Ehop 016

Liver tissues were harvested from hepatomegaly presented malignant Dnmt3a^–/– mice or similarly aged Dnmt3a^+/+ mice and homogenized by shearing followed by CD45 enrichment using CD45 negative selection kit (MagniSort Mouse CD45 depletion kit, Invitrogen). Magnetic bead–bound cells were assessed for percent CD45 enrichment using flow cytometry. Cell invasion and migration assay was performed as previously described (58 (link)). Briefly, transwell filters (6.5 μM pore filter; Costar) were placed in the lower chamber containing 500 μL of complete medium with or without SCF (100 ng/mL). Enriched CD45 cells (2.5 × 10⁵) from Dnmt3a^–/– liver tissues were resuspended in 500 μL RPMI media in the presence or absence of 100 nM PI3K αβ inhibitor (Bay1082439) or 0.5 μM RAC1/3 inhibitor (EHop-016; Selleckchem) or 200 nM RAC/CDC42 inhibitor (MBQ-167; MCE) and allowed to migrate toward the bottom of the top chamber. After 20 hours of incubation at 37°C, nonmigrated cells in the upper chamber were removed with a cotton swab. The migrated cells that attached to the bottom surface of the membrane were stained with 0.1% crystal violet dissolved in 0.1M borate (pH 9.0) and 2% ethanol for 5 minutes at room temperature. The number of migrated cells per membrane was counted in 10 random fields with an inverted microscope using 20× objective lens.

We used the following reagents: IMB5046, Vincristine and Vinblastine from J&K Scientific Ltd.; Colchicine from SERVA Feinbiochemica; SiR‐tubulin, SiR‐actin and Rho Inhibitor I (CT04) (C3 exoenzyme covalently linked to a cell penetrating moiety) from Cytoskeleton Inc; PF‐573228 (PF‐228), PF‐431396, TAE226, Y‐27632 2HCl, Dasatinib, Bosutinib, EHop‐016, SB203580, ML141, Paclitaxel, Epothilone B and cRGD (Arg‐Gly‐Asp) peptide from Selleck Chemicals; Blebbistatin, ML‐7, ML‐9 and Nocodazole from Sigma Aldrich; all chemicals used were of analytical grade. Following antibodies were used: p‐FAK(Tyr397)(D20B1), FAK(D2R2E), p‐MLC(Ser19)(3675), MLC(D18E2), p‐HSP27(Ser82)(D1H2F6), HSP27(D6W5V) and GEF‐H1(55B6) from Cell Signaling Technology; β‐Actin(TA‐09), FITC‐conjugated anti‐Mouse IgG, HRP‐conjugated anti‐Mouse IgG and HRP‐conjugated anti‐Rabbit IgG from Zhongshan Golden Bridge Biotechnology.

GGTI2133 was bought from Santa Cruz Biotechnology (Dallas, USA, #1217480-14-2). FTI was from Selleck (Texas, USA, #S7465). CsA was from Selleck (#S2286). NAC was from Sigma (#A7250). Necrosulfonamide (NSA, # 432531) and Necrostatin-1 (Nec-1) (Sigma #480065) were from Sigma. Nec-1s (VWR, Pennsylvania, USA, #852391-15-2), zvad-fmk (Sigma #V116), CT04 (Cytoskeleton, Denver, USA, #CT04), Ehop016 (Selleck #S7319). During the experiments, cells were pretreated with inhibitors for 30 minutes and then went for following stimulations.

Specific JNK inhibitor, SP600125 (Selleck Chemicals, Houston, TX, USA), was injected at a dose of 1 mg/kg/day i.p. for 2 weeks or 1 mg/kg twice a week for 3 months. Rac1-specific small-molecular inhibitors, NSC23766 and EHop-016 (Selleck Chemicals), were injected intraperitoneally at a dose of 2 mg/kg/day for 4 weeks.

ARPE-19 or MDMs were seeded in six-well plates the day prior to infection and challenged with DENV at a multiplicity of infection (MOI) = 1 for ARPE-19 or MOI = 3 for MDMs, in serum-free medium. Cells were incubated with the inoculum for 90 min at 37°C in 5% CO₂ with rocking of the plates every 15 min. After incubation, the inoculum was removed, and cell monolayers washed once with phosphate-buffered saline (PBS, Gibco) then cultured in complete media. For experiments with drug treatment, cells were pre-treated for 2 h with 50 µM azathioprine (Selleck Chemicals) or 1 µM EHop-016 (Selleck Chemicals) prior to infection as above, with subsequent culture of the cells in the presence of drug. Uninfected or no drug treatment (vehicle, 0.01% dimethyl sulfoxide [DMSO]) controls were performed in parallel, and samples were collected at the indicated time point post-infection (pi) for analysis.

Liver cancer cell lines SNU-387, SNU-449, SNU-475, Hep3B, HepG2 and SNU-423, and HEK293T cells were purchased from American Type Culture Collection (ATCC). Liver cancer cell lines HuH-6 and HuH-7 were purchased from RIkagaku KENkyusho/Institute of Physical and Chemical Research (RIKEN). All cell lines were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% penicillin/streptomycin (Hyclone, USA) at 37 °C with 5% CO₂ in a humidified atmosphere. To evaluate the potential influence of hypoxia, cells were cultured with 1.0% O₂ in a hypoxic chamber (Thermo Fisher Scientific, USA). MK-2206 (Selleck #S1078) and EHop-016 (Selleck #S7319) were purchased from Selleck Chemicals (USA) and dissolved in DMSO for later use. Thus, DMSO was used as vehicle control.