Fig. 1.

Characteristics of expression of CAST EST. (A) Effect of doxycycline (Dox) on the susceptibility of clone 3-12. Cells were preincubated in the presence or absence of 1 µg/mL Dox for 2 d and then were seeded at a concentration of 2 × 10⁴ cells/mL in a 96-well plate containing or lacking 1 µg/mL of Dox. Cells were treated with a mixture containing the indicated concentrations of PA plus 250 ng/mL LF for 2 d, and an MTT assay was performed as described in Materials and Methods. Cell viability is shown as the percent survival relative to treatment without toxin and represented as mean ± SD. Data are from one representative experiment (n = 3) performed in quadruplicate. LD₅₀ values of PA for clone 3-12 in the absence and presence of Dox are 172 ± 13.8 ng/mL and 29.5 ± 2.2 ng/mL, respectively. P < 0.01. (B–D) The CAST EST identified in clone 3-12 was reintroduced into the pLEST lentiviral vector, yielding pLEST-CAST, which then was used to infect RAW264.7tTA cells. Selective growth of cells containing pLEST-CAST or the empty lentiviral vector (pLEST) was accomplished by treating cultures with 800 µg/mL G418. (B) Effect of CAST EST expression on the cytotoxicity of PA-LF. Pooled pLEST-CAST–infected cells were treated with a mixture of PA plus LF, as in A. An MTT assay was performed 1 d after toxin treatment. The percent of cell viability is represented as mean ± SD. Data are from one representative experiment (n = 3) carried out in triplicate. LD₅₀ values of PA in pLEST-CAST and pLEST are 10.2 ± 0.8 ng/mL and 2.6 ± 0.1 ng/mL, respectively. P < 0.01. (C) Effect of CAST EST on CAST mRNA expression. Relative mRNA abundance was quantified by real-time RT-PCR using primers corresponding to a segment of exons 1–3, as indicated in Materials and Methods and normalized to β-actin. Data represent mean ± SD of three independent experiments. **P < 0.01. (D) Abundance of CAST protein. Cell lysates were analyzed by Western blotting using anti-CAST antibody. The numbers below the Western blots indicate mean ± SD of relative CAST level (upper band) normalized to actin from two independent experiments. (E) Calpain activity in cells infected by pLEST-CAST or pLEST was determined in total cell lysates using synthetic fluorogenic substrates as described in Materials and Methods. Data represent mean ± SD of three independent experiments. **P < 0.01. RFU, relative fluorescence units.