EDTA

To sort CD4⁺ T cells from the spleen cell surface staining was performed in PBS, 1% BSA (Sigma), 1 mM EDTA (AppliChem). Sorting was performed on MoFlo Astrios. Annexin V binding buffer, fluorochrome-conjugated Annexin V and 7-AAD were purchased from Biolegend and used according to manufacturer’s instructions. Briefly, cells were washed once with PBS and once with Annexin V binding buffer. Cells were stained with Annexin V (1:200 dilution) for 15 min at 4 °C in Annexin V binding buffer. Data were acquired on an Accuri C6 (BD) flow cytometer and analysed in FlowJo. The induced cell death was calculated as [(% death with CMA treatment − % death without CMA treatment)/(100 − % death without CMA treatment)]. The FACS gating strategies are shown in Supplementary Fig. 10.

Knee joints (both the femur and tibia) were isolated and fixed in 4% phosphate-buffered formalin for 48 h. Two embedding techniques were used to prepare the knee samples, which required two different methods of sample preparation: (1) for paraffin-embedded sections the samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) (AppliChem, Darmstadt, Germany) for a period of 8 weeks. Samples were then embedded in paraffin and sectioned at 5 μm thickness; (2) For Technovit® 9100 New-embedded sections (Heraeus Kulzer, Hanau, Germany) the samples were dehydrated in increasing concentrations of ethanol for 24 h prior to xylene for 12 h at room temperature (RT). Samples were subjected to pre-infiltration before the infiltration solution (see manufacturer’s instructions) was added to the samples for a 48-h incubation period at 4°C. A freshly prepared polymerization solution was used to embed the knee samples that were then evacuated at 200 mbar for 5 minutes. The polymerization process took place between −2 and −20°C and was completed within 24 h. The embedded samples were cut using a special carbide blade at 5 μm thickness. Subsequently, the mounted sections were covered by parafilm and pressed overnight (ON) at 60°C.

GA (purity ≥ 98%) was a gift from Dr. Willmar Schwabe GmbH & Co. KG (Karslruhe, Germany). Zileuton [N-(1-benzo[b]thien-2-ylethyl)-N-hydroxyurea] was from Sequoia Research Products (Oxford, UK); PGH₂ was from Larodan (Malmö, Sweden); IL-1β was from ReproTech (Hamburg, Germany); RSC-3388 was from Calbiochem (Darmstadt, Germany); EDTA and Nonidet P-40 were from AppliChem (Darmstadt, Germany); p-anisidinium chloride was from Merck (Darmstadt, Germany); Insect Express Sf9-S2 and RPMI media, glutamine, penicillin, and streptomycin were from PAA (Coelbe, Germany); Bac-to-Bac baculovirus expression system was from Invitrogen (Karlsruhe, Germany); Ni-NTA agarose was from Qiagen (Hilden, Germany). AA, Ca²⁺-ionophore A23187, dextrane, dithiothreitol, fetal calf serum, indomethacin, lipopolysaccharide (LPS), Triton X-100, and all other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), unless stated otherwise. HPLC/UPLC solvents were from VWR (Darmstadt, Germany).

Cells were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA (AppliChem), 10 mM NaF, 1% (v/v) NP40, 0.1% sodium deoxycholate, 2 μg/ml aprotinin and 200 μg/ml AEBSF). Cell extracts were cleared by centrifugation at 18,000 g for 10 min at 4 °C and the protein concentration of each sample was measured by the BCA protein assay (Pierce). Equal amount of lysates were subjected to 10% SDS – PAGE and subsequently transferred to nitrocellulose membrane (Millipore). Blots were blocked with blocking buffer for infrared immunoblotting (LI-COR #927-40000) for 1 h and co-incubated with primary antibodies against vimentin (Cell Signaling #5741) and actin (Sigma Aldrich #A5441) overnight at 4 °C. Secondary antibodies coupled to IRDye infrared dyes (LI-COR #926-32211 and #926-68070) were used for detection with infrared Odyssey imager (LI-COR). Signal quantification was performed using an ImageQuant system (GE Healthcare). Replicates from different membranes were scaled and averaged using methods described in^{63 (link)}. Vimentin blot images were acquired with both short and long exposure times. Thereby, no significant dependency of vimentin expression measurements on duration of exposure was observed.

DMEM, OptiMEM, penicillin-streptomycin, PBS and sodium pyruvate were obtained from Gibco by Life Technologies (Carlsbad, CA, United States), SYBR green MicroAmp Fast Optical 96-well Reaction Plate 0.1 ml and StepOnePlus thermocycler were obtained from Applied Biosystems (Foster City, CA, United States). FCS was purchased from Capricorn (Ebsdorfergrund, Germany). New born calf serum was purchased from Biochrom (Berlin, Germany). All primer pairs were purchased from Eurofins (Friedrichsdorf, Germany). Cell culture flasks (T75, Cell+, vented cap) and 6-well plates for sub-culturing 3T3-L1 cells were obtained from SARSTEDT (Nümbrecht, Germany). Rosiglitazone, pioglitazone, zafirlukast, montelukast, IBMX and (±)14(15)-EET-d₁₁ were purchased from Cayman Chemical (Ann Arbor, MI, United States). Insulin, dexamethasone and Oil Red O were obtained from Sigma Aldrich (St. Louis, Missouri, MO, United States). Tris, Triton-X-100, NP-40, NaCl, EDTA and SDS were purchased from AppliChem (Darmstadt, Germany).

For RhoA GTPase expression, HEK293 cells were transfected with the corresponding constructs and cultured for 16 h. For drug treatments, DIV 14 mouse hippocampal neurons were incubated with 50 μM Rho-Kinase-II-Inhibitor (Calbiochem), 50 μM bicuculline (Sigma), 50 μM GABA (Sigma) or 4 μM AMPA (Sigma). The solvent DMSO (Sigma-Aldrich, Taufkirchen, Germany) was used as a control. The following steps were performed at 4 °C: cells were washed once in PBS and harvested in PERM-lysis buffer, containing 1% Triton X-100, 50 mM NaF (Applichem, Darmstadt, Germany), 2 mM Na₃OV₄ (Applichem), 2 mM EDTA (Applichem), 10 mM β-glycerol phosphate (Sigma) and 10 mM sodium pyrophosphate (Sigma) in PBS. After incubation for 30 min, lysates were centrifuged at 1,000g for 10 min. The resulting supernatants were boiled in SDS sample buffer after adjustment of protein concentrations using a BCA assay (Pierce Biotechnology). Samples were analysed by WB. For figure displays, blots were cropped. Full scans of uncropped blots are presented in Supplementary Fig. 8.