Wortmannin

Mice were anesthetized with ketamine/xylazine (65/6 mg/kg, i.p), and their body temperature was maintained at 37 °C by a heating pad and feedback control system (FHC, Bowdoin, ME, USA). A laser Doppler probe was fixed on the skull 5 mm lateral and 2 mm posterior to the bregma. A coated filament was placed on the right middle cerebral artery (MCA) with concurrent recording of laser Doppler cerebral blood flow to ensure that the cerebral blood flow decreased to below 25 % of the baseline. After 45 min, the filament was removed (Additional file 2: Figure S2). Either Ex-4 (10 mg/kg) or saline was injected through the tail vein immediately after reperfusion. In the Ex-4 + wortmannin group, we intravenously injected 15 μL/kg wortmannin (Sigma-Aldrich), a non-specific, covalent inhibitor of PI3K immediately after reperfusion.

The human SCLC cell line NCI-H446 (Cell Bank of Shanghai Institutes for Biological Sciences, China Academy of Sciences, Shanghai, China) was cultured as in a previous study (1 (link)). Once passaged to the fifth generation, the cells were grown in 75 cm² culture flasks and harvested in a solution of trypsin-EDTA at the logarithmic growth phase. A cytometry assay was used for cell counting (2 (link)). For the demethylation treatment, NCI-H446 cells (60–70% confluence) were treated with 5 μm 5-aza-2′-deoxy-cytidine for six days and the medium was replaced every other day. DNA, RNA and protein were extracted for analysis using kits obtained from Sangon Biotech Co., Ltd. (Shanghai, China). For blocking Akt expression, wortmannin (Sigma-Aldrich, St. Louis, MO, USA) was used as an inhibitor of the PI3K/Akt signaling pathway. wortmannin was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) and final concentrations of 20 μM were used to treat the cells. Equal volumes of DMSO were used for the vehicle control (17 (link)). CPA-7, which is a specific inhibitor of the PI3K/Akt signaling pathway, was used for blocking STAT3 expression and dissolved in DMSO to achieve a final concentration of 10 μM (18 (link)). For plasmid transfection, pcDNA3-STAT3 and pcDNA3-Akt were received from the Department of Viral-Gene Therapy, Shanghai Eastern Hepatobiliary Surgery Hospital (Shanghai, China).

DsRed-expressing CD8⁺ T cells were activated in vitro and rested in the RPMI medium containing 1% fetal bovine serum at 37 °C on a 35-cm dish (NEST) pre-coated with 7.5 μg ml⁻¹ recombinant mouse ICAM-2 (R&D) in PBS for 1 h before imaging. To inhibit PI3K activities, the T cells were pre-treated with 100 nM Wortmannin (Sigma) for 1 h before imaging and maintained in Wortmannin-containing media during imaging. Images were acquired using a Nikon A1RMP microscope equipped with Mai Tai DeepSee laser (Spectra-Physics), a 561-nm laser (Coherent) and × 60 oil objective lens (Nicon). Acquisition was controlled by NIS-Elements software (Nikon). Data were processed by NIS-Elements Analysis software (Nikon) and Imaris (Bitplane).

For AvrPto treatment, protoplasts expressing eGFP-Adi3 for 16 hr were transformed with pTEX:avrPto:FLAG as previously described [8] (link). Samples were collected at 0.5, 1.5, 3 and 4 hr for confocal imaging, immunoblotting, and determination of cell viability. For other treatments, eGFP-Adi3 expressing protoplasts were incubated with H₂O for 4h (no treatment), at 42°C for 30 min (heat), –20°C for 1 min and thawed at room temperature (cold), or shaken at 120 rpm at room temperature for 30 min (wounding) followed by confocal microscopy imaging. For nuclei staining, endosomal compartment identification, and wortmannin treatment, protoplasts were treated with 10 µM hoechst 33342 for 2 h, 20 µM FM4-64 for 2 h, and 33 µM wortmannin (Sigma) for 1 h, respectively, followed by confocal microscopy imaging. For colocalization studies and endosome fractionation, soybean α-1,2-mannosidase-mCherry [22] (link), 2xFYVE-DsRed, GFP-Ara7, and GFP-RHA1 were coexpressed in protoplasts for 16 hrs. All experiments were carried out a minimum of three times and images presented are representative of each experiment.

Unless indicated otherwise, all reagents were obtained from Sigma-Aldrich. All reagents were of the highest available grade and lowest possible endotoxin level. Tissue culture media and buffers were obtained from Life Technologies, and dextran and Percoll were from GE Healthcare. The antibodies used were as follows: β-actin (rabbit polyclonal) from Abcam; Bax (clone B-9) and Mcl1 (rabbit polyclonal) from Santa Cruz Biotechnology; phospho-Akt Thr 308 (clone L32A4), phospho-Akt Ser 473 (clone D9E biotinylated), phospho-Erk Thr 202/Tyr 204 (clone E10), phospho-Mek1/2 Ser 217/221 (rabbit polyclonal), phospho-p38 Thr 180/Tyr 182 (rabbit polyclonal), and phospho-Pak1 Ser 144 (rabbit polyclonal) from Cell Signaling Technology; and CD62L-PC5 conjugate from Immunotech.
The inhibitors and final concentrations used were as follows: pan-PI3K, wortmannin (50 nM), and LY2940002 (10 μM, used for prolonged inhibition because wortmannin has a very short half-life in aqueous solutions); PI3Kα and A66 (10 μM); PI3Kβ and TGX221 (40 nM); PI3Kδ and IC87114 (1 μM); and PI3Kγ, AS252424 (30 μM), and CZC24832 (10 μM) (all from Sigma); the Raf inhibitor AZ628 (5 μM); the Mek inhibitors AZD6244 (1 μM) and trametinib (1 μM); the Pak inhibitors PF3758309 (5 μM) and IPA3 (10 μM); the Erk inhibitor FR180204 (10 μM); and BVD523 (5 μM) (all from Selleckchem).