Ab120638

Interferon-gamma (IFN-γ, 300-02) and tumor necrosis factor alpha (TNF-α, 300-01A) were purchased from Peprotech. AMPK agonists Metformin hydrochloride (PHR1084), AICAR (A9978), Resveratrol (R5010); SIRT1 agonist SRT1720 (SRT1720) and NRF-2/HO-1 agonist Protoporphyrin IX cobalt chloride (C1900) were purchased from Sigma-Aldrich. Inhibitors of NF-κB Emodin (E7881) and JSH23 (J4455); IKKβ inhibitor IKK16 (SML1138); STAT1 inhibitor Fludarabine (F2773) and NOS2 inhibitor 1400W (W4262) were purchased from Sigma-Aldrich. Inhibitors of MEK1 and MEK2 PD98059 (ab120234), JNK SP600125 (ab120065) and MAPK SB202190 (ab120638) were purchased from Abcam. Reagents were dissolved in DMSO or water (Metformin and AICAR) at 100x stock concentrations and maximum DMSO concentration used in cells was 1%. Monoclonal antibody against 3-nitrotyrosine (ab61392) was purchased from Abcam. Monoclonal antibody against NF-κB p65 (sc-8008) was purchased from Santa Cruz Biotechnology.

After overnight serum starvation, cells were treated with dimethyl sulfoxide (DMSO; 276855), 10 μM Yoda1 (Tocris, 5586, reconstituted in DMSO) or EGF (10 ng/ml; PeproTech, 400-25, reconstituted in deionized water) for indicated durations. Experiments involving inhibitors included a 30-min pretreatment with EGFR kinase inhibitor 1 μM PD153035 (Sigma-Aldrich, SML0564), SFK inhibitor 200 nM PP2 (Sigma-Aldrich, P0042), or p38 inhibitor 10 μM SB202190 (Abcam, ab120638), all reconstituted in DMSO. All treatments were prepared reusing starvation medium.
Shear stress was delivered to cells seeded on microfluidic chambers (Ibid 80166, I Luer 0.2 mm height, polymer coating) using a peristaltic pump (Thermo Fisher Scientific, 16609762) with a starvation medium flow rate of 4 ml/min. According to the manufacturer’s instructions, shear stress (τ) is the product of flow rate (ϕ, 4 ml/min), medium’s dynamical viscosity [η ≈ 0.0073 dynes·s/cm² for serum-free DMEM (87 (link))] and a chamber-dependent correction factor (512.9), yielding ≈ 15 dynes/cm², a shear stress value shown to activate Piezo1 (9 (link), 10 (link)).