Figure 5. B. anthracis-induced ATP release and inflammasome activation depend on connexin-43.

(A-C) C57BL/6 macrophages were infected with B. anthracis as above and 45 min later the connexin-43 inhibitor carbenoxolone (CBX, 2.5 μM) was added to the culture medium. ATP release was measured 1 hr later (A) and LDH release was measured after 2 hr (B). Secretion of active caspase-1 (p10) and IL-1β (p17) to the culture medium was examined 12 hr after CBX addition. Cellular actin was used as a control. This experiment was repeated 3 times and the results of one representative experiment are shown.

(D) RAW264.7 cells were transduced with lentiviruses expressing connexin-43 shRNA or control shRNA (Ctrl.). Connexin-43 expression in the transduced cells was analyzed by immunoblotting. This experiment was repeated 2 times and the results of one representative experiment are shown.

(E,F) RAW264.7 cells were transduced with lentiviruses expressing connexin-43 shRNA or control shRNA. Transduced cells were left uninfected or infected with B. anthracis. ATP release was measured 2 hr later (E). Caspase-1 (p10) and IL-1β (p17) secretion was examined after 12 hr (F). This experiment was repeated twice and the results of one representative experiment are shown. Results are means ± SD, **p < 0.01 denote significant differences between the groups.

(G) C57BL/6 macrophages were treated as above and at the indicated times cell lysates were prepared and analyzed by immunoblotting for phosphorylation of connexin-43 at Ser279 and Ser282. This experiment was repeated 3 times and the results of one representative experiment are shown.

(H) Myr-AKT-expressing or parental RAW264.7 cells were infected as above. After 2 hr, cell lysates were prepared and analyzed by immunoblotting for connexin-43 phosphorylation and tubulin content. This experiment was repeated 3 times and the results of one representative experiment are shown.