EMSA showed the activity of NF-κB p65 in ACC-M, ACC-M/IκBαM, ACC-2 and ACC-2/IκBαM cell lines

<p><b>Copyright information:</b></p><p>Taken from "In vitro angiogenesis and expression of nuclear factor κB and VEGF in high and low metastasis cell lines of salivary gland Adenoid Cystic Carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/7/95</p><p>BMC Cancer 2007;7():95-95.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903362.</p><p></p> indicate the migration of the induced NF-κB DNA-binding complexes. Migration of the free probe is not shown. The Oct-1 motif was used as a control for quality and quantity of cell extract

RT-PCR showed the mRNA expression of VEGF in ACC-M, ACC-2, ACC-M/IκBαM and ACC-2/IκBαM cell lines

<p><b>Copyright information:</b></p><p>Taken from "In vitro angiogenesis and expression of nuclear factor κB and VEGF in high and low metastasis cell lines of salivary gland Adenoid Cystic Carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/7/95</p><p>BMC Cancer 2007;7():95-95.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903362.</p><p></p

Immuofluorescence double staining and semi-quantitative confocal laser scanning analysis showed NF-κB p65 and VEGF expressed in ACC-M and ACC-2 cell lines

<p><b>Copyright information:</b></p><p>Taken from "In vitro angiogenesis and expression of nuclear factor κB and VEGF in high and low metastasis cell lines of salivary gland Adenoid Cystic Carcinoma"</p><p>http://www.biomedcentral.com/1471-2407/7/95</p><p>BMC Cancer 2007;7():95-95.</p><p>Published online 1 Jun 2007</p><p>PMCID:PMC1903362.</p><p></p> As figure 3A showed, the rate of NF-κB p65 nuclear localization (a) (white arrow) and VEGF staining intensity (b) in ACC-M was higher than that in ACC-2 (e) and (f). As figure 3B shows, bars represent the mean value of immunofluorescence intensity of VEGF and nuclear staining rate of NF-κB p65 in two cell lines, < 0.01(*)

DataSheet_1_Construction of a DDR-related signature for predicting of prognosis in metastatic colorectal carcinoma.docx

BackgroundColorectal cancer (CRC) is the third most prevalent malignancy and the one of most lethal cancer. Metastatic CRC (mCRC) is the third most common cause of cancer deaths worldwide. DNA damage response (DDR) genes are closely associated with the tumorigenesis and development of CRC. In this study, we aimed to construct a DDR-related gene signature for predicting the prognosis of mCRC patients.MethodsThe gene expression and corresponding clinical information data of CRC/mCRC patients were obtained from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. A prognostic model was obtained and termed DDRScore by the multivariate Cox proportional hazards regression in the patients with mCRC. The Kaplan-Meier (K-M) and Receiver Operating Characteristic (ROC) curves were employed to validate the predictive ability of the prognostic model. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway were performed for patients between the high-DDRscore and low-DDRscore groups.ResultsWe constructed a prognostic model consisting of four DDR-related genes (EME2, MSH4, MLH3, and SPO11). Survival analysis showed that patients in the high-DDRscore group had a significantly worse OS than those in the low-DDRscore group. The area under the curve (AUC) value of the ROC curve of the predictive model is 0.763 in the training cohort GSE72970, 0.659 in the stage III/IV colorectal cancer (CRC) patients from The Cancer Genome Atlas (TCGA) data portal, and 0.639 in another validation cohort GSE39582, respectively. GSEA functional analysis revealed that the most significantly enriched pathways focused on nucleotide excision repair, base excision repair, homologous recombination, cytokine receptor interaction, chemokine signal pathway, cell adhesion molecules cams, ECM-receptor interaction, and focal adhesion.ConclusionThe DDRscore was identified as an independent prognostic and therapy response predictor, and the DDR-related genes may be potential diagnosis or prognosis biomarkers for mCRC patients.</p

Caspase-8 and Caspase-9 Dependent Apoptosis of HepG2 Cells Induced by DMQ and H-EtOAc Fraction.

<p>A. HepG2 cells were incubated with caspase-8 inhibitor (Z-IETD-FMK: 20 μmol/L) or caspase-9 inhibitor (Z-LEHD-FMK: 20 μmol/L) for 30 min followed by DMQ at 0, 50, 125 and 250 μmol/L for 24 h, the inhibitory rates were detected by SRB assays. B. HepG2 cells were incubated with Z-IETD-FMK or Z-LEHD-FMK for 30 min and subsequently treated with H-EtOAc fraction at 0, 12.7, 31.8 and 63.5 μg/mL for 24 h, and the inhibitory rates were determined by SRB assays. C. HepG2 cells were pre-incubated with Z-IETD-FMK or Z-LEHD-FMK for 30 min before treated with DMQ (79 μmol/L) or H-EtOAc fraction (100 μg/mL) for 24 h, the cellular apoptotic rates were measured by flow cytometry. Values are means ± SD of three independent experiments. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Death Receptor Pathway Related to HepG2 Cells Treated with DMQ and H-EtOAc Fraction.

<p>A. Western blot analysis of FasL and Fas. Lane 1: Control; Lane 2–4: 79, 157 and 315 μmol/L of DMQ for 24 h, respectively; Lane 5: 250 μg/mL of H-EtOAc fraction for 24 h. B. The protein expression levels of FasL (a) and Fas (b). Values are means ± SD of three independent experiments. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Mitochondrial Apoptotic Pathway Related to DMQ and H-EtOAc Fraction Treated HepG2 Cells.

<p>A. The MMP changes of HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). B. The production changes of intracellular ROS in HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). C. Western blot analysis of proteins expression levels of p53, Bax, Bcl-2, mitochondrial cyto C, cytoplasmic cyto C and β-actin. Lane 1: Control; Lane 2–4: 79, 157 and 315 μmol/L of DMQ for 24 h, respectively; Lane 5: 250 μg/mL of H-EtOAc fraction for 24 h. D. The protein expression levels of p53 (a) Bax (b) Bcl-2 (c) the ratio of Bax/Bcl-2 (d) mitochondrial cyto C (e) cytoplasmic cyto C (f) and the ratio of cytoplasmic cytoC/mitochondrial cyto C (g). The protein expression strength were analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Inc. China) with ScanWizard 5 software. E. RT-PCR results of p53, Bax, Bcl-2 and β-actin. Lane 1: Control; Lane 2–4: 79 μmol/mL for 12 h, 157 μ/L for 12 h and 157 μmol/L for 24 h of DMQ, respectively; Lane 5: 100 μg/mL for 24 h of H-EtOAc fraction. F. The calculated mRNA expression levels of p53 (a) Bax (b) Bcl-2 (c) and the ratio of Bax/Bcl-2 (d). The mRNA expression strength were analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Inc. China) with ScanWizard 5 software. Values are means ± SD of three independent experiments. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

Cell Cycle Arrest of HepG2 Cells Treated with DMQ and H-EtOAc Fraction.

<p>A. The cell cycle distribution of treated HepG2 cells. B. Contents of each phase of HepG2 cells treated with DMQ (a), H-EtOAc fraction (b). C. Western blot analysis of p21, CDK 2 and cyclin E. Lane 1: Control; Lane 2–4: 79, 157 and 315 μmol/L of DMQ for 24 h, respectively; Lane 5: 250 μg/mL of H-EtOAc fraction for 24 h. D. The protein expression levels of p21 (a), CDK 2 (b) and cyclin E (c). The protein expression strength were analyzed by scanning densitometry using a Microtek ScanMaker 8700 (Zhongjing Inc. China) with ScanWizard 5 software. Values are means ± SD of three independent experiments. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p

<i>In vitro</i> antitumor activity of H-EtOAc fraction against six human tumors (±SD).

<p><i>In vitro</i> antitumor activity of H-EtOAc fraction against six human tumors (±SD).</p

Apoptosis Induced by DMQ and H-EtOAc fraction of HepG2 Cells.

<p>A. Apoptosis was measured on HepG2 cells treated with gradient DMQ (a)-(d) and H-EtOAc fraction (e)-(g) by flow cytometry. B. The apoptosis rates were calculated of DMQ (a) and H-EtOAc fraction (b) on HepG2 cells. C. DNA ladder. Lane 1: Control; Lane 2–4: 79 μmol/L at 24 h, 157 μmol/L at 12 h and 157 μmol/L at 24 h for DMQ; Lane 5: 400 μg/mL at 24 h for H-EtOAc fraction; Lane 6: Negative group. D. The activities of caspase-3, -8, -9 proteases of HepG2 cells treated with DMQ (a) and H-EtOAc fraction (b). Values are means ± SD of three independent experiments. * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (<i>p</i><0.001) represented significant differences compared to the control.</p