Journal: Molecular Therapy. Nucleic Acids

Article Title: Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

doi: 10.1016/j.omtn.2023.102031

Figure Lengend Snippet: Nim inhibits DNMTs expression and restores SFRP1 expression in BCSCs (A) Boxplot showing the expression of SFRPs in breast cancer patients (GEPIA 2 database). The y axis log scale is represented as log 2 (TPM + 1), and the method for differential analysis is the one-way ANOVA, using disease state (tumor or normal) as the variable for calculating differential expression and statistical significance, with each dot representing a distinct tumor or normal sample. Red boxplot, tumor tissues (n = 1085); blue boxplot, normal tissues (n = 291). TPM, transcript per million. (B) The correlation between the gene expression level of SFRP1 and methylation rate of SFRP1 promoter from TCGA database, obtained by cBioportal Browser. (C) Real-time PCR assay showing mRNA expression of DNA methyltransferases (DNMTs) such as DNMT1 , DNMT3A , and DNMT3B upon treatment with Nim, Nim NPs, and DAC. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (D) Immunoblotting analysis demonstrating protein expression of DNMTs following treatment with Nim, Nim NPs, and DAC (5 μM) respectively in BCSCs. (E) Real-time PCR assay showing mRNA expression of SFRP1 upon treatment with Nim, Nim NPs, and DAC. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (F) Immunoblotting analysis demonstrating protein expression of SFRP1 following treatment with Nim, Nim NPs, and DAC. (G and H) pcDNA3/Myc-DNMT1 and pcDNA3/Myc-DNMT3A were transiently overexpressed and cells were treated with Nim or Nim NPs (5 μM) for 48 h and subjected to immunoblotting with indicated antibodies respectively. (I–K) Bar graph demonstrates quantification of methylation level at different regions of SFRP1 CpG island (SCpGR1, SCpGR2, and SCpGR3) in bisulfite-converted DNA with or without treatment with Nim, Nim NPs, or DAC (5 μM) measured by methylation-specific PCR (MSP) using methylation and unmethylation-specific primers. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. SCpGR1, SFRP1 CpG island region 1; SCpGR2, SFRP1 CpG island region 2; SCpGR3, SFRP1 CpG island region 3.

Article Snippet: The sheared chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and soluble chromatin was incubated overnight with blocking using 5% BSA followed by 15 μL of magnetic beads (Pierce ChIP-grade Protein A/G Magnetic Beads, Thermo Fisher Scientific, 26162) and 1 μg of antibody against DNMT1 (Thermo Fisher Scientific, PA5120552), and DNMT3A (Thermo Fisher Scientific, PA1882), and immunoglobulin (Ig) G as a negative control (Diagenode, C15410206).

Techniques: Expressing, Methylation, Real-time Polymerase Chain Reaction, Comparison, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

doi: 10.1016/j.omtn.2023.102031

Figure Lengend Snippet: Nim inhibits SFRP1 promoter hypermethylation and consequently downregulates Wnt/β-catenin signaling in BCSCs (A) Schematic representation of SFRP1 gene locus and regions analyzed before and after Nim or Nim NP treatment. The solid box (green) represents SFRP1 CpG island, lines (red) represent the regions evaluated to estimate methylation status before and after treatment of Nim or Nim NPs compared to the positive control DAC, and lines (blue) represent negative controls selected from upstream and downstream of non-CpG region. (B–I) Chromatin immunoprecipitation (ChIP)-qPCR represents DNMT (DNMT1 and DNMT3A) enrichment in different CpG island regions such as transcription start site (TSS 1–6) and promoter (PRM 7–8) of the SFRP1 gene before and after treatment with Nim, Nim NPs, and DAC (5 μM) respectively in BCSCs. IgG pull-down was used as the control. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (J) Immunoblotting analysis demonstrating protein expression of Wnt/β-catenin signaling associated markers following treatment with Nim, Nim NPs, and DAC (5 μM), respectively, in BCSCs.

Article Snippet: The sheared chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and soluble chromatin was incubated overnight with blocking using 5% BSA followed by 15 μL of magnetic beads (Pierce ChIP-grade Protein A/G Magnetic Beads, Thermo Fisher Scientific, 26162) and 1 μg of antibody against DNMT1 (Thermo Fisher Scientific, PA5120552), and DNMT3A (Thermo Fisher Scientific, PA1882), and immunoglobulin (Ig) G as a negative control (Diagenode, C15410206).

Techniques: Methylation, Positive Control, Chromatin Immunoprecipitation, Comparison, Western Blot, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Nimbolide-based nanomedicine inhibits breast cancer stem-like cells by epigenetic reprogramming of DNMTs-SFRP1-Wnt/β-catenin signaling axis

doi: 10.1016/j.omtn.2023.102031

Figure Lengend Snippet: In vivo anti-tumor and anti-metastatic efficacy of Nim NPs in zebrafish xenograft (A) Experimental layout for therapeutic and anti-metastatic studies in tumor-bearing zebrafish xenograft model. (B) ALDH high BCSCs were stained with Dil and transplanted into transgenic zebrafish embryos (Tg(kdrl: EGFP)) at the perivitelline space 48 h post fertilization (hpf) under the microscope. Further, 48 h post-injection (hpi) zebrafish were imaged by stereomicroscope and then treated with Nim or Nim NPs (1 or 2 μM). Zebrafish were again imaged 48 h post treatment (hpt) to estimate the tumor reduction after treatment (n = 6). Whole-fish images were taken at 25× objective. Scale bar, 1.1 mm. Zoomed images of the head were taken at 75× objective. Scale bar, 368.4 μm. (C) Graphical representation showing reduction of tumor volume after treatment with Nim NPs compared to Nim. Data represented as mean ± SEM (n = 6), one-way ANOVA with Tukey’s multiple comparison test. (D) Effect on tumor cell metastasis was detected upon treatment with Nim or Nim NPs (1 or 2 μM) using a stereomicroscope at 48 hpt in zebrafish xenograft. Zoomed images of the tail were taken at 75× objective. Scale bar, 368.4 μm. (E) Graphical representation of metastatic cell clusters before and after treatment with Nim or Nim NPs. Data represented as mean ± SEM (n = 3), one-way ANOVA with Tukey’s multiple comparison test. (F and G) Representative immunofluorescence images by stereomicroscope shows the expression of DNMT3A and β-catenin before and after treatment of Nim or Nim NPs (2 μM) in zebrafish xenograft. Images were taken at 126× objective. Scale bar, 172.1 μm.

Article Snippet: The sheared chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and soluble chromatin was incubated overnight with blocking using 5% BSA followed by 15 μL of magnetic beads (Pierce ChIP-grade Protein A/G Magnetic Beads, Thermo Fisher Scientific, 26162) and 1 μg of antibody against DNMT1 (Thermo Fisher Scientific, PA5120552), and DNMT3A (Thermo Fisher Scientific, PA1882), and immunoglobulin (Ig) G as a negative control (Diagenode, C15410206).

Techniques: In Vivo, Staining, Transgenic Assay, Microscopy, Injection, Comparison, Immunofluorescence, Expressing

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A Co-immunoprecipitations using fusion-positive leukemic megakaryoblasts confirm the association of ETO, CtBP1, and p300 with the fusion. All immunoprecipitations were repeated twice, representative blots are shown. Source data are provided as a Source Data file. B CUT&RUN-seq has performed in fusion-positive megakaryoblasts from two secondary transplants and fusion negative megakaryoblasts cultured in vitro for ETO, CtBP1, and p300. Distribution of CUT&RUN-seq peak locations for ETO, CtBP1, and p300. CBFA2T3-GLIS2 is included as a comparison. TSS transcription start site, TTS transcription termination site, UTR untranslated region. Source data are provided as a Source Data file. C Summary of ETO, CtBP1, and p300 bound genes retained, gained, and lost in fusion-positive megakaryoblasts compared to fusion-negative megakaryoblasts. Source data are provided as a Source Data file. D Heatmaps of enrichment for CBFA2T3-GLIS2 (CG-TY), H3K27ac, ETO, CtBP1, and p300 at genes bound by the fusion. E Venn diagram showing the overlap of genes bound at the promoters of ETO, CtBP1, and p300 exclusively in fusion-positive megakaryoblasts and their overlap with fusion-bound genes (Supplementary Data ).

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Cell Culture, In Vitro, Comparison

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: Impact of co-factor binding on over-representation analysis

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A–E Co-immunoprecipitation using 293T cells transfected with empty vector (MIG), wild-type fusion, or a fusion containing one or more of the mutations outlined in Fig. . All immunoprecipitations were repeated twice, representative blots are shown. Source data for all blots are provided as a Source Data file. A Immunoprecipitation of ETO followed by staining with the TY-1 tag to detect CBFA2T3-GLIS2 and the reciprocal immunoprecipitation of CBFA2T3-GLIS2 via the TY-1 tag followed by staining for ETO is shown. B TY-1-tagged wild-type CBFA2T3-GLIS2 and one of three FLAG-tagged mutant constructs were co-transfected into 293T cells to evaluate the ability of the mutant constructs to dimerize with the wild-type fusion. Immunoprecipitation by TY-1 followed by staining for FLAG and the reciprocal immunoprecipitation of FLAG followed by staining for TY-1 is shown. C 293T cells were co-transfected with two mutant constructs, one FLAG-tagged and one TY-1-tagged, to verify the results shown in ( B ). Immunoprecipitation of TY-1-tagged NHR2 deletion mutant followed by staining for FLAG again revealed a reduction of dimerization. D TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for CtBP1 followed by staining for the fusion via TY-1 and the reciprocal immunoprecipitation of the fusion followed by staining for CtBP1. E TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for p300, followed by staining for the fusion via TY-1, and the reciprocal immunoprecipitation of the fusion followed by staining for p300. F , G Transplantation of immunodeficient NSG-SGM3 mice with primary megakaryoblasts transduced with the mutant fusion constructs ( N = 5 per mutant construct, N = 9 wild type in panel F and 12 wild type in panel G ). p < 0.0001 for NHR2 deletion and NHR1-2 (DL380,487AS) mutant constructs compared to wild type. p -values determined by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Staining, Mutagenesis, Construct, Transplantation Assay, Transduction