Rh-endostatin decreases microvascular density (MVD) and promotes vessel normalization of A549 lung carcinoma.

<p>A549 tumor-bearing mice were treated with rh-endostatin (5 mg/kg, s.c.) for consecutive 7 days with normal saline as control. On days 3, 6 and 9, mice (n = 4, each group and each time point) were sacrificed and tumor samples were stained by anti-CD31 antibody and anti-α-SMA antibody. A, typical fluorescence images of tumors showed CD31-positive endothelial cells (red), α-SMA positive pericytes (green) and merged images (orange) in A549 lung carcinoma in control group and rh-endostatin treated group on days 3, 6, and 9, respectively. B, columns represented microvascular densities at different time points in two groups. C, columns represented pericyte coverages at different time points in the two groups. Representative sections are shown from all groups with a magnification of 400×. Columns, mean; Bars, SE; *p<0.05, indicating significant difference.</p

Rh-endostatin decreases tumor hypoxic area and hypoxia inhibits the activity of CIK cells in vitro.

<p>A549-bearing mice were divided into two groups and were given rh-endostatin or normal saline respectively for 7 days. On days 3, 6 and 9, mice (n = 4) in the two groups were administrated with pimonidazole. Tumor hypoxic areas were stained by monoclonal antibody (Mab1) against protein adducts of pimonidazole. Hypoxic areas were stained dark yellow. In vitro experiments were conducted by culturing CIK cells under normoxia or hypoxia for 48 h. Proliferation of CIK cells were measured by counting cells with a hemocytometer. A549 lung cancer cells were cocultured with the indicated number of CIK cells under normoxia or hypoxia for 48 h in 96-well plates. The killing rate of CIK cells against the A549 lung cancer cell was measured by LDH release assay. Transmigration assay was performed with Transwell™ inserts containing HUVECs culture. CIK cells were added upon the HUVECs layer and were incubated under normoxic or hypoxic conditions for 48 h. CIK cells that migrated into the lower chamber were harvested and counted by hemocytometry. A, Individual fields at 40×magnification were chosen to represent hypoxic areas in tumor samples on days 3, 6 and 9 in two groups. B, representative pictures of CIK cells cultured under normoxia or hypoxia at 100×magnification. C, bar graphs depicting the density of CIK cells cultured under normoxia or hypoxia. D, bar graphs depicting hypoxic fraction in rh-endostatin treated group or control group. E, bar graphs depicting the cytotoxicity of CIK cells towards A549 cells at different E-to-T ratio. F, bar graph depicting the number of CIK cells migrating across the HUVECs layer. Similar results were observed in three independent experiments. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance.</p

Rh-endostatin depletes the accumulation of MDSCs in the tumor.

<p>C57BL/6 mice were injected s.c. with Lewis lung carcinoma cells and when tumor volume reached 100 mm³ treatment were initiated. After administration of rh-endostatin for consecutive 7 days, tumor-bearing mice were sacrificed and single cell suspensions of spleen, lymph node and tumor tissue were prepared to analyze frequency of MDSCs by flow cytometry. A, representative flow cytometry analysis data showing the frequency of CD11b⁺Gr1⁺ MDSCs in two groups. B, bar graph depicting the percentages of MDSCs in the spleen, lymph node or the tumor. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance.</p

Rh-endostatin treated tumors show increased K^trans value for Gd-DTPA and delayed Evans blue extravasation.

<p>DCE-MRI and intravital microscopy were performed to test tumor vascular permeability to small molecules and macromolecules, respectively. A, representative images of DCE-MRI with Gd-DTPA well infiltrating in the A549 tumor. B, the columns represent mean ± SE of K^trans for A549 tumors in control group and rh-endostatin treated group at day3, 6 and 9. C, the columns represent mean ± SE of Evans blue extravasation time in control group and rh-endostatin treated group at day 3, 6 and 9. Columns, mean; Bars, SE; *p<0.05 indicating statistical significance. Results shown are representative of 3 independent experiments.</p

Tumor vascular is normalized by rh-endostatin assessed by intravital microscopy.

<p>A549 tumor-bearing mice were treated with rh-endostatin (5 mg/kg, s.c.) for consecutive 7 days with normal saline as control. On days 3, 6 and 9, intravital microscopy were performed to test tumor vascular permeability to macromolecules. A, representative figures showing the exposure of tumor surface and intravenous injection of Evans blue in to BALB/c mice. B, a representative figure of Evans blue infused tumor vessels of control group at 100× magnification. C, a representative figure of Evans blue infused tumor vessels of rh-endostatin treated group at 100× magnification.</p

Additional file 1: Figure S1. of MiR-143-3p functions as a tumor suppressor by regulating cell proliferation, invasion and epithelial–mesenchymal transition by targeting QKI-5 in esophageal squamous cell carcinoma

The relative expressions of five candidate genes of miR-143-3p in ESCC cell line.QKI is one of the most upregulated candidate genes of miR-143-3p in ESCC cell lines. Real time RT-PCR analysis of five candidate genes in three human ESCC cell lines and the normal esophageal epithelial cell line (HEEC). (TIF 42 kb

DNMT1 and MBD2 expression determined by realtime-PCR (A,B) and Western blot (C,D).

<p>Data are representative of at least 3 independent experiments. Early effects mean 2(A,C); Delay effects mean 1 month postirradiation (B,D). PBMC, peripheral blood mononuclear cell. 1, control; 2, acute exposed to 0.5 Gy; 3, chronic fractionated exposure. ^*<i>P</i><0.05 versus control.</p

E2 and IGF-I quickly stimulate GFP-ERα translocation from nucleus to cytoplasm in NWTB3 cells.

<p>ER was mostly located in the nucleus in the non-treated control group, however, only a small part in the cytoplasm after E2 treatment (20 min). ER was significantly increased in the cytoplasm in IGF-I and IGF-I plus ER treatment groups (20 min).</p

Rad23b and Ddit3 methylation determined by BSP.

<p>Bisulfite treated DNA was amplified by PCR with BSP primers. PCR products were cloned into the pUC57 vector, and five clones selected and sequenced from each sample. Methylation level was defined as the ratio of methylated CpG sites in all clones. (A) Typical sequencing results; (B) early effects, tissues were collected 2 h postirradiation; (C) delay effects, 1 month postirradiation. ^*<i>P</i><0.05 versus control.</p

Functional and pathway analysis of the 811 genes with hypermethylated promoter identified by MeDIP-chip.

<p>Gene ontology (GO) analysis by three domains: Biological Process (A), Cellular Component (B) and Molecular Function (C). (D) KEGG Pathway analysis.</p