Figure 2.

Effect of glycogen synthase 1 (GYS1) knockdown on rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) function. shGYS1-2 was used in the subsequent experiments. (A,B) Effect of GYS1 knockdown on pro-inflammatory cytokines and matrix metalloproteinase (MMP) expression induced by TNF-α (A) or hypoxia (B). Transfected FLSs were unstimulated or stimulated with TNF-α (10 ng/ml) or cultured under normoxic (21% O₂) or hypoxic conditions (1% O₂) for 24 h. mRNA expression was determined by qRT-PCR. The data are presented as the mean ± SD of five independent experiments. (C) Cell proliferation was determined by EdU assay. Data are presented as the mean ± SD of five independent experiments (right panel). Original magnification 400×. (D,E) Effect of GYS1 knockdown on the migration (D) and invasion (E) of RA FLSs. Cells transfected with GYS1 shRNA (ShG) or scramble (Scr) were serum starved overnight, seeded in a Boyden chamber, allowed to migrate for 8 h, fixed, and stained with crystal violet. TNF-α was used as a chemoattractant. An in vitro invasion assay was performed using inserts coated with a Matrigel basement membrane matrix in Boyden chambers. The data are presented as the mean ± SD of five independent experiments. The images are representative of migration or invasion through the membrane after staining. Original magnification 200×. *P < 0.05, **P < 0.01 vs. scramble; ^#P < 0.05 vs. scramble + TNF-α or scramble + hypoxia.