Disease modification and symptom relief in osteoarthritis using a mutated GCP‐2/CXCL6 chemokine

A

3D homology model of human GCP‐2 based on the NMR structure of human CXCL5 dimer (PDB: 2MGS) generated using SWISS‐MODEL. The GCP‐2 monomers are colored blue and green with Lys100, Lys101 and Lys105 shown in space filling representation for the latter.

B

Comparison of 1D NMR spectra of WT and mutant GCP‐2 proteins (all human) made in this study.

C

SDS–PAGE analysis of GCP‐2 WT and mutants under reducing (R) and nonreducing (NR) conditions. Protein samples (1 μg) were either reduced and alkylated (R) or alkylated (NR). MW size from protein markers is displayed on the left. WT—wild‐type GCP‐2; K101E—K101E GCP‐2_single mutant; K105E—K105E GCP‐2_single mutant; D—K101E_K105E GCP‐2_double mutant; T—K100E_K101E_K105E GCP‐2_triple mutant.

D, E

Dose‐dependency of WT GCP‐2‐induced (D) chemotaxis and (E) and transendothelial migration (TEM) of CXCR2‐expressing 300‐19 pre‐B cell line (mean values ± SEM). N = 4.

F

Representative images of immunostaining for the Ly6G neutrophil marker in the intercondylar notch of mice injected intra‐articularly with GFP, GCP‐2, or GCP‐2‐T adenovirus. Time point: 2 days. White arrowheads indicate neutrophils. Scale bar—50 μm. F—femur; CL—cruciate ligament.

G

Average thickness of the synovial membrane 4 days after the intra‐articular injection of adenovirus encoding GFP, GCP‐2 or GCP‐2‐T. The thickness of the synovial membrane was assessed by histomorphometry using ImageJ. P‐values were determined with ANOVA n = 4 per group.

H

Caliper measurement of knee size 4 days after the injection of GFP, GCP‐2 and GCP‐2‐T adenovirus; P‐values were determined by one‐way ANOVA (n = 4).

Figure EV2. GCP‐2 model and characterization of WT and mutants.