Figure 3.

Increased expression of PLCβ1 abolishes siRNA (GAPDH) activity. A) Top panels: HEK293-TAP-PLCβ1 cells were treated with siRNA(GAPDH) and/or tetracycline (tet) to induce PLCβ1 expression. Cells treated only with siRNA(GAPDH) had diminished protein expression, which was reversed in cells also treated with tetracycline. All bands were normalized according to the actin loading control, n = 3. Error bars = sd. Bottom left panel: Representative Western blot showing the rescue of GAPDH levels in SKNSH cells with PLCβ1-eYFP overexpression (n=4). Bottom right panel: degree of cell survival of siRNA(GAPDH)-treated cells as normalized by untreated controls, showing the rescue of cell death by PLCβ1 overexpression and the reversal of this effect by cotransfection with the constitutively active Gα_q(RC). B) A similar study, except cells were transiently transfected with eCFP-TRAX with tetracycline treatment to restore siRNA(GAPDH) activity, as seen by a reduction in GAPDH expression. Inclusion of 10 μM of the PLC inhibitor U73122 did not affect the reversal of siRNA(GAPDH) by PLCβ1 overexpression. Bands were normalized using the actin loading control, n = 3. Error bars = sd. C) Left panel: relative levels of GAPDH mRNA, as determined by RT-PCR (see Materials and Methods) for showing a recovery of GAPDH mRNA with PLCβ1 overexpression in siRNA treated cells where the levels were normalized to ubiquitin mRNA (n=3, P=0.016). Right panel: change in the levels of small RNAs (i.e., <1600 nt) with PLCβ1 overexpression either by tetracycline treatment of HEK293-β1 cells or transient transfection of HeLa cells (36 h). Bands were quantified by Image J and normalized to the highest value. Each set was compared using a paired t test; n = 6.