Cell experiment:

The murine macrophage cell line RAW264.7 and peritoneal macrophages from both types of mice are maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum (FCS). Cultures are maintained at 37℃ in a humidified incubator in a 95% O2 plus 5% CO2 atmosphere. Cells are treated with varied concentrations of TNF-α and used IC-87114 (IC87114) to inhibit PtdIns(3,4,5)P3-dependent phosphorylation of Akt before TNF-α stimulation at early time points (30 min)[2].

Animal experiment:

Mice[3]BALB/c mice are immunized once by i.p. injection of 10 μg ovalbumin (OVA) in 0.2 ml of alu-Gel-S on day 0. Ten days later, mice are intranasally (i.n.) challenged with OVA (30 μg in 50 μL PBS) or PBS, once daily, over four consecutive days. To investigate if ERK1/2, PI3Kδ and NF-κB are signaling effectors downstream of EGFR transactivation, six treatment groups (A-F, 10-30 animals per group) are established. Mice in groups A and B are pretreated intranasally with 0.2 mL of the vehicle for the drugs. Groups C, D and E are pretreated with the same volume of three different drugs (PD 98059, IC-87114 and BAY 11-7085, respectively) at 10 mg/kg, 10 mg/kg and 0.3 mg/kg respectively, and group F with Dexamethasone (1 mg/kg), 1 h before each i.n. challenge with OVA. These doses are chosen from previous studies where they are shown to be effective.

IC87114 is a PI(3)Kδ selective inhibitor with IC50 of 50 nM.

It has been reported that IC87114 reduced FMLP-induced PIP3 synthesis and chemotaxis in neutrophils[1]. IC87114 also blocked TNF1α-stimulated elastase exocytosis from neutrophils in a mouse model of inflammation. A role for PI3Kδ in TNFα-induced signaling was demonstrated by a reduction in Akt-phosphorylation and PDK1 enzyme activity upon treatment of this cell type with IC87114 [2]. In human memory T cells, IC87114 largely inhibited T-cell receptor-induced PI(3)K signaling by both naive and effector. Cytokine production in memory T cells was blocked by IC87114 from healthy and allergic donors, or from inflammatory arthritis patients [3].

References:
[1]. Workman P, van Montfort RL. PI(3) kinases: revealing the delta lady. Nat Chem Biol. 2010 Feb;6(2):82-3.
[2]. Puri KD, Doggett TA, Douangpanya J, Hou Y, Tino WT, Wilson T, Graf T, Clayton E, Turner M, Hayflick JS, Diacovo TG. Mechanisms and implications of phosphoinositide 3-kinase delta in promoting neutrophil trafficking into inflamed tissue. Blood. 2004 May 1;103(9):3448-56.
[3]. Soond DR, Bjorgo E, Moltu K, Dale VQ, Patton DT, Torgersen KM, Galleway F, Twomey B, Clark J, Gaston JS, Taskén K, Bunyard P, Okkenhaug K. PI3K p110delta regulates T-cell cytokine production during primary and secondary immune responses in mice and humans. Blood. 2010 Mar 18;115(11):2203-13.