Kg 501

LN229 and U-87MG were plated in cell culture dishes or well plates 24-48h before treatment. Chemicals were dissolved in ddH₂O or DMSO (max. 0.2%). All antagonists (MK801, memantine, ifenprodil, BAPTA-AM (Tocris, Cologne, Germany) and KG-501 (Sigma-Aldrich) were applied in presence of the agonist Glu or NMDA (Sigma-Aldrich) and glycine (Roth, Karlsruhe, Germany) (if not stated otherwise) and maintained during the whole assay. All chemicals were added prior to irradiation. X-ray irradiation was performed at 90 kV and 19 mA with an aluminum filter with single doses of 2, 4 and 6 Gy using an x-ray tube equipped with a tungsten-anode (Philips, Amsterdam, Netherlands) as described previously [55 (link)]. X-ray treatment was performed using a power of 19 mA, 90 kV voltage and 30 cm distance to the IR source, which makes an applied dose of 1.96 Gy/min, established by Ficke-dosimetry. To all radiated samples equal control samples were performed and placed in the deactivated x-ray tube for the same time.

The following compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich): 666-15 (inhibitor of CREB-mediated gene transcription; C₃₃H₃₀ClN₃O₅.HCl; CAS#: 1,433,286-70-4; TOCRIS #5661); KG-501 (Naphthol AS-E phosphate) (binds KIX-domain of CBP and prevents interaction between CREB and CBP; C₁₇H₁₃ClNO₅P; CAS#: 18,228-17-6; Sigma-Aldrich #70,485); NASTRp (Naphthol AS-TR phosphate disodium salt) (modified from Naphthol analog KG-501, binds KIX-domain of CBP and disrupts association between KIX and KID domain; C₁₈H₁₃ClNNa₂O₅P; CAS#: 4264-93-1, Sigma-Aldrich #N6125); Ro 31-8220 (Bisindolylmaleimide IX) (pan-PKC inhibitor, C₂₅H₂₃N₅O₂S·CH₄O₃S; CAS#: 138,489-18-6; Selleckchem #S7207); and BMS-754807 (IGF-IR/IR ATP antagonist; C₂₃H₂₄FN₉O; CAS#: 1,001,350-96-4; Biomol #LKT-B5072.1). The final DMSO concentration did not exceed 0.2% (v/v) for all in vitro and in vivo applications.

GLP-1, SB203580 and KG-501 were purchased from Sigma-Aldrich. Liraglutide was purchased from Novo Nordisk (Tianjin, China). LY294002 and U0126 were purchased from Cell Signaling Technology. H89 was from Adipogen Life Science. Anti-LC3A/B, anti-pERK1/2, anti-ERK1/2, anti-pAKT, and anti-AKT antibodies were purchased from Cell Signaling Technology. Anti-FNDC5 (ab174833) and anti-ATGL were from Abcam, while anti-UCP-1 and anti-β-actin antibodies were purchased from Sigma-Aldrich. HRP-conjugated secondary antibodies for western blotting were purchased from Cell Signaling Technology. Irisin-competitive ELISA kit (AG-45A-0046YEK-KI01) was from Adipogen Life Science.

Cell lines and patient derived samples were treated with the following prior to downstream assays: 50 ng/ml DKK1 (GF170, Milipore), 50 µg/ml Vantictumab (Oncomed), 100 nm Wnt3A (5036-WN, R&D systems), 100 µM LGK974 (S7143, Selleckchem), 10 ng/ml rIL15 (247-ILB, R&D systems), 10 ng/ml rIL1β (201-LB, R&D systems), 5 µg/ml IL1β neutralising antibody (MAB201, R&D systems), 5 µg/ml IL15 neutralising antibody (MAB-274, R&D systems), 5 µg/ml IL6 neutralising antibody (MAB2061, R&D systems), 5 µg/ml IL8 neutralising antibody (MAB208, R&D systems), 10 µg/ml Anakinra (Amgen, Cambridge, UK), 5 mM Sulfasalazine (Sigma), 10uM KG-501 (Sigma). Treatment times for individual assays are detailed below.

5-Fluorouracil was obtained from the Hospital Pharmacy of Lille and treatment of the different cell lines was done accordingly to previous work of the laboratory [1 (link)]. KG-501 and Verteporfin (VP) were purchased from Sigma Aldrich and cells were treated for 48 hours at micro molar concentrations. For VP treatment, cells were treaded in the dark to avoid any issue due to photosensitivity of this molecule. Cellular toxicity (CT) of VP (5 and 10 μM) was < 5% in HCT116 cells and 7% in 5F31 cells, respectively. Clonogenic assays were performed in 6 wells-plate with low density cell seeding (5,000 cells per well). After two weeks of growing, cells were fixed with 4% paraformaldehyde and stained with a 0.4% violet crystal solution. The number of colonies was counted and the experiment is represented as a percentage of Colony Forming Unit (number of colonies/number of cells seeded per well).

For oxygen-glucose deprivation (OGD), the cells were discarded from the original medium and rinsed twice with phosphate-buffered saline, glucose-free DMEM was added and then cultured in a humidified anaerobic incubator (containing 1% O₂, 5% CO₂, and 94% N₂) at 37°C to simulate the ischemic environment in vitro. In this study, HUVECs and SH-SY5Y cells were maintained under OGD conditions for 2, 4, 6, or 8 h and 0.5, 1, 2, or 4 h, respectively. The culture medium was then replaced with complete DMEM and the cells were placed in a normal incubator (containing 95% O₂ and 5% CO₂) at 37°C for reoxygenation for 12 h to mimic reperfusion in vitro. At the beginning of the reoxygenation phase, metformin (0, 5, 10, 20, 50, and 100 μM), AICAR (an AMPK agonist, 500 μM; MedChemExpress, China), Compound C (10 μM), and KG-501 (a CREB inhibitor, 25 μM; Sigma-Aldrich, United States) were added to the culture medium to treat HUVECs, and BDNF (0, 10, 20, 50, and 100 ng/ml; R&D Systems, United States) was added to treat SH-SY5Y cells in different groups. All cells were collected for later experiments at the end of the reoxygenation period.