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. Author manuscript; available in PMC: 2014 Jul 8.
Published in final edited form as: Science. 2010 Oct 15;330(6002):390–393. doi: 10.1126/science.1194598

Fig. 3.

Fig. 3

SipA causes activation of caspase-3 and its release at the apical surface of the epithelium. (A) Time course of caspase-3 activation during S. Typhimurium infection of T84 cell monolayers. Cells were infected with either wild-type S. Typhimurium (WT) or the ΔSipA mutant. Casp3 refers to pro-caspase-3 while A-Casp3 represents the activated form of the enzyme; Soluble and insoluble refers to the respective membrane fractions. (B) The ΔSipA mutant strain complemented with a vector expressing SipA (ΔSipA/pSipA) rescued the ability to induce the activation of caspase-3 following a 2 hour infection. Buffer refers to the buffer control. (C) Infection of T84 cells with different isogenic mutant strains of S. Typhimurium. Immunoblots were performed on cell extracts 3 hour post infection with anti-caspase-3 antibody. Casp3 refers to pro-caspase-3; A-Casp3 represents the activated form of the enzyme. (D) Activated caspase-3 was released at the apical but not basolateral surface during in vitro infection of T84 cells with wild-type S. Typhimurium (WT). No caspase-3 was detected in ΔSipA mutant infected wells or in the buffer control, uninfected wells. (E) Cleaved SipA was detected in apical media but not in the cell cytosol following infection with the ΔSipA mutant complemented with SipA (ΔSipA/pSipA). The arrow shows the N-terminal fragment generated by cleavage. No SipA is detected in the SipA negative mutant infected cells. (F) Caspase-3 (black bars) or caspase-1 (open bars) activity was measured in mucosal extracts post in vivo infection of wild-type mice. Infection was carried out with wild-type Salmonella (WT), a ΔSipA mutant, and ΔSipA complemented with either SipA (ΔSipA/pSipA) or pCSM-SipA (ΔSipA/pCSM-SipA). Buffer refers to the uninfected buffer control. (G) Immunoblots showing cleavage of SipA after addition of mucosal extract isolated from mice infected with various indicated strains. WT refers to wild-type Salmonella and ΔSipA is the ΔSipA strain complemented with either wild-type SipA (SipA/pSipA) or the pCSM-SipA (ΔSipA/pCSMSipA). Buffer refers to the negative control.