Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 1 Ring1b is associated with breast cells metastasis in vitro and in vivo. Ring1b was stably overexpressed or knocked down in cells using lentivirus. A Heatmap of PRC1-genes expression influenced by TGF-β. Total RNAs isolated from 10 A and TGF-β-induced (15 ng/ml, 24 h) 10 A cells were analyzed by qRT-PCR. B Effect of TGF-β on H2Aub1, H2AK119ub, and Ring1b expression. Total protein extractions from 10 A, TGF-β-induced (24 h) 10 A cells were analyzed by western blot. C Effect of Ring1b on H2AK119ub expression. Total protein extractions were analyzed by western blot. D Effect of TGF-β and Ring1b on H2AK119ub expression. Total protein extractions from 10 A and TGF-β-induced (15 ng/ml, 24 h) 10 A cells were analyzed by western blot. E, F Function of Ring1b in cell migration and invasion. 10 A cells were stimulated with TGF-β (15 ng/ml) for 24 h. Statistical analysis of cells migration and invasion by Transwell assays are presented in the bar graphs. Scale bar, 60 μm. G Immunofluorescent staining of Ring1b (red; TRITC) and E-cadherin (red; TRITC) in 10 A cells. 10 A cells were stimulated with TGF-β (15 ng/ml) for 24 h. Scale bar, 25 μm. H Bright field images and H&E staining of lungs at 6 weeks after mouse tail vein injection. Scale bar, 75 μm. I, J Flow cytometry analysis of GFP+ 231 cells in lungs at 6 weeks after tail vein injection. Statistical analysis shows the percentage of GFP+ 231 cells in the lung. Error bars represent the means ± SEM. Unpaired t-test is performed to indicate a statistically significant difference. ***P < 0.001.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: In Vitro, In Vivo, Stable Transfection, Expressing, Isolation, Quantitative RT-PCR, Western Blot, Migration, Staining, Injection, Flow Cytometry

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 2 Verification of Ring1b-interacting proteins. A, B Ring1b interacts with DDX3X and DDX5, or with Snail1 and Twist2. The 293 T cells were transfected with plasmids as indicated for 24 h. Proteins were captured by IP and analyzed by western blot. C Heatmap of interaction between Ring1b-associated proteins. D Schematic depiction of Ring1b complexes. E–G Ring1b complexes verified in breast cell lines. Lysates from 10 A, 10A- Ring1b, MCF-7, and 231 cells were analyzed by IP followed by western blot. H Expression of endogenous Ring1b-associated proteins in breast cell lines. Total protein extractions from cells were analyzed by western blot.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Transfection, Western Blot, Expressing

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 3 Ring1b cooperates with DDXs or EMT TFs to strengthen the inhibition of E-cadherin. Proteins as indicated were stably overexpressed or knocked down in cells using lentivirus. A Clustering analysis of EMT-related genes’ expression influenced by Ring1b. Total RNAs isolated from 10A-control and 10A-Ring1b cells were analyzed by qRT-PCR. B Gene Ontology (GO) term analysis of EMT-related genes influenced by Ring1b. C Effect of Ring1b on epithelial and epithelial genes expression. Total RNAs isolated from 10 A and 231 cells were analyzed by qRT-PCR. D–K Effect of Ring1b complexes on E-cadherin expression in 10 A and 231 cells. Total protein and RNAs isolated from cells were analyzed for E-cadherin expression by western blot and qRT-PCR. Error bars represent the means ± SEM. Unpaired t-test is performed to indicate a statistically significant difference. ns, P ≥0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Inhibition, Stable Transfection, Expressing, Isolation, Control, Quantitative RT-PCR, Western Blot

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 4 Ring1b complexes promote invasion in breast cells. Proteins as indicated were stably overexpressed or knocked down in cells using lentivirus. A–D Effect of Ring1b complexes on cell invasion in 10 A and 231 cells. Statistical analysis for cells migration and invasion by Transwell assays are presented in the bar graphs. Scale bar, 60 μm. Error bars represent the means ± SEM. Unpaired t-test is performed to indicate a statistically significant difference. ***P < 0.001.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Stable Transfection, Migration

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 5 Ring1b complexes inhibit E-cadherin expression at transcriptional level. Proteins as indicated were stably overexpressed or knocked down in cells using lentivirus or si-RNA. IgG was used as control antibody. The probability of proteins binding to the E-cadherin promoter was analyzed by ChIP-qRT-PCR. A Schematic representation of E-cadherin promoter region. E-cadherin promoter was divided into five regions to determine the degree of amplification. B, C DNA binding ability of DDXs, EMT TFs, and Ring1b on the E-cadherin promoter. Immunoprecipitated DNA fragments were captured by antibodies specific to DDX3X, DDX5, Flag (Snail1), HA (Twist2), and Ring1b in breast cell lines. D Schematic diagram of Ring1b complexes associated with metastatic features in breast cells. E–H Effect of Ring1b complexes on E-cadherin transcription. Immunoprecipitated DNA fragments from 10 A cells were captured by antibodies specific to Ring1b, DDX3X, DDX5, Flag (Snail1), and HA (Twist2). I, J Effect of three proteins on E-cadherin expression. Total RNAs isolated from 231 and 10 A cells were analyzed by qRT-PCR. K Recruitment of Ring1b at site 1 and 2 in 10 A cells. Immunoprecipitated DNA fragments from 10 A cells were captured by antibody specific to Ring1b. Error bars represent the means ± SEM. Unpaired t-test is performed to indicate a statistically significant difference. ns, P ≥0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Expressing, Stable Transfection, Control, Binding Assay, Quantitative RT-PCR, Immunoprecipitation, Isolation

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 6 Ring1b-dependent epigenetic remodeling is associated with multiple epigenetic markers. Proteins as indicated were stably overexpressed or knocked down in 10 A cells using lentivirus. IgG was used as control antibody. Immunoprecipitated DNA fragments were captured by antibodies as indicated. The probability of proteins binding to E-cadherin promoter was analyzed by ChIP-qRT-PCR. A, B Effect of HDAC1 and Ezh2 on E-cadherin transcription. C–E Effect of Ring1b complexes on epigenetic markers distribution on the E-cadherin promoter. F–H Effect of three proteins on epigenetic markers distribution on the E-cadherin promoter. Error bars represent the means ± SEM. Unpaired t-test is performed to indicate a statistically significant difference. ns, P ≥0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Stable Transfection, Control, Immunoprecipitation, Binding Assay, Quantitative RT-PCR

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 7 Schematic diagram of Ring1b-dependent epigenetic remodeling for E-cadherin transcription in breast cell lines. Distinct Ring1b complexes gradually make breast cells lose the expression of E-cadherin, change epigenetic markers on the E- cadherin promoter and acquire metastatic characteristics.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Expressing

Journal: Cell death & disease

Article Title: Distinct Ring1b complexes defined by DEAD-box helicases and EMT transcription factors synergistically enhance E-cadherin silencing in breast cancer.

doi: 10.1038/s41419-021-03491-4

Figure Lengend Snippet: Fig. 8 Ring1b and associated proteins predict breast cancer metastasis. Mean integrated optical density (IOD) was used to evaluate expression of proteins in tissues. E-cadherin expression was determined by immunoreactive scoring system (−negative, + weak, ++ to +++ moderate and strong, respectively). A Single factor analysis of E-cadherin expression in cancer tissues. B Double factor analysis of E-cadherin expression in cancer tissues. C Correlation analysis of Ring1b and associated proteins in cancer tissues. D Double factor analysis of survival curves in cancer tissues. The data from TCGA atlas were analyzed by Kaplan–Meier method. Paired t-test is performed to indicate a statistically significant difference.

Article Snippet: Primary antibodies were against Vimentin, Fibronectin, DDX3X, DDX5, Snail1 (sc-6260, sc-18825, sc-365768, sc365164, sc-271977; Santa Cruz, USA), H2A (129418; GeneTex, USA), Ring1b, E-cadherin, H2AK119ub, Ezh2, H3K27me3, IgG, H3, HDAC1, H3K27ac, β-actin (#5694, #3195, #8240, #5246, #9733, #2729, #4620, #34589, #8173, #3700; Cell Signaling Technology, USA), HA, Flag (H9658, F1804; Sigma–Aldrich), IgG, CBX4 (AC011, A6221; ABclonal; China), and Twist2 (4173 R; Bioss, China).

Techniques: Expressing

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet: BAP1 binds active gene promoters and is excluded from Polycomb repressive domains (A) Heatmaps representing ChIP-seq intensity of the indicated proteins in wild-type ESCs. (B) Venn diagram of HA-BAP1, SUZ12, and RING1B target genes in ESCs. (C) Genome-wide functional annotation of peaks generated from the indicated ChIP-seq analyses. (D) Boxplots showing the expression levels obtained from RNA-seq analyses in WT mESCs for the clusters of target genes generated in (A). (E) Genome browser snapshot of ChIP-seq tracks showing an example of mutual exclusivity of PRC1/2 and PR-DUB target genes. (F) Western blot analysis with the indicated antibodies on total protein extracts from the indicated rescue ESC cell lines (E14 WT + empty vector, Bap1 KO + empty vector, Bap1 KO + BAP1 WT, Bap1 KO + BAP1 C91S). (G) Volcano plots of −log10 (p value) against log2 fold change representing the differences in gene expression in the indicated cell lines. (H) Percentage overlap of differentially expressed genes (DEGs) from (G) with either HA-BAP1, RING1B, or SUZ12 ChIP-seq targets. See also Figure S1 and , , and .

Article Snippet: Rabbit monoclonal anti-RING1B-pS168 , Abcam , Cat #ab234421.

Techniques: ChIP-sequencing, Genome Wide, Functional Assay, Generated, Expressing, RNA Sequencing Assay, Western Blot, Plasmid Preparation

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet: BAP1 loss causes global increases in H2AK119ub1 and displacement of PRC1 from target loci (A) Metaplots and heatmaps representing normalized ChIP-seq intensity for H2AK119ub1 or RING1B in the indicated cell lines. (B) Boxplot of normalized intensity profiles for H2AK119ub1 and RING1B ChIP-seq in the indicated cell lines. (C) Boxplot representing H2AK119ub1 ChIP-seq RPKM levels in the indicated cell lines at intergenic sites (n = 38,068). (D) Representation of the log2 fold change CPM in H2AK119ub1 ChIP-seq signal in the indicated cell lines across chromosome 19 using 10 kb windows. (E) Schematic of experimental plan to biochemically characterize PR-DUB in chromatin and nucleosol fractions. (F) Western blot of the indicated cell lines in either nucleosol or chromatin fractions. (G) Comparison of stoichiometry (IBAQ relative to BAP1) of PR-DUB subunits in FLAG/HA-BAP1 IP mass spectrometry purifications from nucleosol and chromatin fractions. Data are represented as mean ± SD. (H) Model of activity of PR-DUB complex at both its bound target genes and in “hit-and-run” model of highly mobile nucleosolic complex throughout the genome. See also Figure S2 .

Article Snippet: Rabbit monoclonal anti-RING1B-pS168 , Abcam , Cat #ab234421.

Techniques: ChIP-sequencing, Western Blot, Mass Spectrometry, Activity Assay

Journal: Molecular Cell

Article Title: BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation

doi: 10.1016/j.molcel.2021.06.020

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-RING1B-pS168 , Abcam , Cat #ab234421.

Techniques: Recombinant, Transfection, Purification, Sequencing, Imaging, Plasmid Preparation, Software

Journal: The EMBO Journal

Article Title: The microcephaly-associated transcriptional regulator AUTS2 cooperates with Polycomb complex PRC2 to produce upper-layer neurons in mice

doi: 10.1038/s44318-024-00343-7

Figure Lengend Snippet: Reagents and tools table

Article Snippet: The following primary antibodies were used, rabbit anti-AUTS2 (HPA000390; Sigma-Aldrich), rabbit anti-RING1B (5694S; Cell Signaling Technology), rabbit anti-H2AK119ub (8240S; Cell Signaling Technology), rabbit anti-H3K27me3 (9733S; Cell Signaling Technology), rabbit anti-H3K4me3 (9751S; Cell Signaling Technology), rabbit anti-H3K27ac (8173S; Cell Signaling Technology), rabbit anti-EZH2 (5246S; Cell Signaling Technology), and rabbit anti-SUZ12 (3737S; Cell Signaling Technology).

Techniques: Recombinant, Immunofluorescence, Western Blot, FACS, Sequencing, Imaging, SYBR Green Assay, In Situ, Software