u6 sgrna co expression vector backbone px458  (Addgene inc)

Journal: bioRxiv

Article Title: Synergic homology directed recombination by PRDM9 meiotic factor

doi: 10.1101/2022.12.05.519167

Figure Lengend Snippet: A) Scheme of synergistic HDR Enhacer techonology: Cas9 (in blue) is fused to PRDM9 (green). Cas9 is associated with different HDR enhacers via sgRNA loops that include MS2 phage aptamer sequences bound by fusion proteins of HDR enhancers and the MS2 coat protein. B) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9, Cas9-PRDM9, Cas9 co-deliver with PRDM9 and Cas9-PRDM9 together with a HDR enhancing factors alone or in combination. C) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9-PRDM9 and Cas9-PRDM9 together with the best HDR enhancing factors. D) HDR/NHEJ ratio of the Traffic Light Reporter, upon transfection with Cas9 or Cas9-PRDM9 together with MS2-CtlP. E) SHE performance for HDR in human iPS cells for HDR and NHEJ comparison, F) and relative measurements.

Article Snippet: Similarly, MS2 expressing vector (Addgene #61423) was also amplified by PCR.

Techniques: Transfection, Comparison

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: a RT-qPCR and ( b ) western blot analysis of ERβ mRNA and protein expression in a panel of TNBC cell lines. Effects of E2, LY, and ICI on proliferation of ERβ− ( c ) and ERβ+ ( d ) TNBC cell lines. e Pie graph and ( f ) volcano plot depicting the number and magnitude of gene expression changes detected by RNA-seq of MDA-MB-231-ERβ cells following E2 treatment relative to vehicle (ethanol) control in the presence of dox. IPA analysis of the ERβ transcriptome depicting the ( g ) top 15 canonical pathways regulated by ERβ (NFκB/p65-related pathways shaded black) and ( h ) top 15 upstream regulators (NFκB/p65-associated regulators highlighted in blue). i Network analysis of RNA-seq data revealed the NFκB/p65 signaling pathway as centrally down-regulated by ERβ. j GSEA of differentially expressed genes identified negative correlations between the ERβ transcriptome and NFκB/inflammatory responses. k GREAT and ( l ) motif analysis of ERβ ChIP-seq peaks identified following 3 h of dox + E2 treatment of MDA-MB-231-ERβ cells relative to veh + dox treatment. ERE estrogen response element, NRE NFκB/p65 response element. Venn diagrams of all ERβ binding sites and ERβ binding sites that encode ( m ) an NFκB/p65 consensus motif or ( n ) an NFκB/p65 consensus motif with no ERE within 50 kb. All data is presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 relative to vehicle or between indicated treatments (one-way ANOVA). See also Figure S .

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Quantitative RT-PCR, Western Blot, Expressing, RNA Sequencing Assay, ChIP-sequencing, Binding Assay

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: a Expression of NFκB/p65 target genes shown to be suppressed by ERβ in TNBC cell lines in ERβ+ ( n = 32) versus ERβ− ( n = 225) TN breast tumors from the Mayo Clinic Cohort as assessed by RNA-seq. Dashed line indicates median expression. b GSEA of differentially expressed genes in ERβ+ tumors versus ERβ− tumors revealed negative correlations with NFκB/p65 pathways. c Protein levels of NFκB/p65 target genes in conditioned medium isolated from MDA-MB-231-ERβ cells treated with E2+ dox for five days relative to vehicle + dox as determined via a cytokine array. d Co-culture real-time proliferation assays of parental (red) and ERβ-expressing (green) MDA-MB-231 cells treated with veh + dox or E2+ dox using an IncuCyte S3 system. Representative images of wells are shown in bottom panels. Scale bars are equivalent to 200 µm. e Western blot indicating knockdown of p65 protein following transfection of p65-specific siRNAs relative to scramble control (scr). f Proliferation rates of dox treated MDA-MB-231-ERβ cells following p65 knockdown and E2 treatment. g Western blot depicting expression of the HA-tagged constitutively active p65 expression vector (CA p65) in MDA-MB-231-ERβ cells. h Proliferation rates of dox treated parental and CA p65 cells following vehicle and E2 treatment. All data is presented as mean ± SEM. ( c , f , h ), * P < 0.05, ** P < 0.01, *** P < 0.001 relative to vehicle (one-way ANOVA). ( d ) * P < 0.05, ** P < 0.01, *** P < 0.001 as determined via two-way ANOVA.

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Expressing, RNA Sequencing Assay, Isolation, Co-Culture Assay, Western Blot, Transfection, Plasmid Preparation

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: a Venn diagram depicting the overlap of genes differentially expressed in dox treated MDA-MB-231-ERβ cells following TNFα, E2+ TNFα, or E2 stimulation relative to vehicle control. The number of induced and repressed genes by each treatment is indicated. b Fold change (FC) relative to vehicle of TNFα-induced and -repressed genes following E2, TNFα (T), or E2+ TNFα (E2+ T) treatment (Wilcoxon rank sum test). c RT-qPCR analysis of NFκB/p65 target gene mRNA expression relative to vehicle in MDA-MB-231-ERβ cells treated as indicated in the presence of dox. NFκB/p65 luciferase reporter activity relative to vehicle in ( d ) MDA-MB-231-ERβ and ( e ) Hs578T-ERβ cells following indicated treatments in the setting of dox. f Migration assay of dox treated MDA-MB-231-ERβ cells and constitutively active p65 cells (CA p65) following veh, T, E2+ T, or E2 treatment. *** P < 0.001 between indicated treatments (non-linear fit modeling). g Western blot analysis of phospho- and total p65 and IκBα following indicated treatments in the presence of dox. Vinculin is shown as a loading control. h Confocal microscopy images depicting cellular localization of total p65 in MDA-MB-231-ERβ cells following indicated treatments in addition to dox. Hoechst stain used to identify nuclei. Scale bars are equivalent to 50 µm. Data represent mean ± SEM. c – e * P < 0.05, ** P < 0.01, *** P < 0.001 relative to vehicle or between indicated treatments (one-way ANOVA). See also Figure S .

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Migration, Western Blot, Confocal Microscopy, Staining

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: a Venn diagram depicting overlap of p65 binding sites following TNFα or E2+ TNFα stimulation in dox treated MDA-MB-231-ERβ cells as determined by ChIP-seq. b Aggregate plots and ( c ) heat maps of p65 binding intensity in the presence of TNFα alone (Unique T), E2+ TNFα (Unique E + T), or both (Common) following indicated treatments. d GIGGLE analysis of identified p65 binding sites assessing their similarity with other known protein binding sites in publicly available datasets. e Venn diagram depicting overlap of ERβ and p65 binding sites identified via ChIP-seq. f Bar graph of all p65 binding sites and their distribution relative to ERβ binding sites. Note the 936 overlapping sites from ( e ) are at the exact same genomic location. g Co-immunoprecipitation experiments using nuclear lysates from dox treated MDA-MB-231-ERβ cells demonstrating protein interactions between ERβ and p65, but not RELB. h ChIP-seq tracks from IGV for ERβ, p65, and RNA Polymerase II phospho-Ser2 ChIP-seq following dox plus veh, E2, T, and E2+ T treatment at NFκB/p65 target gene loci. See also Figure S .

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Binding Assay, ChIP-sequencing, Protein Binding, Immunoprecipitation

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: a Heat maps, aggregate plots, and ( b ) box plots of H3K27me3 and H3K27ac peak intensity at TNFα-induced p65 binding sites in dox treated MDA-MB-231-ERβ cells following vehicle (veh), E2, TNFα (T) or E2+ TNFα (E2+ T) stimulation. * P < 0.05, ** P < 0.01, *** P < 0.001 relative to vehicle or between indicated treatments (Mann–Whitney test). c Co-immunoprecipitation experiments using nuclear lysates from MDA-MB-231-ERβ cells treated with or without dox and veh or E2. Lysates were immunoprecipitated with an ERβ antibody or a p65 antibody followed by western blotting for indicated proteins. d ChIP-PCR depicting the relative binding of ERβ, p65, and EZH2, as well as enrichment of H3K27me3 (me3), at ERβ binding sites nearby NFκB/p65 target genes ( ALOX5AP , CXCL2, and IL11) following dox + vehicle or dox + E2 treatment. RT-qPCR analysis of NFκB/p65 target gene expression in dox treated MDA-MB-231-ERβ cells exposure to: ( e ) veh, E2, 5 µM GSK126, or E2 + GSK126 (E2 + G), or ( f ) a non-targeting siRNA (scr) or p65-targeting siRNA in the presence of veh or E2. g RT-qPCR analysis of NFκB/p65 target gene expression in WT and CA p65 MDA-MB-231-ERβ cells treated with veh or E2 relative to WT cells + vehicle and dox. h ChIP-PCR assessing relative association of ERβ and EZH2, as well as H3K27me3 (me3), at ERβ binding sites near NFκB/p65 target genes following treatment with non-targeting siRNA (scr) or p65-targeting siRNA in the presence of dox + vehicle or dox + E2. All data is presented as mean ± SEM. except for ( b ) which depicts the minimum, maximum, and median values. * P < 0.05, ** P < 0.01, *** P < 0.001 relative to vehicle or between indicated treatments. See also Figure S .

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Binding Assay, MANN-WHITNEY, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Expressing

Journal: NPJ Breast Cancer

Article Title: Estrogen receptor beta repurposes EZH2 to suppress oncogenic NFκB/p65 signaling in triple negative breast cancer

doi: 10.1038/s41523-022-00387-0

Figure Lengend Snippet: In ERβ− TNBC cells (left), EZH2 associates with p65 in a PRC2-independent manner and functions to enhance p65 transcriptional activity and promote aggressive cancer phenotypes. In ERβ+ TNBC cells (right), ERβ induces formation of a co-repressor complex involving p65, EZH2, and the PRC2 complex (including SUZ12, EED, and EZH2). This repressive complex results in trimethylation of H3K27, chromatin compaction, and subsequent blockade of NFκB/p65 target gene expression, ultimately resulting in anti-cancer effects and less aggressive cancer phenotypes.

Article Snippet: MDA-MB-231-ERβ cells stably expressing a constitutively active form of NFκB (S276G) were generated by transfection of the HA-tagged pCMV3 NFκB p65 expression vector (Sino Biological, Beijing, China) after point mutation generation via Quikchange® PCR.

Techniques: Activity Assay, Expressing