FIG. 3.
Transient and specific inhibition of Hsp70 in Caco-2 cells. (a) Kinetics of inhibition of the Hsp70 protein level after specific siRNA targeting (0 to 14 days). A Caco-2 cell suspension of 5 × 105 cells was supplied with 2 μg of siRNA-Hsp70 and then electroporated. Cells were plated and cultured at the indicated times after electroporation. Cells were harvested, and lysates were analyzed by immunoblotting as described in Materials and Methods. Hsp70 levels were quantified by immunoblot scanning. Results of this quantification are means ± SEM (n = 4). Note that the maximal inhibition of the Hsp70 level was observed 4 days after siRNA electroporation. (b) Specificity of siRNA-Hsp70. Caco-2 cells were plated and cultured for 96 h in the presence of 100 pmol scrambled siRNA in 100 μl of T solution from Amaxa, and 5 × 105 cells were subjected to electroporation and 2 μg of siRNA-Hsp70. Cell lysates were harvested and analyzed by immunoblotting as described in Materials and Methods, using monoclonal anti-Hsp70 or anti-Hsc70 antibodies. A representative experiment of four total experiments is shown. Only the Hsp70 level was affected by siRNA treatment, whereas the Hsc70 level remained unaffected. (c) Quantitative analysis of Hsp70 levels in siRNA-Hsp70-transfected cells. Caco-2 cells were treated as described for panel b, and the resulting immunoblots (four independent experiments) were quantified using immunoblot scanning. Hsc70 (white histograms) and Hsp70 (black histograms) were measured. *, P < 0.05. Note that at this time (4 days after electroporation) only the Hsp70 level decreased, whereas the Hsc70 level remained unaffected.