Article Figures & Data
Figures
- Figure 1.
CuB inhibits cellular proliferation and induces apoptosis as well as alters the expression of key tumor-related genes in human NSCLC cells. A, IC50 values of CuB (0.01–40 μmol/L)-treated NSCLC cells at 24, 48, and 72 hours. B, treatment with CuB at indicated doses for 48 hours induced cellular apoptosis in H1299 cells. The graphical representation shows the percentage of total apoptotic cells. C, CuB-induced G2–M phase cell-cycle arrest in H1299 cells at 48 hours. The values are mean ± SE of cells in G2–M phase. D and E, effect of CuB treatment (0–860 nmol/L) on the mRNA and protein expressions of p16INK4A, p21CIP1/WAF1, c-MYC, and K-RAS at 48 hours in H1299 cells. F, effect of CuB treatment on the mRNA expressions of hTERT at 48 hours. The values were plotted as relative fold mRNA expression and were normalized to GAPDH. The protein expressions at similar treatment conditions are shown under respective real-time mRNA expression results with β-actin as an equal loading control. Results represent mean of 3 independent experiments (mean ± SE). Significance against respective control. *, P < 0.05.
- Figure 2.
CuB inhibits epigenetic modulatory enzymes. A, effect of CuB treatment on the mRNA expressions of DNMTs in H1299 cells after 48 hours. The values are normalized to GAPDH. B, treatment of H1299 cells with CuB for 48 hours altered the levels of protein expression of DNMTs. Graphical representations are indicative of relative band intensities. C, effect of CuB treatment on the protein expressions of HDACs, SIRTs, and HATs at 48 hours in H1299 cells. β-Actin was used as an equal loading control. D, H1299 cells were treated with indicated concentrations of CuB for 48 hours and nuclear extracts were assessed for HDACs activity. The values are mean ± SE. *, P < 0.05 against respective control.
- Figure 3.
CuB induced histone modification changes at the p16INK4A promoter. A, schematic representation of different CpG-rich regions (represented as ChIP 1–5) of the p16INK4A promoter, upstream of the transcription start site. B, H1299 cells were treated with the indicated concentrations of CuB for 48 hours and analyzed by ChIP-PCR assays for the enrichment of different chromatin markers at the p16INK4A promoter. Input DNA was used as control. IgG control was used as negative control. The gel images are representatives of 3 independent experiments. C, The x-axes represent CuB concentrations in nmol/L, and the y-axes represent the relative enrichment of individual histone modifications and binding factors, the percentage of untreated immunoprecipitates compared with the corresponding input samples (defined as 1) at different primer positions. Each point indicates the mean ± SE. *, P < 0.05 against respective control.
- Figure 4.
CuB induced histone modification changes at the p21CIP1/WAF1 promoter. A, schematic representation of amplicons for 2 different CpG-rich regions (represented as ChIP1 and ChIP2) of the p21CIP1/WAF1 promoter. B, H1299 cells were treated with the indicated concentrations of CuB for 48 hours and analyzed by ChIP-PCR for the enrichment of different chromatin markers at the p21CIP1/WAF1 promoter. Input DNA was used as control. IgG control was used as a negative control. The gel images are representatives of 3 independent experiments. C, the x-axes represent CuB concentrations in nmol/L, and the y-axes represent the relative enrichment of individual binding factors. Each point indicates the mean ± SE. *, P < 0.05 against respective control.
- Figure 5.
CuB alters the enrichment of different histone modification marks at the hTERT promoter. A, schematic representation of amplicon for different CpG-rich regions (represented as ChIP 1–5) of the hTERT promoter, spanning from distal promoter to the first exonic region. B, H1299 cells were treated with the indicated concentrations of CuB for 48 hours and analyzed for the binding of various chromatin marks and transcriptional factors in the promoter region of hTERT. Input DNA samples were used as control. IgG control was used as a negative control. The gel images are representatives of 3 independent experiments. C, the x-axes represent CuB concentrations in nmol/L, and the y-axes represent the relative enrichment of individual binding factors compared with the corresponding input samples (defined as 1). Each point indicates the mean ± SE. *, P < 0.05 against respective control.
- Figure 6.
CuB inhibited NNK-induced lung tumorigenesis in A/J mice. A, schematic representation of the experimental protocol used to study the effects of CuB on NNK-induced lung carcinogenesis in A/J mice. B, representative lung histology (10× and 20×) of different animal groups using H&E staining. The arrows represent angiogenesis; circles represent microadenomas; and squares represent tumor hyperplasia. C, the percentages of tumor incidence and tumor multiplicity of indicated animal groups are plotted. Columns, mean (n = 8); bars, SD. D, percentage of PCNA- and TUNEL-positive cells in the lung sections of different animal groups. Columns, mean (n = 8); bars, SD. *, P < 0.05; **, P < 0.01, significant against the NNK-induced control group. E, effect of CuB treatment on the protein expressions of different DNMTs and HDACs in mouse lung tissue. β-Actin was used as an equal loading control. The blots are representative of 3 independent experiments. F, effect of CuB treatment on the mRNA expressions of p16INK4A, p21CIP1/WAF1, and Tert as well as protein expressions of c-MYC and RAS in the mouse lung tissue. For mRNA expressions, the values were plotted as relative fold mRNA expression and were normalized to Gapdh. The results are means ± SE. *, P < 0.05, significant against the vehicle-treated control group.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Tables S1-4 - Supplementary Tables S1-4. The isolation and spectral data of CuB, list of Primers used for qRT-PCR, MSP, ChIP analysis and histopathological grading of CuB-treated NNK-induced lung tumors
- Supplementary Fig S1 - Supplementary Fig S1. Anti-proliferative and pro-apoptotic effects of CuB against different NSCLC cells
- Supplementary Fig S2 - Supplementary Fig S2. CuB alters the global as well as gene-specific pattern of methylation in H1299 cells
- Supplementary Fig S3 - Supplementary Fig S3. Average body weights and representative lung histology of different animal groups
- Supplementary Figure legends - Supplementary Figure legends. Supplementary Figure S1-S3 legends
Volume 8, Issue 6
