Glx351322

To detect ROS levels, FLS were stained with CellROX or MitoSOX™ Red mitochondrial superoxide indicator (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. To determine mitochondrial membrane potentials, TMRM (Tetramethylrhodamine, Methyl Ester, Perchlorate; Invitrogen, Thermo Fisher Scientific) was used according to the manufacturer’s instructions. FLS were analyzed on a FACSCanto2 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data were processed using FlowJo software v 10.8 (Tree Star, Ashland, OR, USA).
GLX351322 (MedKoo Biosciences, Morrisville, NC, USA) was used to inhibit NOX4 in these cellular processes, and MitoTEMPO (Sigma-Aldrich) was used as a specific scavenger of mitochondrial superoxide. Human recombinant TNF-a (10 ng/mL) and IL-17 (10 ng/mL) were obtained from PeproTech (Cranbury, NJ, USA). A769662 (Sigma-Aldrich) was employed for activating AMPK-mediated cellular signaling.

FLS were cultured in serum-free media for 5 h. Following pre-incubation with or without NOX4 inhibitor (GLX351322, MedKoo Biosciences) for 1 h, RA FLS were stimulated with recombinant IL-17 (10 ng/ml, Peprotech) and TNF-α (10 ng/ml, Peprotech) for 1 h. For the transwell migration assay, cells were centrifuged and loaded onto transwell filters with an 8-μm pore (Corning) positioned on top of the migration chamber for 23 h. DMEM containing 10% FBS was transferred to the bottom chamber of the transwell plate as a chemoattractant. For the invasion assay, transwell filters were pre-incubated with Matrigel (Corning) at 37 °C for 1 h. Then, transwells were incubated at 37 °C for 3 days, fixed with 100% methanol, and stained with crystal violet (Sigma-Aldrich). Non-migrating cells on the top membrane surface were removed by washing with PBS and cotton swabs. Invaded cells were counted in five random fields per sample under an inverted microscope (magnification, × 100; 0.55 numerical aperture dry objective; scale bar, 100 μm; Olympus). For quantification, the crystal violet dye was eluted with 0.1% sodium dodecyl sulfate (SDS) and quantitated using a Sunrise absorbance reader (Tecan) at 595 nm.