Fig. 4 |. Mutant p85 mice expressing an LT-resistant variant are more resistant to toxin challenge, and LT-induced lethality in mice requires targeting of both MeK and Pi3K pathways.

a, Sequence chromatograms from Pik3r1^GVAA/GVAA (KI/KI), heterozygote (KI/WT) and wild-type mice. b, The survival of these mice after toxin challenge (LT, 25 or 35 μg, intravenous) were compared to wild-type mice. P = 0.0434 (25 μg) and P = 0.0014 (35 μg); log-rank (Mantel–Cox) test. Studies represent six experiments (KI survival range, 36–67%). c,d, LT-treated (1 μg ml⁻¹, 18 h) C57BL/6J BMDMs (c) or organs (d) from LT-challenged mice (35 μg, intraperitoneal, 24 h) were analysed by western blot with antibodies against the N termini of p85α, MeK1 and MKK3. Loss of reactivity indicates cleavage. LC (loading control, blue arrowheads) indicates cross-reactive species showing equal protein loading. e, C57BL/6J mice (n = 5 per group) were treated with PI3K and p38 inhibitors independently (to ensure inhibitor doses were not lethal) or in combination with the nonlethal mutant LT(W271A). Drugs were given by gavage at −18 h, −4 h and +20 h relative to LT(W271A) challenge at time zero. Two control groups received the vehicle and were challenged with either LT(W271A) or LT at time zero. Toxin challenge dose (80 μg, intraperitoneal) is not lethal for LT(W271A) but causes full cleavage of MeK1 (extended Data Fig. 3f). In both panels, groups treated with the PI3K–mTOR inhibitor dactolisib (indicated as PI3K inh1, 50 mg kg⁻¹; red closed circle, drug; green squares, drug + LT(W271A)) or vehicle (open orange squares, LT(W271A); open red triangle, LT) were similarly treated. For p38 inhibition, a mixture of p38 inhibitors (losmapimod + PH-797804) (top) or losmapimod only (bottom) was used. These treatments (25 mg kg⁻¹ per drug per application) are indicated in both panels as p38 inh (black closed triangle, drugs; blue inverted triangle, drugs + LT(W271A)). For inhibition of both PI3K and p38, mice were treated with dactolisib + PH-797804 + losmapimod (PI3K inh1 + p38 inh; open black diamond, drugs; blue star, drugs + LT(W271A)) (top). For PI3K inhibition, mice were treated with GDC-0941 (PI3K inh2, 75 mg kg⁻¹) (open black circle, drug; black X, drug + LT(W271A)). Log-rank (Mantel–Cox) test shows no statistical difference between groups with 100% mortality (P > 0.1). All groups with survivors are significantly different from non-surviving groups (P < 0.05).