferase

Journal: Aging (Albany NY)

Article Title: Caspase-3 knockout attenuates radiation-induced tumor repopulation via impairing the ATM/p53/Cox-2/PGE 2 pathway in non-small cell lung cancer

doi: 10.18632/aging.103984

Figure Lengend Snippet: Casp3 KO attenuates the DDR via ATM/p53 signaling in irradiated NSCLC cells. ( A ) Immunofluorescence analysis of 8 Gy-irradiated wild-type and Casp3 KO NSCLC cells co-stained for EndoG and γH2AX foci at 48 h. Scale bars: 25 μm. ( B ) Western blot analysis of EndoG in the cytoplasmic and nuclear fractions of 8 Gy-irradiated wild-type and Casp3 KO NSCLC cells at 48 h. β-tubulin and Histone H3 were used as cytoplasmic and nuclear loading controls, respectively. ( C ) Levels of DNA damage response (DDR)-related proteins ATM, pATM (S1981), Chk2, pChk2 (T68), p53, and pp53 (S15) were measured by western blotting at indicated times after 8 Gy irradiation of wild-type and Casp3 KO NSCLC cells. GAPDH was used as the loading control.

Article Snippet: Cells were incubated with antibodies against γH2AX (#80312, Cell Signaling Technology), caspase-3 (#9662, Cell Signaling Technology), or EndoG (#ab9647, Abcam) overnight at 4°C, followed by incubation with an Alexa Fluor 488- or 594-conjugated secondary antibody (Proteintech) for 1 h at room temperature.

Techniques: Irradiation, Immunofluorescence, Staining, Western Blot

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B modifications in nascent chromatin mirror parental chromatin states (A) Scheme outlining the question addressed in this study. Do recycling of modified H2A-H2B contribute to transmission of chromatin states during replication? Flags represent post-translational modifications. (B) Overview of the ChOR-seq workflow. (C–F) (Left) Nascent chromatin (ChOR-seq, blue) and total chromatin (ChIP-seq, gray) occupancy of H3K27me3, H2AK119ub1, H2A.Z, and H2BK120ub1 (C–E). (Right) Heatmap and average profile across peaks of H3K27me3, H2AK119ub1, and H2A.Z (F). (Right) Metagene analysis across H2BK120ub1-decorated genes. TSS: transcription start site; TES: transcription end site. Signal is sorted according to total ChIP-seq intensity and quantified with reads per million (RPMs). Data are represented as average of two replicates. See also Figure S1 .

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Modification, Transmission Assay, ChIP-sequencing

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B modifications in nascent chromatin mirror parental chromatin states, related to Figure 1 (A–C) Barplot of mapped reads (mouse) relative to spike-in reads ( Drosophila ) in noEdU or nascent samples for H2AK119ub1 (A, n = 4), H2A.Z (B, n = 2), and H2BK120ub1 (C, n = 2). (D) Western blot analysis of aniPOND signal with indicated antibodies. A representative image is shown. nE: no EdU control. (E) Quantification of aniPOND signal of nascent (T0) relative to no EdU samples for the indicated antibodies. Average of three replicates. (F) Pearson correlation plot of replicates and conditions of the marks examined. RPM-normalized values were used to probe correlation. (G) Heatmap and average profile across peak boundaries (H3K27me3, H2AK119ub1, and H2A.Z, left to right). RPM scale. (H) Heatmap and average profile across TSS for H2BK120ub1-decorated genes. RPM scale. (I and K) Barplot of mapped reads (human) relative to spike-in reads ( Drosophila ) in noEdU or nascent samples for H2A.Z and H2BK120ub1. (J and L) (Left) Nascent chromatin (ChOR-seq, blue) and total chromatin (ChIP-seq, gray) occupancy of H2BK120ub1 and H2A.Z. (Right) Metagene analysis across H2BK120ub1-decorated genes (J) and heatmap and average profile across peaks of H2A.Z, (L) RPM scale. (G–L) Data are represented as average of two replicates.

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Western Blot, ChIP-sequencing

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B is recycled symmetrically during DNA replication independent of H3-H4 (A) Illustration of the question addressed. Is H2A-H2B segregated symmetrically to both daughter strands? (B) Average SCAR-seq profile, showing replication fork directionality (RFD, measured by OK-seq), and asymmetry (measured as partition to leading and lagging strand), for the indicated histone PTMs across all replication initiation zones with a H2AK119ub1 peak. (C and D) Average SCAR-seq profile for H3K27me3 or H2AK119ub1 in WT or POLE4KO cells. (E and F) Average SCAR-seq profile for H3K27me3 or H2AK119ub1 in WT or MCM2-2A cells. (G and H) Average SCAR-seq profile for H3K27me3 or H2AK119ub1 in WT or POLA1-3A cells. (B), (D), (F), and (H): Note the change in scale to visualize smaller biases. Individual replicates shown in Figure S3 . Data in (C), (E), and (G) are represented as average of n = 2–4 replicates. N(IZ): number of initiation zones analyzed . See also Figure S3 .

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques:

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B is recycled symmetrically during DNA replication independent of H3-H4, related to Figure 3 (A) Asymmetry plot for H2AK119ub1, H3K27me3, and H4K20me0 across H2AK119ub1 peaks (n = 6–8). Positive values indicate leading strand bias. Negative values indicate bias toward lagging strand. (B) Asymmetry plot for H2AK119ub1, H3K27me3, and H4K20me0 across H2AK119ub1 peaks (n = 6–8). Individual replicates. (C) Asymmetry plot for H3K27me3 across called H3K27me3 peaks in different cell lines (n = 3–6). (D) Asymmetry plot for H2AK119ub1 across H2AK119ub1 peaks in POLE4KO cells, individual replicates (n = 2 for WT, n = 4 for 2 POLE4KO clones). (E) Asymmetry plot for H2AK119ub1 across H2AK119ub1 peaks in MCM2-2A cells, individual replicates (n = 3 for WT and 2 MCM2-2A clones). (F–H) Average SCAR-seq profile for H4K20me0 in WT and POLE4KO (F), MCM2-2A (G), or POLA1-3A (H) cells. (I) Asymmetry plot for H2AK119ub1 across H2AK119ub1 peaks in POLA1-3A cells, individual replicates (n = 3 for WT, n = 4 for 2 POLA1-3A clones). (J) Asymmetry plot for H4K20me0 in different cell lines (n = 3–4). (K–M) Average xSCAR-seq profile for H2A.Z in WT and POLE4KO (K), MCM2-2A (L), or POLA1-3A (M) cells (n = 2). (N) Asymmetry plot for H2A.Z in different cell lines (n = 2). (D, E, I, and N) Statistics by unpaired Wilcoxon test.

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Clone Assay

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B marks restore accurately and rapidly after DNA replication (A) Experimental outline of the quantitative ChOR-seq time course. (B–E) Average profile of RRPM-normalized occupancy signal for H2BK120ub1, H2A.Z, H2AK119ub1, and H3K27me3 signal across 3 kb centered on the TSS. Only TSSs occupied by the respective mark were included. Log 2 scale. (F) Average profile of RPM-normalized occupancy signal of nascent or total pan-histones (combined pan-H2A and pan-H3) across all TSSs (n = 30,025). Log 2 scale. (G) Restoration curve for relative abundance of H2BK120ub, H2A.Z, H2AK119ub1, and H3K27me3 post-replication. Data points in gray were excluded from regression analysis . (H) Kinetic parameters for investigated marks. t(90% restored): relative time (in hours) needed to restore 90% of the total signal. %recycled: estimated abundance at nascent chromatin (T0) across peaks. Data are represented as average of two replicates. See also Figure S4 .

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques:

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A-H2B marks restore accurately and rapidly after DNA replication, related to Figure 4 (A–D) Average profile of RRPM-normalized occupancy signal across peak centers (top) or boundaries (bottom) for H2BK120ub1, H2A.Z, H2AK119ub1, and H3K27me3. (E) Average profile of RPM-normalized occupancy signal of nascent or total pan-H2A or pan-H3 across all TSS, (n = 30,025). Z score normalized. (F) Kinetic parameters for investigated marks. t(90% restored): relative time (in hours) needed to restore 90% of the total signal. % recycled: estimated abundance at nascent chromatin (T0) across the entire genome. Considering all regions may not represent as accurate parameters as in contrast to Figure 4 H. Data are represented as average of two replicates.

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques:

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A.Z and H2BK120ub1 restoration correlate with transcription (A) Outline of restoration categories for H2A.Z peaks. Peaks are classified as restored once their signal show no further increase in all subsequent timepoints (for example, R60: restored at 60 min) or classified as unstable if their signal is decreasing in subsequent time points . (B) Restoration kinetics of peaks overlapping enhancers or promoters (within 1 kb). (C) Promoter signature (within 1 kb from TSS) for the different restoration categories for H2A.Z. Log 2 (RPKM + 1) scale. Statistics by unpaired Wilcoxon test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. H3K4me3 data from Sethi et al., RNAPII-Ser5p data from Stewart-Morgan et al. (D) Restoration curve for H2A.Z peaks at promoters stratified according to gene expression level quartiles. (E) As in (D) but for H2BK120ub1 peaks. No peaks overlapped promoters at low- or not-expressed genes. (F) Relative restoration categories according to distance of H2BK120ub1 peaks to TSS. Data are represented as average of two replicates. See also Figure S5 .

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Expressing

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: H2A.Z and H2BK120ub1 restoration correlate with transcription, related to Figure 5 (A) Box plot showing distance to TSS for H2A.Z peak restoration categories. Log 10 scale. (B) Restoration lineplot for H2A.Z peaks mapping to promoters stratified according to overlap with H2BK120ub1 peaks. (C) Restoration lineplot for H2A.Z peaks mapping to promoters stratified according to overlap with H3K27me3 or H2AK119ub1 peaks. (D) Genomic occupancy of H2BK120ub1 across the time course. RRPM scale. Data are represented as average of two replicates.

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques:

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: Cross talk with H2AK119ub1 drives H3K27me3 restoration, related to Figure 7 (A) (Top) ChOR-seq restoration categories for H3K27me3 in WT cells, classified identically as in Figure 5 A . (Bottom) Fraction of H3K27me3 peaks (parsed into 1 kB windows) overlapping H2AK119ub1ub peaks. (B) Western blot analysis of RING1B and H2AK119ub1 levels in DMSO or Auxin-treated cells (using H2A as a loading control). (C) Quantification of (B) relative to H2A. Average of two independent replicates. Quantification using ImageJ. (D) Barplot of mapped reads relative to spike-in for H3K27me3 in 2 h DMSO or Auxin-treated cells. (E) Average profile of RRPM-normalized occupancy signal for H3K27me3 across peak centers in DMSO or Auxin-treated cells. (F) Asymmetry plot showing the restoration of H3K27me3 asymmetry in DMSO-treated MCM2-2A cells over time in across H3K27me3 peaks stratified according to overlap with H2AK119ub1. (G) Asymmetry plot showing the restoration of H3K27me3 asymmetry in DMSO-treated MCM2-2A cells over time across all regions with significant signal (RPM > 0.6) but outside Polycomb domains (neither H2AK119ub1 nor H3K27me3 peaks). (H) Heatmap and average profile of total ChIP JARID2 occupancy across peak centers in genome-wide chromatin in 2.5 h DMSO or Auxin-treated cells. RRPM scale. (I) Asymmetry plot showing the restoration of H2AK119ub1 asymmetry in WT or MCM2-2A cells over time across all H2AK119ub1 peaks stratified according to overlap with H3K27me3 peaks. (F, G, and I) Statistics by unpaired Wilcoxon test. Data are represented as average of three replicates. (D) Statistics by Student t test (unpaired, two replicates).

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Western Blot, Genome Wide

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet: Cross talk with H2AK119ub1 drives H3K27me3 restoration (A) Illustration of the questions addressed. Does H2AK119ub1 stimulate H3K27me3 restoration on the lagging strand? Does H3K27me3 increase H2AK119ub1 deposition on the leading strand? (B) Experimental outline to assess H2AK119ub1-dependence of H3K27me3. AID-RING1B RING1A −/− MCM2-2A cells were treated for 2 h with Auxin or DMSO prior to 10 min EdU labeling and indicated chase time course. (C) Asymmetry plot showing restoration of H3K27me3 asymmetry across H3K27me3 peaks in DMSO and Auxin-treated cells over time. Positive values indicate leading strand bias. Negative values indicate bias toward lagging strand. (D) Asymmetry plot showing restoration of H3K27me3 asymmetry stratified by H2AK119ub1 co-occupancy in Auxin-treated cells over time. (E) JARID2 occupancy across JARID2 peaks on nascent chromatin in untreated and Auxin-treated cells. RRPM scale. (F) Average SCAR-seq profile of JARID2 in untreated or Auxin-treated cells. (G) Asymmetry plot showing restoration of H2AK119ub1 asymmetry across H2AK119ub1 peaks in WT or MCM2-2A cells over time. (H) As in (E) but focusing on H2AK119ub1 peaks overlapping cPRC1 sites (RING1B, PCGF2). (I) As in (E) but focusing on H2AK119ub1 peaks overlapping vPRC1 sites (RING1B, no PCGF2). (C, D, and G–I) Statistics by unpaired Wilcoxon test. Data are represented as average of three replicates. (J) Model. (Left) Recycling of H2A-H2B at replication forks transmits modifications on parental H2A-H2B to nascent chromatin. Parental H2A-H2B dimers are recycled to both daughter strands independent of H3-H4 tetramers through a pathway involving POLA1 on the lagging strand. Upon reincorporation on daughter strands, parental-modified H2A-H2B can form nucleosomes with new and parental H3-H4 tetramers and new H2A-H2B dimers. Fast restoration kinetics of H2A-H2B modification together with intra- and inter-nucleosomal PTM cross talk then contribute to establishment of modifications on new H3-H4 and H2A-H2B and maintenance of chromatin states across replication. (Right) We propose that recycled parental H2A-H2B PTMs guide the restoration of newly deposited H3-H4 and H2A-H2B in nascent chromatin. Vice versa, stable H3-H4 PTMs reinforce H2A-H2B modifications in mature chromatin responding to dynamic exchange of H2A-H2B. Replisome components involved in histone recycling are colored in blue for H3-H4 and yellow for H2A-H2B. Yellow arrows depict the path of H2A-H2B at the replication fork, while black arrows indicate positive feedback. See also Figure S7 .

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Labeling, Modification

Journal: Cell

Article Title: Recycling of modified H2A-H2B provides short-term memory of chromatin states

doi: 10.1016/j.cell.2023.01.007

Figure Lengend Snippet:

Article Snippet: H2A.V (mouse) , Active Motif , Cat no. 61751; RRID: AB_2793757.

Techniques: Recombinant, Purification, Western Blot, Software