Mk 2206

When neonatal rat cardiomyocytes reached ∼80% confluence, growth media were changed to serum-free media for 24 h. Cells were then incubated with palmitate (Sigma–Aldrich) pre-conjugated to BSA (Sigma–Aldrich) in 0.5% FBS–DMEM, in the absence or presence of GLP1 (Abcam, Cambridge, UK) alone, or in combination of GLP1 with exendin-(9–39) (Sigma–Aldrich), a GLP1 receptor (GLP1R) antagonist or MK2206 (Selleck, Trenton, NJ, USA), an Akt inhibitor, for up to 24 h. For drug interference, cardiomyocytes were pre-treated with the indicated reagents (GLP1, exendin-(9–39), MK2206) 1 h prior to palmitate treatment. The stock solution of 5 mM palmitate bound to 10% BSA was prepared using the methods described before (Cousin et al. 2001 (link)). The final concentration of palmitate in the stock solutions was determined before and after sterile filtration with a commercially available kit (Wako Chemicals, Neuss, Germany). For control incubations, 10% BSA was also prepared.

MDCK cells were grown in Eagle’s minimum essential medium (MEM; Thermo Scientific, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, GE Healthcare, Pittsburgh, PA), 1.2% penicillin-G/streptomycin and 2 mM l-glutamine, and maintained in a humidified incubator at 37 °C with 5% CO₂. MDCK cells were seeded and incubated in a 6-well or 24-well plate overnight, and treated with COM crystals for 48 h, and then subjected to subsequent investigations. For COM-treated group, COM crystals were added to complete Eagle’s MEM medium to obtain a final concentration of 1 mM. For the pretreatment of two inhibitors, NAC (inhibitor of ROS, Sigma-Aldrich, St. Louis, MO) was added to the complete medium of MDCK cells and incubated for 2 h at the final concentration of 10 mM²⁰ (link); and MK-2206 (inhibitor of Akt, Selleck) was added to the medium and incubated for 24 h at the final concentration of 5 μM.³² (link) After pretreatment with NAC or MK-2206, MDCK cells were treated with or without 1 mM COM crystals for 48 h.

Before the recruitment assay, fibroblasts were pre-cultured with a CCR1 inhibitor (BX471, 100 nM, MedChemExpress, HY-12080A), CCR5 inhibitor (Maraviroc, 100 nM, Selleck, S2003), or Akt inhibitor (MK-2206, 2 μM, Selleck, S1078) for 48 h. During the recruitment assay, BX471, Maraviroc, or MK-2206 was added to both the upper and lower chambers.

Fully confluent fibroblasts plated in T162 flasks were treated overnight with either fresh media or fresh media supplemented with 100 ng/ml human recombinant IL-1β (PeproTech). The next morning, the cells were incubated in fresh media for 5 h, which was subsequently added to melanoma cells plated in either 6- or 12-well plates for 48 h with various inhibitors. The reagents used for these experiments were 1% DMSO (Sigma-Aldrich), 1 µM PLX4032 (Selleck Chemicals), 1 µM RAF265 (Selleck Chemicals), or 0.5 µM both PLX4032 and selumetinib (Selleck Chemicals). When the duotherapy treatment (PLX4032 and selumetinib) was also used in combination with either MK-2206, SB 225002, Bay 11-7082, or obatoclax, the concentration of each drug used was: 1 µM MK-2206 (Selleck Chemicals), 0.5 µM SB 225002 (Alfa Aesar), 0.2 µM Bay 11-7082 (Sigma-Aldrich), and 0.2 µM obatoclax (Selleck Chemicals). These concentrations were also used when these inhibitors were used as single agents. For each drug treatment, melanoma cells were cultured in nonconditioned media, conditioned media taken from unstimulated fibroblasts, or conditioned media taken from fibroblasts previously stimulated with IL-1β. Then, cell survival was assayed by crystal violet staining (outlined in the next section).

CRLF1-overexpressing IHH-4 cells were grown as described above until they reached 40% confluence. Then, the media were replaced with RPMI-1640 containing vehicle (0.1% (v/v) DMSO), U0126 (10 μM) (S1102, Selleck, Houston, TX), MK-2206 (10 μM) (S1078, Selleck, Houston, TX), or U+M (10 μM U0126+10 μM MK-2206). Samples were collected at different time intervals as indicated for each experiment.

Normal PC12 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (Shanghai) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma, USA) supplemented with 5% heat-inactivated fetal calf serum (FCS, Sigma, USA), 10% heat-inactivated horse serum, penicillin (50 U/mL), and streptomycin (50 mg/L). PC12 cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ and 95% air.
PC12 cells were subjected to OGD-R as previously described [22 (link)]. Briefly, the original medium was removed, and the cells were washed with Earle’s balanced salt solution (EBSS) at pH 7.4 and placed in fresh glucose-free EBSS supplemented with Na₂S₂O₄. The culture dishes were then introduced in a mixture of 5% CO₂ and 95% air at 37°C for 2 h, after which the medium was replaced with fresh DMEM for another 24 h. The control culture was maintained in normal EBSS and incubated under normal conditions. The cells were divided into five groups: the control group; the OGD-R group; the OGD-R+rLj-RGD3 group, the OGD-R+MK-2206 (5 μM, Selleck, USA) group and the OGD-R+MK-2206+rLj-RGD3 group.