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