Journal: Molecular Cancer Research

Article Title: 5-Hydroxy-2-Methyl-1,4-Naphthoquinone, a Vitamin K3 Analogue, Suppresses STAT3 Activation Pathway through Induction of Protein Tyrosine Phosphatase, SHP-1: Potential Role in Chemosensitization

doi: 10.1158/1541-7786.mcr-09-0257

Figure Lengend Snippet: FIGURE 6. A. Overexpression of constitutive STAT3 rescues A293 cells from plumbagin-induced cytotoxicity. First, A293 cells were transfected with constitutive STAT3 plasmid. After 24 h of transfection, the cells were treated with 2.5 μmol/L plumbagin for 24 h, the cytotoxicity was determined by Live/Dead assay, and 20 random fields were counted. B. Plumbagin potentiates the apoptotic effect of thalidomide and bortezomib. U266 cells (1 × 106) were treated with 2.5 μmol/L plumbagin, and 10 μg/mL thalidomide or 20 nmol/L bortezomib alone or in combination for 24 h at 37°C. Cells were stained with a Live/Dead assay reagent for 30 min and then analyzed under a fluorescence microscope. The results shown are representative of two independent experiments.

Article Snippet: Bortezomib (Velcade, PS-341) was obtained from Millennium, and thalidomide was from Tocris Cookson.

Techniques: Over Expression, Transfection, Plasmid Preparation, Live Dead Assay, Staining, Fluorescence, Microscopy

Journal: Nature Genetics

Article Title: Targeting histone H2B acetylated enhanceosomes via p300/CBP degradation in prostate cancer

doi: 10.1038/s41588-025-02336-6

Figure Lengend Snippet: a . Immunoblot analysis of p300, CBP, and indicated histone marks in 22Rv1 wild type (WT), p300 knockout (KO), CBP KO, and p300 KO with siCBP cells. b . ChIP-seq read-density heatmaps of H3K27ac at top 5000 AR binding sites on non-promoter regions in VCaP cells with 4 h of 1 µM GNE-049 or 1 µM CCS1477 treatments. c . Structure of CBPD-409 and schematic of p300 and CBP domains. d . Immunoblot analysis of p300 and CBP in LNCaP cells treated with 100 nM CBPD-409 for the indicated durations. e . Immunoblot analysis of p300 and CBP in VCaP cells treated with 10 and 100 nM CBPD-409 for 4 h. f . Immunoblot analysis of p300 and CBP in non-neoplastic prostatic cells treated with 10 nM or 100 nM CBPD-409 for 4 h. g . Immunoblot analysis of p300 and CBP in Myc-Cap and TRAMPC2 treated with 10 nM or 100 nM CBPD-409 for 4 h. h . Tandem mass tag (TMT) mass spectrometry (MS) assay to evaluate effects of CBPD-409 (100 nM, 4 h) on the proteome of 22Rv1 cells. N = 3 independent experiments. All t-tests performed were two-tailed t-tests assuming equal variances. i . Heatmap of relative abundance of all bromodomain-containing proteins detected in TMT-based quantitative MS after 4 h CBPD-409 treatment of VCaP cells. j . Immunoblot analysis of bromodomain and extraterminal (BET) proteins (bromodomain-containing 2 (BRD2), bromodomain-containing 3 (BRD3), and BRD4) in VCaP cells treated with 100 nM CBPD-409 for the indicated durations. k . Immunoblot analysis of cereblon (CRBN) neo-substrates (GSPT1 and Ikaros) in LNCaP cells treated with 100 nM CBPD-409 for 24 h. l . Immunoblot analysis of p300 and CBP in VCaP cells pre-treated with thalidomide (Thali), then treated with CBPD-409 at the indicated concentrations for 4 h. m . Immunoblot analysis of p300 and CBP in VCaP cells pre-treated with carfilzomib (Carfil), then treated with CBPD-409 at the indicated concentrations for 4 h. n . Schematic of CBPD-409-Me structure. o . Immunoblot analysis of p300 and CBP in VCaP cells treated with CBPD-409 and CBPD-409-Me at indicated concentrations for the noted durations. p . Immunoblot analysis of p300 and CBP in VCaP cells treated with 10 nM CBPD-409, 10nM dCBP1 and 100nM JQAD1 for 4 h.

Article Snippet: GNE-049, CCS1477, A485, JQAD1, enzalutamide, carfilzomib and thalidomide were purchased from Selleck Chemicals. dCBP-1 was purchased from MedChemExpress.

Techniques: Western Blot, Knock-Out, ChIP-sequencing, Binding Assay, Mass Spectrometry, Two Tailed Test

Journal: Nature Genetics

Article Title: Targeting histone H2B acetylated enhanceosomes via p300/CBP degradation in prostate cancer

doi: 10.1038/s41588-025-02336-6

Figure Lengend Snippet: a , GSEA plots for AR, MYC, E2F and G2M checkpoint pathway-related genes from the CBPD-409-treated VCaP cells. n = 2 biological replicates. Statistical significance was assessed using a two-sided GSEA permutation test with adjustment for multiple comparisons. NES, normalized enrichment score; DEGs, differentially expressed genes. b , Immunoblot analysis of indicated proteins and histone marks in VCaP cells treated with 100 nM CBPD-409 for the indicated times. c , RNA-seq heatmaps for AR target genes in VCaP cells treated with 100 nM CBPD-409, 1 µM GNE-049, 1 µM CCS1477 or 1 µM A485 for 24 h. n = 2 biological replicates. d , RNA-seq heatmaps of AR target genes in LNCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049. Cells were cultured in 10% charcoal-stripped serum (CSS) medium, pre-treated for 1 h and then stimulated with 1 nM R1881 for 12 h. n = 2 biological replicates. e , GSEA plots of AR signaling pathway based on nascent RNA (left, AR target genes; right, AR-associated enhancer RNAs) expression in VCaP cells treated with 100 nM CBPD-409 for 4 h. Statistical significance was assessed using a two-sided GSEA permutation test with adjustment for multiple comparisons. f , Comparison of whole-transcriptome in VCaP cells treated with 100 nM CBPD-409 and 1 µM GNE-049 for 24 h. n = 2 biological replicates. CCND1 , NKX3-1 and CITED2 are highlighted in red. g , GSEA plots for G2M checkpoint, AR, E2F and MYC signaling pathways in VCaP cells: comparison of 24 h treatment with 100 nM CBPD-409 versus 1 µM GNE-049. Statistical significance was assessed using a two-sided GSEA permutation test with adjustment for multiple comparisons. h , Uniquely downregulated genes in CBPD-409 relative to GNE-409-treated VCaP cells analyzed for overlap with molecular signatures database (MSigDB) hallmark gene sets. Statistical significance was assessed using a two-sided hypergeometric test with adjustment for multiple comparisons. i , Immunoblot analysis of indicated proteins in VCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049 for the indicated times. j , Immunoblot analysis of labeled proteins and histone marks in VCaP cells pre-treated with different concentrations of thalidomide (Thali) for 1 h, then treated with CBPD-409 at indicated concentrations for 4 h. k , Immunoblot analysis of indicated proteins and histone marks in VCaP cells treated with CBPD-409 or CBPD-409-me (inactive analog) for indicated times.

Article Snippet: GNE-049, CCS1477, A485, JQAD1, enzalutamide, carfilzomib and thalidomide were purchased from Selleck Chemicals. dCBP-1 was purchased from MedChemExpress.

Techniques: Western Blot, RNA Sequencing, Cell Culture, Expressing, Comparison, Protein-Protein interactions, Labeling

Journal: Nature Genetics

Article Title: Targeting histone H2B acetylated enhanceosomes via p300/CBP degradation in prostate cancer

doi: 10.1038/s41588-025-02336-6

Figure Lengend Snippet: a . Venn diagrams showing overlaps between genes suppressed by CBPD-409 and those suppressed by GNE-049 or CCS1477 in VCaP cells. Data obtained from RNA-seq with two independent samples for each condition. b . Comparison of whole-transcriptome in VCaP cells treated with 100nM CBPD-409 and 1 µM CCS-1477 for 24 h. N=2 biological replicates. NKX3-1 , cyclin D1 (CCND1) and CBP/p300-interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 ( CITED2) are highlighted in red. c . GSEA net enrichment score (NES) plot comparing hallmark pathway alterations in VCaP cells: 100 nM CBPD-409 vs. 1 µM GNE-049 treatment for 24 h. d . Compute overlaps of ranked top 300 CBPD-409 unique down-regulated genes with gene ontology (GO) and Reactome gene sets. P-values were calculated using a two-sided statistical test with adjustment for multiple comparisons. e . qPCR of indicated genes expression in LNCaP cells treated with CBPD-409, GNE-049, or CCS1477 for 24 h, n=3 biological replicates, data are presented as mean ± SD. f . Immunoblot analysis of NKX3-1, CCND1, and CITED2 in VCaP cells treated with 100 nM CBPD-409 for the noted durations. g . Immunoblot analysis of indicated proteins and histone marks in LNCaP cells treated with 100 nM CBPD-409, 1 µM GNE-049, or 1 µM CCS1477 for 24 h. h . qPCR of indicated genes expression in VCaP cells pre-treated with 100 µM thalidomide for 1 h, then treated with CBPD-409 or GNE-409 for 4 h, n=3 biological replicates, data are presented as mean ± SD. i . ChIP-seq tracks of H3K27ac, H2BK20ac, and Pol II within the NKX3-1 and CCND1 gene loci in VCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049 for 4 h. j . Immunoblot analysis of indicated proteins in VCaP cells treated with 10 nM CBPD-409, 10 nM dCBP-1, 100 nM JQAD1, or 100 nM A485 for noted durations. k . Left: representative images showing invasive cells from the Boyden chamber invasion assay. Each group included three biological replicates (independent transwell inserts). Right: Bar plot quantifying invasive cells. Five random fields were imaged per transwell, yielding n = 15 fields per group. Data are presented as mean ± SD. Statistical significance was assessed using a two-sided t-test. Scale bar = 100 µm.

Article Snippet: GNE-049, CCS1477, A485, JQAD1, enzalutamide, carfilzomib and thalidomide were purchased from Selleck Chemicals. dCBP-1 was purchased from MedChemExpress.

Techniques: RNA Sequencing, Comparison, Expressing, Western Blot, ChIP-sequencing, Invasion Assay

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 1. Mercury orange (HgO) -stained rat (gestation day [GD] 13) and rabbit (GD 12) limbs treated with 300 and 70 mg thalidomide/kg per day, respectively. Black arrows denote the region of the progress zone (PZ); and white arrows denote the area of the apical ectodermal ridge (AER). Rat thalidomide-treated limb buds did not show any relevant decrease in HgO staining compared with control rat limb buds, suggest- ing little or no oxidative stress. In thalidomide-treated rabbit limb buds, there is a clear decrease in HgO fluorescence in the PZ but not the overlying AER compared with control rabbit limb buds, indicating a de- pletion of PZ glutathione and regional oxidative stress.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Staining, Control

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 2. pNF-B-d2EGFP–transfected rat and rabbit limb bud cells show a decrease in GFP fluorescence with thalidomide treatment but are rescued by redox modulating agents, N-acetylcysteine (NAC) and -phe- nyl-N-t-butylnitrone (PBN). Diamide, an oxidant and known inhibitor NF- B/DNA binding, acted as a positive control. Data are represented as percentage of control GFP expression: hatched bars denote transfected rat limb bud cells, and open bars denote transfected rabbit limb bud cells. Asterisks (*) denote a statistically significant (0.05) difference from control GFP expression. Crosses (†) represent a statistically significant (0.05) difference between transfected cells treated with thalidomide (THAL) and thalidomide NAC or PBN. TPA, tetradecanoylphorbol acetate.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: Transfection, Binding Assay, Positive Control, Control, Expressing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 3. In situ hybridization of control and thalidomide-treated rat embryos for Fgf-10, Twist, and Fgf-8 expression in the gestation day (GD) 11.5 rat limb bud. Thalidomide treatment had no effect on the expression of Fgf-10, Twist, and Fgf-8 compared with control. Arrows denote the limb and regions where Fgf-10, Twist, and Fgf-8 expression normally occurs.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: In Situ Hybridization, Control, Expressing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 4. In situ hybridization of control and thalidomide-treated rat embryos for Fgf-10, Twist, and Fgf-8 expression in the gestation day (GD) 13 rat limb bud. Thalidomide treatment had no effect on the expres- sion of Fgf-10, Twist, and Fgf-8 compared with control. Arrows denote regions where Twist expression normally occurs.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: In Situ Hybridization, Control, Expressing

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 5. In situ hybridization of control, thalidomide-treated, and tha- lidomide -phenyl-N-t-butylnitrone (PBN) rabbit embryos for Fgf-10 expression in the gestation day (GD) 10–12 rabbit limb bud. Thalidomide greatly reduced the expression of Fgf-10 at each stage of limb develop- ment. However, gene expression was not affected in embryos that re- ceived both thalidomide and the free radical trapping agent, PBN. Loss of normal limb morphology is evident in GD 12 embryos treated with tha- lidomide where the outer most portion of the limb is not uniformly devel- oped. Arrows denote regions where Fgf-10 expression normally occurs.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: In Situ Hybridization, Control, Expressing, Gene Expression

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 6. In situ hybridization of control, thalidomide-treated, and - phenyl-N-t-butylnitrone (PBN) rabbit embryos for Twist expression in the gestation day (GD) 10–12 rabbit limb bud. Thalidomide greatly reduced the expression of Twist at each stage of limb development. However, gene expression was not affected in embryos that received both thalid- omide and PBN. Loss of normal limb morphology is evident in GD 12 embryos treated with thalidomide where limb bud paddles have failed to develop normally and Twist expression is absent. Arrows denote regions where Twist expression normally occurs.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: In Situ Hybridization, Control, Expressing, Gene Expression

Journal: Developmental dynamics : an official publication of the American Association of Anatomists

Article Title: Misregulation of gene expression in the redox-sensitive NF-kappab-dependent limb outgrowth pathway by thalidomide.

doi: 10.1002/dvdy.10150

Figure Lengend Snippet: Fig. 7. In situ hybridization of control, thalidomide-treated, and - phenyl-N-t-butylnitrone (PBN) rabbit embryos for Fgf-8 expression in the gestation day (GD) 10–12 rabbit limb bud. Thalidomide greatly reduced the expression of Fgf-8 at each stage of limb development. However, gene expression was not affected in embryos that received both thalid- omide and PBN. Loss of Fgf-8 expression is most notable in the region of the zone of polarizing activity. Arrows denote regions where Fgf-8 ex- pression normally occurs.

Article Snippet: Thalidomide was purchased from Toronto Research Chemicals (Toronto, Ontario, Canada).

Techniques: In Situ Hybridization, Control, Expressing, Gene Expression, Activity Assay