Fig. 4. TSG12 is a specific TG2 agonist in ASMCs.

(A) Kinetic analysis of TSG12 by Biacore surface plasmon resonance with TG2 immobilized on the CM5 sensor chip. The response signals were recorded for the indicated time. (B) Schematic interaction of the molecular docking of TSG12 and TG2 (Protein Data Bank code: 1WYM). (C) Comparison of control and TG2-deficient rat ASMCs challenged with acetylcholine without treatment (ACh) or treated with TB (10,000 nM) or TSG12 (50 nM) (*P < 0.05 versus ACh and #P < 0.05 versus TB, n = 4 per group). (D) Pulmonary resistance (R) in OVA-induced asthma WT and TG2-KO mice treated with vehicle (control) or TSG12 (100 ng/kg). (*P < 0.05 versus control and #P < 0.05 versus WT mice treated with TSG12, n = 8 per group). (E) TSG12 organ and serum distribution after inhalation. The distribution of TSG12 in different tissues after inhalation of TSG12 in mice (n = 3 per group) was determined by liquid chromatography-electrospray ionization tandem mass spectrometry and expressed as picograms per milligram of tissue or picograms per microliter of serum. N.D. means not detected or the concentration is lower than the limit of detection (0.1 pg/mg tissue). (F) The changed cross-sectional area (µm²) in precision-cut lung slices (PCLS) of arteries incubated with potassium chloride (KCL, 150 mM), sodium nitroprusside (SNP, 100 µM), or TSG12 (100 nM; n = 4 per group). (G) Decreased cross-sectional area (µm²) in PCLS of airways challenged with acetylcholine without treatment (ACh) or treated with isoproterenol (ISO; 100 µM); TSG12 (10 and 100 nM) or inactive control compound (CC; 100 nM). (*P < 0.05 versus ACh, n = 4 per group). (H) Western blot analyses of TSG12 on the phosphorylation and total protein of MYPT1 in rat tracheal spirals. Lower panel: Corresponding densitometric analyses showing phosphorylated/total protein (%) treated with vehicle (control) or TSG12 (25 µM). (*P < 0.05 versus control, t test; n = 3 per group). (I) Western blot analyses of TSG12 on the phosphorylation and total protein of RhoA, ROCK, MYPT1, and myosin light chain (MLC) in murine ASMCs. Right panels: Corresponding densitometric analyses showing phosphorylated/total protein (%) treated with vehicle (control) or TSG12 (25 µM). (*P < 0.05 versus control, t test; n = 3 per group). All data are expressed as means ± SEM. Statistical analyses were performed by one-way ANOVA with the Games-Howell/LSD post hoc test.