The alterations of modifications occurred in these signaling proteins were quantitated and shown in Figure 6A. factor 2a). Taken together, our findings implicate that ANKRD49 promotes the proliferation of human malignant glioma cells. ANKRD49 maybe an attractive target for malignant glioma therapy. gene were downloaded from the website of The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov). A total of 558 samples, which contained transcriptional expression data of 548 tumor tissues and 10 normal tissues, were available for this analysis. Cell culture Human malignant glioma cell lines U251 and U87 were cultured in Dulbeccos modified Eagles medium (DMEM) (Invitrogen), supplemented with 10% FBS (Corning) and 1% penicillin and streptomycin solution (Corning). Cells were cultured at 37C with 5% CO2. Total mRNA isolation and quantitative real-time PCR Total RNA was isolated from indicated cells using TRIzol reagent (Invitrogen) according to the manufacturers instructions. Moloney murine leukemia virus (M-MLV) reverse transcriptase (Promega) was used for cDNA synthesized. Quantitative real-time PCR was used to analyze gene expression using synergy brands (SYBR) master mixture (Takara). The PCR primers used were as follows: ANKRD49 forward, 5-GGTACTCAAAGTCTTTGGGTAGG-3 and reverse: 5-AGAAGCAATCTGCTTGGGTCT-3; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward: 5-TGACTTCAACAGCGACACCCA-3 and reverse: 5-CACCCTGTTGCTGTAGCCAAA-3. The relative ANKRD49 expression was normalized to GAPDH, and data analysis was conducted using the comparative cycle threshold (tests were used to analyze the differences between the two groups. One-way ANOVA analysis was applied for analyzing the difference for more than two groups. A probability value of less than 0.05 was considered significant. Results ANKRD49 is highly expressed in glioma and significantly correlated with glioma grade and survival Through reanalyzing RNA sequencing data of glioma-related datasets in TCGA database, we found that the expression of ANKRD49 was predominantly higher in glioma samples compared with matched normal samples (Figure 1A) (fold change =2.11, mRNA level in glioma patients. ANKRD49 expression in NVP-BGJ398 phosphate gliomas correlates with overall survival. Table 1 Analysis for ANKRD49 between LGG and GBM in TCGA data (MannCWhitney U-test) mRNA was measured by q-PCR in four human glioma cell lines: U251, U87, U373, and A172. (B,C) Quantitative RT-PCR analysis revealed that ANKRD49 expression was efficiently knocked down in the U251 (B) or U87 (C) cells; ***P<0.001. (D) Western blot analysis revealed NVP-BGJ398 phosphate that ANKRD49 expression was efficiently knocked down in the U251 or U87 cells. GAPDH served as an internal control. Cell proliferation is NVP-BGJ398 phosphate impaired by ANKRD49 knockdown in U251 and U87 cells To examine whether SOCS2 ANKRD49 contributes to the development of human malignant glioma, two different assays were employed to evaluate cell proliferation. First, we monitored the cell number of U251 cells everyday for 5 straight days. Obvious cell proliferation impairment was observed in U251 cells from the second day in the shANKRD49 group as compared with the shCtrl group (Figure 3A,B). Furthermore, the cell proliferation status of ANKRD49 knockdown on U251 cells was also tested by MTT assay. Compared with shCtrl U251 cells, the proliferative rate of U251 cells with ANKRD49 knockdown was significantly decreased from the third day (Figure 3C). Besides, we used MTT assay to test the effect of ANKRD49 silencing on U87 cells. As expected, ANKRD49 knockdown blunted the proliferation rate of U87 cells (Figure 3D). These results suggested that ANKRD49 is indispensable for U251 and U87 cell proliferation. Open in a separate window Figure 3 Knockdown of ANKRD49 inhibited the proliferation of U251 and U87 cells(A) Representative images of U251 cells infected with shCtrl (top) and.