Pd0325901

The effect of PD0325901, trametinib, perifosine (SelleckChem, Houston, TX, USA) or DMF (FUJIFILM Wako) on cell viability was examined using the trypan blue stain exclusion assay as previously described [37 (link),51 (link)]. DLD-1, HT-29, LoVo, Colo-205, LoVo/PR, Colo-205/PR and LoVo/TR cells were seeded onto flat-bottom 96-well plates for 24 h. Next, DLD-1, HT-29, LoVo and Colo-205 cells were treated with various concentrations of PD0325901 or trametinib for 1, 3 or 5 days; combination treatment with PD0325901 or trametinib and perifosine or DMF for 3 days; and LoVo/PR, LoVo/TR and Colo-205/PR cells were treated with various concentrations of PD0325901 or trametinib with or without perifosine for 3 days. A 0.4% trypan blue solution was mixed with the cell cultures and loaded into a hemocytometer. The cell survival rate represented the survival ((unstained cells)/death (stained cells)) rate on each day.

The reagents used in this study were as follows: MEK inhibitor PD0325901 (S1036) and GSK-3 inhibitor CHIR99021 (S2924) were purchased from Selleckchem (Houston, TX), ERK1/2 inhibitor SCH772984 (HY-50846) from MCE (Monmouth Junction, NJ), MG132 (M8699) from Sigma (St. Louis, MO) and Actinomycin D (HY-17559) from MCE(Monmouth Junction, NJ). The antibodies used in this study were as follows: Erk1/2 (4695), P-ERK1/2 (4370), E2F1 (3742) and SP1 (9389) from Cell Signaling Technology (Danvers, MA); DNMT1 (sc-20701) and DNMT3A (sc-20703) from Santa Cruz Biotechnology (Dallas, TX); UHRF1 (21402-1-AP) from Proteintech (Rosemont, IL); β-actin (M1210-2) from HUABIO (Cambridge, MA) and GAPDH (3B3) from Abmart (Berkeley Heights, NJ).

Doxcyclin (Dox) and Jak inhibitor (Jaki) were purchased from Merck Millipore (Billierica, MA, USA). CHIR99021 and PD0325901 were purchased from SelleckChem (Houston, TX, USA). The LIF neutralizing antibody (LIFAb) was from R&D Systems. The retro- and lenti-viral vectors including pMXs-Nanog, and FUW- M2rtTA, and the viral packaging plasmids PUMVC, psPAX2 and pCMV-VSV-G (Stewart et al., 1992 (link)) were all obtained from Addgene (Cambridge, MA, USA). FUW-TetO-Esrrb and pMXs-Stat3C were described previously (Tang et al., 2012 (link), 2014 (link)). Nr5a2 cDNA was PCR amplified using primers (forward primer: 5′-AGTTAATTAAGGATCCATGTCTTCTAATTCAGATACTGGGG-3′ and reverse primer: 5′-ACTGTGCTGGCGGCCGCTTATGCTCTTTTGGCATGCAAC-3′) and cloned into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using the In-Fusion kit (Clontech Inc., Mountain View, CA, USA). Lenti- and retro-viruses were prepared with 293T cells according to the protocol from Addgene and filtered with 0.8 μm filters.

Rhodocytin was purified from Calloselasma rhodostoma venom as previously described 29. Horm collagen was from Takeda (Munich, Germany). Crosslinked CRP was from R. Farndale (Cambridge University, UK). The anti‐phosphotyrosine mAb 4G10 was from Upstate Biotechnology (TCS Biologicals, Buckingham, UK). Anti‐phospho‐Akt (Thr308), anti‐phospho‐p38 (Thr180/182), anti‐phospho‐Syk (Tyr352), anti‐phospho‐PLCγ2 (Tyr1217) and anti‐phospho‐GSK3α/β (Ser21/9) were from Cell Signaling Technology (New England Biolabs, Hitchin, UK). Anti‐Syk, anti‐phospho‐ERK1/2 (Thr202/Tyr204) and anti‐ERK2 were from Santa Cruz Biotechnology (Heidelberg, Germany). MK2206, CHIR‐99021 and PD0325901 were from Selleck Chemicals (Stratech, Newmarket, UK). PRT‐318 was provided by Portola Pharmaceuticals (San Francisco, CA, USA). All other reagents were from Sigma‐Aldrich (Poole, UK) or from previously named sources 30.