Images: Ret receptor knockdown using small interference RNA (siRNA) in podocytes. (A) Transfection efficiency: mouse podocytes were transfected with 100 nM concentrations of Ret siRNA or vehicle alone. (a) When podocytes were exposed to i-Fect alone, there was no toxicity. (a and b) A transfection efficiency of nearly 100% was achieved with 100 nM concentration of Ret siRNA (b). Co-transfection with a fluorescently tagged control siRNA was used to determine the transfection efficiency, and fluorescence microscopy revealed a perinuclear localization of the tagged RNA (b, arrowhead). (B) Western blot analysis of Ret after transfection: Ret immunoblotting (top) of WCL 2, 3, or 4 d after transfection revealed that Ret was downregulated within 2 d after transfection with 100 nM Ret siRNA. Transfection of control siRNA at day 4 served as a negative control, and the maximal knockdown of Ret was observed 4 d after transfection. GAPDH immunoblotting confirmed equal protein loading (bottom). doi:10.1681/ASN.2005080835. (full text publication)
Methods: Ret small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled functionally validated Ret siRNA (SMARTpool mouse RET siRNA; Dharmacon, Lafayette, CO). HSMP cells that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above and transfected using the i-Fect siRNA
transfection reagent (Neuromics, Northfield, MN). For determination
of the transfection efficiency, a Texas Red–labeled siRNA (siGLO RISCFree
siRNA; Dharmacon) was co-transfected with Ret siRNA and visualized
using fluorescence microscopy. For control siRNA samples,
identical conditions were used with the substitution of siGLO-RISCFree
siRNA for Ret siRNA. For determination of the efficiency of Ret
knockdown, Western analysis for Ret was performed on WCL from
cells 24 to 96 h after the transfection. Several concentrations of Ret
siRNA (40, 60, and 100 nM) were tested to determine optimal knockdown
conditions. For apoptosis assays, podocytes were exposed to
UV-C or PA (40 g/ml) on days 3 to 4 after transfection of Ret siRNA
or control siRNA (100 nM). Apoptosis was measured by counting
podocytes with Hoechst-positive pyknotic nuclei 3 h after UV and 5 h
after exposure to PA.
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