Wortmannin

The human glioblastoma cell lines U87 MG, U118 MG, U251 MG, A172, LN-229, and T98G were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA).
The antibodies against NAG-1, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, cytochrome c, p-PI3K(p85 Tyr458)/PI3K(p85), p-Akt(Ser473)/Akt, p-ERK1/2(Thr202/Tyr204)/ERK1/2, p-Smad2(Ser465/467)/Smad2, p-Smad3(Ser423/425)/Smad3, and β-actin were purchased from Cell Signaling Technologies (Beverly, MA, USA). Wortmannin, LY294002, Smad2 siRNA, and Smad3 siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Ac-IETD-FMK and Ac-LEHD-FMK were purchased from KeyGEN Biotech Co., Ltd. (Nanjing, China). Enhanced chemiluminescence (ECL) detection system was purchased from Amersham Life Science (Arlington Heights, Illinois, USA). Human NAG-1 ELISA Kit was obtained from Huamei Biological Company (Wuhan, China). Mitochondrial membrane potential assay kit with JC-1 and protein A/G agarose beads were obtained from Beyotime Institute of Biotechnology (Shanghai, China). X-treme GENE siRNA transfection reagent was purchased from Roche Applied Science.

Where indicated, the following drugs were used: VER‐155008 (Santa Cruz Biotechnology), MG132 (Sigma‐Aldrich), nocodazole (AppliChem), wortmannin (Santa Cruz Biotechnology), sodium arsenate dibasic heptahydrate (Alfa Aesar).

All chemical inhibitors were added to cells 1 hour prior to infection and kept in the culture medium throughout the experiment. To avoid inhibition of bacterial transcription by actinomycin D, cells pre-treated with actinomycin D were washed twice with PBS immediately prior to infection and medium without inhibitor was used for the remainder of the experiment. Inhibitors had no effect on Lm viability, as measured by exposing Lm to each inhibitor for 1 hour in D-10 and enumerating CFU. All inhibitors were dissolved in DMSO except bpV(pic) which was dissolved in water. Inhibitors were used at the following concentrations: actinomycin D (Sigma-Aldrich), 10 μg/mL; cycloheximide (Sigma-Aldrich), 1 μg/mL; bpV(pic) (Cayman Chemical Company), 5 μM; CHIR99021 (Cayman Chemical Company), 10 μM; parthenolide (Sigma-Aldrich), 10 μM; wortmannin (Santa Cruz Biotechnology), 100 nM; INK1117 (GlpBio), 1 μM; TGX221 (Cayman Chemical Company), 10 μM; IC87114 (Cayman Chemical Company), 10 μM; and IPI549 (MedChemExpress), 50 nM.

Chlamydomonas reinhardtii WT 4A+ (CC-4051) was obtained from the Chlamydomonas Resource Center (http://www.chlamycollection.org). Chlamydomonas strain OL-Rps6 expressing OLLAS-tagged RPS6 was generated in this study as described below. Chlamydomonas cells were grown under continuous illumination at 25 °C in Tris-acetate phosphate (TAP) medium as described (Harris, 1989 ). When required, cells in exponential growth phase (10⁶ cells ml^–1) were treated with concanamycin A (Santa Cruz Biotechnology, sc-202111A), wortmannin (Santa Cruz Biotechnology, sc-3505), or 3-methyladenine (3-MA; Sigma, M9281), or subjected to nitrogen or phosphate limitation.

Natural CGL was isolated as described previously^{16 (link)}. Recombinant CGL was prepared according to our previous study^{19 (link)} and further purified using Pierce High Capacity Endotoxin Removal Spin Columns (Thermo Fisher Scientific, USA). LPS (from Escherichia coli 0111:B4), N-acetylcysteine (NAC), PD98059, SP600125, SB203580, PDTC and mouse antibodies against mouse phospho-ERK1/2, phospho-JNK1/2, phospho-p38 and actin were purchased from Sigma-Aldrich (St. Louis, MO). Gö6976, Rottlerin, Wortmannin, LY294002, sc-3060, and antibodies against phospho-PKCα/δ, IL-1β, iNOS, COX-2 and IRAK2, as well as secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). IL-1β, IL-6, TNF-α and MCP-1 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Pierce™ LAL Chromogenic Endotoxin Quantitation Kit was purchased from Thermo Scientific (Rockford, IL).