Journal: International Journal of Clinical and Experimental Pathology

Article Title: Primary microcephaly gene MCPH1 shows a novel molecular biomarker of human renal carcinoma and is regulated by miR-27a

doi:

Figure Lengend Snippet: MCPH1 is decreased expression in renal cancer tissues. A. Up. Normal renal tissue showing strong expression of MCPH1. MCPH1 showing low expression in renal cancer tissue and mainly locating in the cytoplasm. Left (×200). Right (×400). B. Kaplan-Meier plots of MCPH1 expression in 20 cases of renal cancer patients. Overall survival rate was performed by log-rank test. P<0.05 indicate significant differences between two groups.

Article Snippet: The immunohistochemistry for the tissue slides was performed as described previously by Simmons et al. [ 15 ] using a rabbit polyclonal anti-MCPH1 antibody (Abcam, Cambridge, MA) at a dilution of 1:100 and a secondary antibody from Bangalore GeneiH, Bangalore, India.

Techniques: Expressing

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Primary microcephaly gene MCPH1 shows a novel molecular biomarker of human renal carcinoma and is regulated by miR-27a

doi:

Figure Lengend Snippet: Tumor suppressor activity of MCPH1. A. MCPH1 overexpression inhibits cell growth examined by cell proliferation assay in ACHN and 768-O cells, compared with the Lent-ctrl. The curve represents the OD (optical density) measurement at 450 nm. The values are the mean OD450 nm of three independent experiments. *indicates P<0.05. B. Flow cytometry analysis of apoptosis of renal cancer cells. MCPH1 overexpression induces apoptosis of ACHN and 768-O cells, 72 h after infection. C. Matrigel invasion analysis of cell invasion in renal cells. Note: MCPH1 overexpression reduced invasiveness in ACHN and 768-O cells as compared to Lent-ctrl cells. The quantitative representation of the cell invasion assay data. The values are the mean ± SD of the number of invaded cells counted in four random microscopic fields.

Article Snippet: The immunohistochemistry for the tissue slides was performed as described previously by Simmons et al. [ 15 ] using a rabbit polyclonal anti-MCPH1 antibody (Abcam, Cambridge, MA) at a dilution of 1:100 and a secondary antibody from Bangalore GeneiH, Bangalore, India.

Techniques: Activity Assay, Over Expression, Proliferation Assay, Flow Cytometry, Infection, Invasion Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Primary microcephaly gene MCPH1 shows a novel molecular biomarker of human renal carcinoma and is regulated by miR-27a

doi:

Figure Lengend Snippet: Binding of miR-27 family members to the 3’-UTR of MCPH1. A. Scheme of the MCPH1 3’-UTR containing luciferase constructs (pMCPH1-3.1, pMCPH1-3.2 and pMCPH1-3.3). The gray horizontal bar represents the MCPH1 3’-UTR, dashed lines indicate different 3’-UTR fragments fused to Renilla luciferase in psiCHECK2. The black dot indicates the location of the predicted miR-27a binding site. B. Plasmid reporter gene constructs were cotransfected with the indicated miRNA mimics or control siRNA into HEK293 cells. After 48 hours, lysates from transfectants were analyzed for luciferase activity miR-27a and miR-27b as well as miR-27c. Data of normalized Renilla luciferase activity (RLU) represent means (+SEM) of n=3 experiments **, P<0.01.

Article Snippet: The immunohistochemistry for the tissue slides was performed as described previously by Simmons et al. [ 15 ] using a rabbit polyclonal anti-MCPH1 antibody (Abcam, Cambridge, MA) at a dilution of 1:100 and a secondary antibody from Bangalore GeneiH, Bangalore, India.

Techniques: Binding Assay, Luciferase, Construct, Plasmid Preparation, Control, Activity Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: Primary microcephaly gene MCPH1 shows a novel molecular biomarker of human renal carcinoma and is regulated by miR-27a

doi:

Figure Lengend Snippet: miR-27a controls MCPH1 expression in renal cells. A. Left, transfection of miR-27 mimics downregulates ULBP2 expression in melanoma cells. ACHN cells were transfected with miRNA mimics or control siRNA. After 48 hours, lysates of cells were analyzed for MCPH1 expression by Western blot. Actin served as loading control and for normalization. A. Right, fold change in MCPH1 expression was calculated from n=3 experiments. B. The influence of miR-27 mimic transfection on MCPH1 mRNA levels was determined by qRT-PCR. Expression of MCPH1 mRNA was normalized to endogenous GAPDH mRNA. Mean expression levels (+SEM) of n=3 experiments are presented relative to control siRNA-transfected cells. C. Endogenous miR-27a reduces MCPH1 expression in renal cells. ACHN cells were transfected with either anti-miR-27a or control anti-miRNA. After 48 hours, cells were lysed and MCPH1 expression levels were determined by Western blot. Actin served as loading control and for normalization. D. The fold change in MCPH1 expression was calculated from n=3 experiments. *, P<0.05.

Article Snippet: The immunohistochemistry for the tissue slides was performed as described previously by Simmons et al. [ 15 ] using a rabbit polyclonal anti-MCPH1 antibody (Abcam, Cambridge, MA) at a dilution of 1:100 and a secondary antibody from Bangalore GeneiH, Bangalore, India.

Techniques: Expressing, Transfection, Control, Western Blot, Quantitative RT-PCR