Ro 106 9920

N-p-tosyl-L-phenylalanine chloromethyl ketone (TPCK, # T4376), N-α-tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK, #90182), suberoylanilide hydroxamic acid (SAHA, #SML0061), anacardic acid (#A7236), decitabine (#A3656), lipopolysaccharide (LPS from Escherichia coli O111:B4, #L4391) were purchased from Sigma-Aldrich. Carfilzomib (#S2853) and tocilizumab (#A2012) were purchased from Selleckchem. Ro-106-9920 (#1178), TPCA-1 (#2559), PS1145 (#4569), Bay11-7082 (#1744), Ruxolinitib (#7048) and Stattic (#2798) were purchased from TOCRIS bioscience. TAK-242 (# 614316) was purchased from Millipore. Etanercept was supplied by Pfizer.

Primary microglia were isolated from the astrocytic monolayer DIV 14 and 21 by gentle agitation based on the distinct adhesive features of microglia and astrocytes and were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Grand Island, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM l-glutamine and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO₂-containing atmosphere. The harvested cells were plated onto poly-l-lysine-coated dishes or coverslips, and ~99% of cells were IbaI-positive as determined by immunocytochemical staining. Flow cytometry analysis showed that 99.70 ± 0.03% and 99.50 ± 0.05% of WT and R6/2 microglia were CD11b-positive cells, respectively. For experimentation, primary microglia were seeded at 7.5 × 10⁴ cells/well in 24-well plates or 2.0 × 10⁵ cells/well in six-well plates for either transduction, treatment, or cytokine release analyses. All experimental treatments were performed 1 or 2 days after seeding. All comparative experiments between HD and WT groups have been carried out using microglia isolated and cultured simultaneously and under the same conditions. Cells were treated with BAY11-7082 (sc-200615, Santa Cruz, USA), Ro 106-9920 (1778, Tocris Bioscience, USA), and MCC950 (CP-456773, Selleckchem, USA) in respective experiments. TD139 was synthesized and characterized as detailed elsewhere^{33 (link)}.

The following reagents were used: cyclopiazonic acid (CPA, 50 μm, Tocris Bioscience, Bristol, UK); 2‐aminoethoxydiphenylborane (2APB, 100 μm, Tocris); U73122 (2–4 μm, Tocris, Abingdon, UK); U73343 (2–4 μm, Tocris); Edelfosine (ET‐18‐OCH3, 2–4 μm, Tocris); Wortmannin (1 μm, Tocris); Pertussis toxin (PTX, 1 μg·mL⁻¹, Tocris); and 6‐(phenylsulfinyl)tetrazolo[1,5‐b]pyridazine (Ro 106‐9920, 0.01–100 μm, Tocris), Xestospongin D (Xesto, 5 μm, Tocris).