The investigations of SATB1 were carried out mainly in immune cells in the past

The investigations of SATB1 were carried out mainly in immune cells in the past. 2, control-shRNA-GFP U251 cells; lane 3, SATB1-shRNA U251 cells. 1479-5876-10-149-S3.tiff (18M) GUID:?47BE6316-F4DA-443E-969A-7C5C2C37247E Additional file 4 Additional file 4: Figure PF299804 (Dacomitinib, PF299) S4. Cytotoxic effect of SATB1-shRNA in U251 cells. The untransfected U251 cells, control-shRNA-GFP U251 cells and SATB1-shRNA U251 cells were cultured in plastic 96-well plates and quantified using the MTT assay. 1479-5876-10-149-S4.tiff (2.5M) GUID:?AF309B9C-D6D0-4C03-A29D-E136A0BA78CC Abstract Background Special AT-rich sequence-binding protein-1 (SATB1) has been reported to be expressed in several human cancers and may have malignant potential. This study was aimed at investigating the expression and potential role of SATB1 in human glioma. Method The relationship between SATB1 expression, clinicopathological parameters, Ki67 expression and MGMT promoter methylation status was evaluated, and the prognostic value of SATB1 expression in patients with gliomas was analyzed. SATB1-specific shRNA PF299804 (Dacomitinib, PF299) sequences were synthesized, and U251 cells were transfected with SATB1 RNAi plasmids. Expression of SATB1 mRNA and protein was investigated by RT-PCR and immunofluoresence staining and western blotting. The expression of c-Met, SLC22A18, caspase-3 and bcl-2 protein was determined by western blotting. U251 cell growth and adherence was detected by methyl thiazole tetrazolium assay. The apoptosis of U251 cells was examined with a flow cytometer. The adherence, invasion, and angiogenesis assays of U251 cells were done. The growth and angiogenesis of SATB1 low expressing U251 cells was measured in an xenograft model. Results Of 70 tumors, 44 (62.9%) were positive for SATB1 expression. SATB1 expression was significantly associated with a high histological grade and with poor survival in univariate and multivariate analyses. SATB1 expression was also positively correlated with Ki67 expression but negatively with MGMT promoter methylation in glioma tissues. SATB1 shRNA expression vectors could efficiently induce the expression of SLC22A18 protein, increase the caspase-3 protein, inhibit the expression of SATB1, c-Met and bcl-2 protein, the growth, invasion, metastasis and angiogenesis of U251 cells, and induce apoptosis and viral contaminants during the entire study period. Knock down SATB1 by RNAi in U251 cells SATB1-specific shRNA sequences were synthesized according to the one used in Han and inserted into the pGCsi-H1/Neo/GFP/siNEGative vector (Genscript), which coexpresses GFP to allow identification of transfection efficiency. The SATB1 shRNA sequence was: SATB1-shRNA 5′-GTCCACCTTGTCTTCTCTC-3′. The KRT4 non-specific shRNA sequence was: control-shRNA-GFP 5′-ACGTGACACGTTCGGAGAA-3′ [16]. U251 cells were transiently transfected with SATB1 RNAi plasmids or control plasmids using an electroporator. Immunohistochemical analysis Antigen retrieval was performed in boiling citrate buffer for 15 minutes. Peroxide blocking was performed with 0.3% peroxide in absolute methanol. The slides were then incubated with anti-SATB1 polyclonal antibody PF299804 (Dacomitinib, PF299) (diluted 1:100; Sigma, St Louis, MO) or mouse anti-PCNA monoclonal antibody (diluted 1:100; Santa Cruz) or anti-Ki67 (diluted 1:20; clone MIB-1, Dako, Denmark) at 4C overnight and washed twice with PBS before being incubated with the secondary antibody (Santa Cruz, CA) at room temperature for 30 mintes. After washing, sections were incubated with immunoglobulins conjugated with horseradish peroxidase (HRP). Finally, the reaction was developed with 3, 3′-diaminobenzidine substrate. Tissue sections were PF299804 (Dacomitinib, PF299) counterstained with hematoxylin or methyl green [17]. Immunohistochemical staining for CD34 and microvessel counting of CD34-positive vessels were performed as described previously [18]. The total SATB1 immunostaining score was calculated as the sum of the percentage positivity of stained tumor cells and the staining intensity scores. The percentage positivity was scored as follows: 0 (< 5%, unfavorable); 1 (5%-25%, sporadic); 2 (25%-50%, focal); 3 (> 50%, diffuse). The staining intensity was scored as follows: 0 (no staining); 1 (weakly stained); 2 (moderately stained); 3 (strongly stained). Both the percentage positivity of cells and the staining intensity were assessed under double-blind conditions. The final SATB1 expression score ranged from 0 to 9 and was calculated as the percentage positivity scorestaining.