Figure 3. S. Typhimurium modulates P-gp expression through a caspase-3-dependent mechanism.

(a) HCT8 cell monolayers were left untreated (−) or infected with wild-type SL1344 in the presence or absence of pharmacological inhibitors of CASP-3 or CASP-1 (negative control) for 5 h. Normalized whole-cell lysates were then probed with P-gp and GAPDH. The data represent a western blot analysis from an individual experiment performed at least three times. (b) HCT8 cell monolayers transfected with a nonspecific siRNA vector control or with siRNA aimed at decreasing CASP-3 expression were left untreated or infected with wild-type SL1344. Whole-cell lysates were then probed as in a. (c) Three-dimensional structure of mouse P-gp (PDB ID, 3G5U) depicted as cartoon and transparent surface. The cytoplasmic Caspase-3 cleavage site (⁴⁵⁴DGQD⁴⁵⁷) is shown in red. The putative CASP3 site ¹⁶⁴DVHD¹⁶⁷ is not shown. Numbers refer to the position of the amino acids in the protein sequence. (d) HCT8 cell monolayers were infected with wild type SL1344 for 1, 3 or 5 h, and then probed using a P-gp antibody capable of detecting P-gp cleavage products. Progressive P-gp modulation was accompanied by the occurrence of 90- and 60-kDa cleavage products. (e) Anexin-V staining was used as measurement of apoptosis in HCT8 cells 3 h post incubation with 160 or 320 μg ml⁻¹ of purified SipA. Staurosporine treatment served as the positive control. Shown is the average of three independent experiments with error bars indicating s.d.; *P=0.0076, **P=0.0009 (Student’s t-test).