Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software

Journal: eLife

Article Title: Distinct and diverse chromatin proteomes of ageing mouse organs reveal protein signatures that correlate with physiological functions

doi: 10.7554/eLife.73524

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-RNF20 (rabbit polyclonal) , Cell Signaling Technology , Cat# 9425S;RRID: AB_2797700 , WB (1:1000).

Techniques: Software

Journal: Nucleic Acids Research

Article Title: Bimodal regulation of the PRC2 complex by USP7 underlies tumorigenesis

doi: 10.1093/nar/gkab209

Figure Lengend Snippet: The assembly of the USP7/EZH2 complex on transcriptional targets. ( A and B ) qChIP analysis of selected promoters in the A375 cells after co-transfection with indicated shRNA and Vector or FLAG-EZH2 using the indicated antibodies. The knockdown efficiencies of USP7 and EZH2 were verified by western blotting. ( C ) Western blotting analysis of the expression of indicated proteins in A375 cells that transfected with different sets of USP7 siRNAs. ( D ) Western blotting analysis of the expression of H3K27me3 and H2AK119ub in A375 cells transfected with the indicated siRNAs. ( E ) A375 cells were transfected with indicated siRNAs for qChIP analysis on the selected promoters using antibodies against the indicated histone modification. ( F ) A375 cells transfected with FLAG-RNF20 or FLAG-RNF40 for the measurement of the indicated histone modification by western blotting. ( G ) A375 cells were transfected with the indicated expression vectors for qChIP analysis on the selected promoters using antibodies against the indicated histone modification. In A, B, E and G, data represent the mean ± SD from biological triplicate experiments. * P < 0.05 and ** P < 0.01, one-way ANOVA.

Article Snippet: The sources of antibodies against the following proteins were as follows: HA (sc-805) from Santa Cruz Biotechnology; PCGF1 (PA5-49390, for WB), β-actin (A1978), EZH2 (AV38470, for IHC), RYBP (PRS2227, for WB) and FLAG (F3165) from Sigma; USP7 (05-1946, for WB, IF and IP) and H2AK119ub1 (ABE569, for WB and ChIP) from Millipore; Ubiquityl-Histone H2B (K120) (#5546, for WB, IF and ChIP), SKP1 (#2156, for WB), Histone H2A (#2578, for WB and ChIP), RNF20 (#11974, for WB), RNF40 (#12187, for WB) and FOXO1 (#2880, for WB and IHC) from Cell Signaling Technology; KDM2B (ab234082, for WB), SUZ12 (ab175187, for WB and IP), H3K27me3 (ab6002, for WB, IF, ChIP, and ChIP-seq), H2B (ab64165, for WB and ChIP), DDDDK-tag (ab1257, for IF) and histone H3 (ab1791, for WB and ChIP) from Abcam; BCOR (12107-1-AP, for WB) from Proteintech; USP7 (A300-033A, for IP, IF, IHC and ChIP), RING1A (A303-552A, for WB) and RING1B (A302-869A, for WB) from Bethyl Lab; Myc (M047-3) from MBL; EED (GTX33168, for WB and IP) and YAF2 (GTX115355, for WB) from GeneTex; EZH2 (612667, for WB, IP and IF) from BD Transduction Laboratories; and EZH2 (39901, for ChIP and ChIP-seq) from Active Motif.

Techniques: Cotransfection, shRNA, Plasmid Preparation, Western Blot, Expressing, Transfection, Modification

Journal: Nucleic Acids Research

Article Title: Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination

doi: 10.1093/nar/gkaa606

Figure Lengend Snippet: Physical and functional interaction of Egr2 and Rnf40/Rnf20. ( A ) Venn diagram depicting the overlap of Egr2 target genes with lost H2Bub1 marks in Rnf40 ΔSC nerves. ( B , C ) GO analysis (B) and list (C) of Egr2 target genes with reduced H2Bub1 marks in Rnf40 ΔSC nerves. ( D ) Co-immunoprecipitation (IP) of Rnf40 and Rnf20 with antibodies directed against Egr2 and pre-immune serum (PI) from extracts of HEK293T cells that were transfected with various expression plasmids as indicated below the panels. Western blot was used to detect precipitated Rnf40 (upper panel), Rnf20 (middle panel) and Egr2 (lower panel). Numbers on the right indicate position of co-electrophoresed size markers in kDa. ( E ) Scheme of Egr2 proteins and their ability to interact with myc-tagged Rnf40 in extracts from transfected HEK293T cells. DUF3446, domain of unknown function. ( F ) Scheme of myc-tagged Rnf40 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. ( G ) Scheme of HA-tagged Rnf20 proteins and their ability to interact with Egr2 in extracts from transfected HEK293T cells. ( H , I ) Co-immunoprecipitation (IP) of Egr2 with antibodies directed against Rnf40 (H), Rnf20 (I) and IgG control (H, I) from extracts of differentiating SC cultures. Western blot was used to detect precipitated Egr2 (upper panels), Rnf40 (lower panel, H) and Rnf20 (lower panel, I). Experiments were repeated three times. Uncropped western blots for D–I are presented as . ( J – M ) Determination of endogenous expression levels of Mbp (J), Mpz (K), Mag (L) and Prx (M) by qRT-PCR in Neuro2a cells transfected with various combinations of expression plasmids for Egr2, Rnf40-specific shRNA (shRnf40), scrambled shRNA (shScr) or various combinations of these as indicated below the lanes after FACS. Transcript levels in cells transfected with empty expression vectors (ctrl) were set to 1 and all other values were expressed in relation to it ( n = 3; mean values ± SEM). Statistical significance was determined by one-way ANOVA with Bonferroni's multiple comparisons test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Exact values are provided in the Supplementary Tables. ( N ) Proposed model for the action of the Rnf40/Rnf20 E3 ligase and H2Bub1 modification in SC development.

Article Snippet: For stainings, the following primary antibodies were used: chicken anti-Mpz antibodies (Aves Labs, #PZO, Lot #PZO8767965, 1:500 dilution), goat anti-Sox2 antiserum (Santa Cruz, #sc17319, Lot #I1115, 1:500 dilution), goat anti-Sox10 antiserum (home-made, generated against a bacterially expressed and purified peptide corresponding to amino acids 181–233 and 308–400 of rat Sox10 according to accession number NM_019193.2, validated on control and knockout tissue, 1:3000), guinea pig anti-Sox10 antiserum (home-made, validated on control and knockout mouse tissue, 1:1000 dilution) , guinea pig anti-Egr2 antiserum (home-made, validated on mouse sciatic nerve, 1:1000 dilution) , rabbit anti-Egr2 antiserum (Covance, #PRB-236P, Lot #D13BF00486, 1:200 dilution), rabbit anti-Rnf40 monoclonal (Abcam, #ab191309, Lot #11974, 1:100 dilution), rabbit anti-Rnf20 monoclonal (Cell signaling, #11974, Lot #1, 1:100 dilution), rabbit anti-Oct6 antiserum (home-made, validated on control and knockout mouse tissue, 1:2000 dilution) , rabbit anti-Nav1.6 antiserum (Alomone Labs, #ASC-009, Lot#ASC009AN2425, 1:50 dilution), rabbit anti-Caspr antiserum (Abcam, #ab34151, Lot #GR86230, 1:1000 dilution), rabbit anti-Iba1 antiserum (Wako, #019–19741, Lot #SAE6921, 1:250 dilution), rabbit anti-Ki67 antiserum (Thermo Fisher Scientific, #RM-9106, Lot#9106S906D, 1:500 dilution), rat anti-Ki67 antiserum (Thermo Fisher Scientific, #14–5698-82, Lot #2056928, 1:500 dilution), rat anti-MBP monoclonal (Bio-Rad, #MCA409S, Lot #210610, 1:300 dilution), mouse anti-H2Bub1 monoclonal (gift of D. Eick, Helmholtz Zentrum München, 1:5 dilution) ( ).

Techniques: Functional Assay, Immunoprecipitation, Transfection, Expressing, Western Blot, Quantitative RT-PCR, shRNA, Modification

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet: In Vivo Screen Identifies CHD1 as Top Candidate Responsible for Resistance to Antiandrogen (A) Graphical representation of analyzed results of the library screen, using RIGER-E method. –Log10 of p value is presented and the area of p < 0.0001 is highlighted. The top eight candidate genes are presented as large red dots with gene symbol. Negative control gene TBC1D4 is presented as a large green dot. (B) Graphical representation of the percentage of tumors which have shRNAs targeting a specific gene and are enriched in resistant tumors. (C) Graphical representation of the number of genes which have multiple independent shRNAs enriched in resistant tumors. (D) Bee swarm plot of the normalized shRNA read counts of a representative pool in the plasmid, pregraft, and resistant tumors, median is presented as a red line (medians below 1 are not presented on log2 scale). shCHD1s are presented as large red dots. See also .

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: In Vivo, Negative Control, shRNA, Plasmid Preparation

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet: CHD1 Loss Confers Significant Resistance to Antiandrogen In Vitro and In Vivo (A) Western blot of CHD1 in LNCaP/AR cells transduced with annotated guide RNAs. (B) Relative cell number of LNCaP/AR cells transduced with annotated guide RNAs, normalized to sgNT + Veh group. Cells were treated with 10 μM enzalutamide (Enz) or DMSO (Veh) for 7 days and cell numbers were counted. p values were calculated using multiple t tests, three biological replicates in each group. (C) Histograms of representative fluorescence-activated cell sorting-based competition assay showing the distribution of shNT LNCaP/AR cells (GFP-negative) compared with cells transduced with cis -linked shCHD1-GFP or shNT-GFP shRNAs (GFP positive). The distribution on day 0 is shown in red and day 7 is shown in blue. (D) Relative cell number fold change compared with shNT group, based on the results of (C). Enz denotes enzalutamide of 10 μM and Veh denotes DMSO. p values were calculated using two-way ANOVA, three biological replicates in each group. (E) Tumor growth curve of xenografted LNCaP/AR cells transduced with annotated guide RNAs. Enz denotes enzalutamide treatment at 10 mg/kg from day 1 of grafting. Veh denotes 0.5% CMC + 0.1% Tween 80. p values were calculated using two-way ANOVA. For all panels, mean ± SEM is presented. ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. See also and .

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: In Vitro, In Vivo, Western Blot, Transduction, Fluorescence, FACS, Competitive Binding Assay

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet: CHD1 mRNA Level Is Correlated with Clinical Outcome of Antiandrogen Treatment (A) Pearson correlation analysis of CHD1 mRNA and time of treatment on abiraterone (Abi)/enzalutamide (Enz)/apalutamide (Apa) of a 52 mCRPC patient cohort. (B) CHD1 expression distribution in all patients of the cohort in (A). (C) Probability of treatment duration of the top quartile compared with bottom quartile of all patients treated with abiraterone (Abi)/enzalutamide (Enz)/apalutamide (Apa); p value was calculated using Mantel-Cox test. (D) Cox hazard ratio analysis of the top and bottom quartile of all patients, p value was calculated using log rank test. (E) Probability of treatment duration of the above median compared with below median of patients who received enzalutamide (Enz)/apalutamide (Apa), p value was calculated using Mantel-Cox test. (F) Probability of treatment duration of the above median compared with below median of patients who received abiraterone (Abi), p value was calculated using Mantel-Cox test. (G) Pearson correlation analysis of CHD1 mRNA and time of treatment on patients who received enzalutamide (Enz)/apalutamide (Apa), n = 21 (2 patients received both apalutamide and abiraterone). (H) Pearson correlation analysis of CHD1 mRNA and time of treatment on patients who received abiraterone (Abi), n = 33.

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: Expressing

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet: CHD1 Loss Enhanced Prostate Cancer Cell Heterogeneity and Lineage Plasticity (A) Heatmap represents the expression fold changes (qPCR) of the top four resistance driver genes and CHD1 in different xenografts derived cell lines, three technical replicates for each line. (B) Heatmap represents the expression fold changes of the top four resistant driver genes (qPCR) in shCHD1 cell line treated with 10 μM enzalutamide (Enz) in charcoal-stripped serum medium, three biological replicates for each line. (C) Unsupervised clustering of 212 patients based on the gene expression level ( Z score) of CHD1 and the 4 TFs. (D) Relative gene expression level (qPCR) of lineage-specific markers and EMT genes in selective shCHD1-XE and sgCHD1-XE cell lines, three technical replicates for each line. See also Figure S7 .

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: Expressing, Derivative Assay

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet: GR Inhibition Has Significant Antitumor Effect on Antiandrogen-Resistant Tumors with CHD1 Loss (A) Relative gene expression of NR3C1 and GR target genes in tumors collected from LNCaP/AR xenografts, all normalized and compared with shNT + Veh group. Mean ± SEM is presented. p values were calculated using two-way ANOVA, and numbers of biological replicates are presented. (B) Western blot showing AR, GR, and their downstream target genes in xenografted LNCaP/AR tumors. For (A) and (B), Enz denotes enzalutamide at 10 mg/kg from day 1 of grafting. Veh denotes 0.5% CMC + 0.1% Tween 80. (C) Histograms of representative FACS-based competition assay showing the distribution of shCHD1-XE-1 cells (RFP-negative) versus shCHD1-XE-1 cells transduced with shGR (RFP-positive). The distributions on different days are presented in different colors. (D) Relative cell number of shCHD1-XE-1 cells transduced with annotated inducible shRNAs, normalized to shCHD1-XE-1 + Veh. Cells were treated with 250 ng/mL doxycycline for 48 h, and then 7 days of 10 μM enzalutamide (Enz) or DMSO (Veh) before cell numbers were counted. Mean ± SEM is presented, and p values were calculated by two-way ANOVA, with three biological replicates in each group. (E) Model depicting the chromatin dysregulation (plasticity) and antiandrogen resistance in mCRPC due to CHD1 loss. For all panels, ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05. See also Figure S8 .

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: Inhibition, Expressing, Western Blot, Competitive Binding Assay, Transduction

Journal: Cancer Cell

Article Title: Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation

doi: 10.1016/j.ccell.2020.03.001

Figure Lengend Snippet:

Article Snippet: Antibodies used for western blot are (also listed in ): (1) CHD1 (D8C2) Rabbit mAb, Cell Signaling, Cat #4351 (2) AR Antibody (N-20), Santa Cruz, sc-816 (3) KLK3 (D6B1) XP® Rabbit mAb, Cell Signaling, Cat # 5365 (4) PMEPA1 Antibody (P-15), Santa Cruz, Cat # sc-85829 (5) STEAP Antibody (B-4), Santa Cruz, Cat # sc-271872 (6) β-Actin (13E5) Rabbit mAb, Cell Signaling, Cat # 4970 (7) Glucocorticoid Receptor (D6H2L) XP® Rabbit mAb, Cell Signaling, Cat #12041 (8) SGK1 (D27C11) Rabbit mAb, Cell Signaling, Cat #12103 (9) c-Myc (D84C12) Rabbit mAb, Cell Signaling, Cat #5605

Techniques: Recombinant, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Transfection, Purification, Cell Viability Assay, Software