Bisindolylmaleimide 1

The human colon carcinoma Caco-2 cell line and the human embryonic kidney 293T cell line were kindly provided by Professor Weiqi He and Professor Ying Xu respectively (Soochow University, Suzhou, China). Cells from 25 to 35 passages were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) (SH30243.01B; Hyclone, South Logan, UT, USA) supplemented with 10% FBS and 1% Penicillin-Streptomycin Solution (C0222; Beyotime Biotechnology, Shanghai, China) at 37°C with 5% CO₂. For infection, Bacteria were washed three times in PBS and resuspended in DMEM-FBS (10%) at a multiplicity of infection (MOI) of ~100. One hour later, infected cells were washed three times with PBS and replaced by DMEM-FBS (10%) containing 100 µg/ml amikacin to eliminate extracellular bacteria. For inhibition of actin polymerization, Caco-2 cells were pretreated with 2 µM Cytochalasin D (ab143484; abcam, Cambridge, MA, USA) for 1 h. For inhibition of PKC activation, Caco-2 cells were pretreated with 1 µM Bisindolylmaleimide I (S7208; Selleck, Houston, TX, USA) for 1 h.

Protein phosphatase PP2A inhibitor LB-100 (S7537), PKC-θ inhibitor (S6577), Darovasertib (S6723), Rottlerin (S7862), and Bisindolylmaleimide I (S7208) were purchased from Selleckchem, Houston, TX, USA. MAP4K4 inhibitor GNE-495 was kindly provided by Genentech Inc.^{66 (link)}. EGF (100-47) and bFGF (100-18B) were purchased from PeproTech, London, UK. If not otherwise indicated, the cells were treated with growth factors at these concentrations: EGF: 30 ng/ml, bFGF: 100 ng/ml.