Fig 1: GRK2 overexpression reverses the inhibitory effects of ZBTB16 overexpression on LPS-induced C-28/I2 cell viability inhibition and apoptosis. The transfection efficiency of Ov-GRK2 plasmids was examined using (A) reverse transcription-quantitative PCR and (B) western blotting. (C) Cell Counting Kit-8 assay of the viability of LPS-challenged C-28/I2 cells overexpressing both ZBTB16 and GRK2. (D) TUNEL assay estimating the apoptosis of LPS-exposed C-28/I2 cells overexpressing both ZBTB16 and GRK2. Scale bar, 50 µm. Magnification, x200. (E) Protein expression levels of apoptosis-associated factors were analyzed by western blotting. ***P<0.001 vs. Control. ###P<0.001 vs. LPS. ∆∆P<0.01 and ∆∆∆P<0.001 vs. LPS + Ov-ZBTB16 + Ov-NC group. GRK2, G protein-coupled receptor kinase type 2; ZBTB16, zinc finger and BTB domain containing 16; LPS, lipopolysaccharide; Ov-GRK2, GRK2 overexpression vector; Ov-NC, empty overexpression vector; Ov-ZBTB16, ZBTB16 overexpression vector.
Fig 2: GRK2 overexpression reverses the inhibitory effects of ZBTB16 overexpression on the LPS-evoked inflammatory response and ECM degradation by C-28/I2 cells. (A) ELISA and (B) western blotting were used to measure inflammatory factor levels and expression in the cell culture supernatant of LPS-treated C-28/I2 cells. (C) Reverse transcription-quantitative PCR and (D) western blotting were used to examine the expression levels of ECM degradation-associated proteins in LPS-treated C-28/I2 cells. ***P<0.001 vs. Control. ###P<0.001 vs. LPS. ?P<0.05, ??P<0.01 and ???P<0.001 vs. LPS + Ov-ZBTB16 + Ov-NC. GRK2, G protein coupled receptor kinase type 2; ZBTB16, zinc finger and BTB domain containing 16; LPS, lipopolysaccharide; ECM, extracellular matrix; Ov-ZBTB16, ZBTB16 overexpression vector; Ov-NC, empty overexpression vector; Ov-GRK2, GRK2 overexpression vector; ADAMTS-5, a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs-5; ACAN, aggrecan; COL2A1, collagen type II a1.
Fig 3: Proposed model of the present study. The main finding of the present study was that ZBTB16 overexpression alleviated lipopolysaccharide-induced inflammation, apoptosis and degradation of the ECM in chondrocytes to suppress the development of OA by suppressing GRK2 transcription. ZBTB16, zinc finger and BTB domain containing 16; ECM, extracellular matrix; GRK2, G protein coupled receptor kinase type 2; TSS, transcription start site.
Fig 4: ZBTB16 transcriptionally suppresses GRK2 expression. (A) Cistrome DB database predicted the binding of ZBTB16 with GRK2 promoter. (B) RT-qPCR and (C) western blotting were used to examine GRK2 expression in LPS-challenged C-28/I2 cells. ***P<0.001 vs. LPS 0 µg/ml. (D) RT-qPCR and (E) western blotting were used to examine GRK2 expression following ZBTB16 overexpression in LPS-challenged C-28/I2 cells overexpressing ZBTB16. ***P<0.001 vs. Control. ##P<0.01 and ###P<0.001 vs. LPS + Ov-NC. (F) Chromatin immunoprecipitation assay was used to assess if ZBTB binds to the GRK2 promoter using the ZBTB16 antibody. ***P<0.001 vs. IgG. (G) Luciferase reporter assay of the luciferase activity of the GRK2 promoter following transfection with Ov-ZBTB16 and Ov-NC plasmids. ***P<0.001 vs. Ov-NC. (H) ZBTB16 binding site on the GRK2 promoter. ZBTB16, zinc finger and BTB domain containing 16; GRK2, G protein coupled receptor kinase type 2; RT-qPCR, reverse transcription-quantitative PCR; LPS, lipopolysaccharide; Ov-NC, empty overexpression vector; Ov-ZBTB16, ZBTB16 overexpression vector; GRK2-WT, GRK2-wild-type; GRK2-MUT, GRK2-mutant; TSS, transcription start site.
Supplier Page from Abcam for Anti-GRK2 antibody [EPR22465]