Fig 1: (a) Protein expression of ACTG1, osteogenesis-related markers (OCN, Runx2, ALP), and cell cycle-related markers (Cyclin D1, c-Myc). (b) Quantitative analysis of protein expression following circ_0004496 overexpression or miR-145 mimic intervention. **p < 0.001 vs. control ***p < 0.01 vs. control. ACTG1, actin gamma 1; ALP, alkaline phosphatase; Runx2, runt-related transcription factor 2; OCN, osteocalcin.
Fig 2: Circ_0004496, miR-145, and ACTG1 mRNA expression levels in the hip capsule tissues from the AS and FNF groups were quantified via qRT-PCR. (a) Circ_0004496 was significantly upregulated in AS compared with FNF, while (b) miR-145 was downregulated. (c) ACTG1 expression was markedly increased in AS relative to FNF (n = 15, ***p < 0.001 vs. FNF). (d) ACTG1 levels were found to be positively associated with circ_0004496 levels, while (e) miR-145 levels were negatively correlated with circ_0004496 levels in AS hip capsule tissues. (f) Representative western blot bands of Runx2, ALP, OCN, and ACTG1 are shown, with (g) quantitative analysis revealing significantly higher expression of these osteogenesis-related proteins in the AS group compared to the FNF group. AS, ankylosing spondylitis; FNF, femoral neck fracture; ACTG1, actin gamma 1; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; OCN, osteocalcin; qRT-PCR, quantitative real-time polymerase chain reaction.
Fig 3: Calcification nodules formed in AS FBs across following ACTG1 overexpression or anti-ACTG1 internention. Optical density was measured at the wavelength of 510 nm. (a) Representative images of Alizarin red staining. (b) The activity of ossification was quantified by ARS staining (***p < 0.001 vs. NC). (c) Quantification of ALP activity (***p < 0.001 vs. NC). #p < 0.05 vs. ACTG1 overexpression group; $p<0.05 vs. anti- ACTG1 group. ARS, Alizarin Red S; FBs, fibroblasts; AS, ankylosing spondylitis; ACTG1, actin gamma 1.
Fig 4: (a) Representative images showing the localization of circ_0004496 and microRNA-145 in AS FBs [green: miR-145; red: circ_0004496; blue: 4’,6-diamidino-2-phenylindole (DAPI); yellow: overlap of signals]. Scale bar: 20 μm. (b) RIP assay showing association between miR-145 and circ_0004496/ACTG1. Summarized results show relative expression of miR-145 and circ_0004496 in AS FBs after different treatments (***p < 0.001 vs. anti-IgG). (c) RNA pull-down assay showing association between miR-145 and circ_0004496/ACTG1. ***p < 0.01 vs. bio-NC. RIP, RNA immunoprecipitation; FBs, fibroblasts.
Fig 5: (a, b) Potential miR-145 binding sites on circ_0004496 and ACTG1 were predicted using the Starbase and CircInteractome databases. (c, d) Dual-luciferase reporter assays were performed in AS FBs co-transfected with either miR-NC or miR-145, alongside WT or MUT constructs of circ_0004496 and ACTG1 3′-UTR. WT, wild-type; MUT, mutant; FBs, fibroblasts; ACTG1, actin gamma 1.
Supplier Page from Abcam for Recombinant Human gamma Actin protein (GST tag N-Terminus)