Figure 3

Reduced PAX8 expression leads to decreased expression of BCL2 and WT1. (A) The PAX8-knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the BCL2 expression levels. Cells lysates were prepared 36 hours after siRNA transfection, and the PAX8, BCL2, p53, and β-actin (loading control) expression levels were measured by western blot. For controls, A172 cells were transfected with mock-treated (Moc), non-targeting siRNAs (NT1, NT2, and NT3) and scrambled s8-1 siRNA (scPAX8). To ensure the reduction in the glioma cell growth rate associated with the PAX8-knockdown was not due to p53 function, p53 was also knocked down in A172 cells (sip53) independently or in combination with a PAX8 siRNA (siPAX8 & p53). (B) The PAX8-knockdown (siPAX8) in the A172 glioma cell line by siRNA (PAX8-1) produced a reduction in the WT1 expression levels. (C) The BCL2-knockdown produced a similar reduction in the cell growth rate compared to PAX8-knockdown in the A172 glioma cell line. Cells were transfected with a BCL2 siRNA (siBCL2) or a PAX8 siRNA (PAX8-1, siPAX8). For controls, A172 cells were mock-transfected (Moc) or transfected with non-targeting siRNAs (NT1 and NT3). The percentage of live cells was determined by the trypan blue exclusion assay every 24 hours post-transfection. (Insert) Western blotting shows the BCL2-knockdown with a BCL2 siRNA and no BCL2-knockdown in controls; the loading control is β-actin.