Fig 1: IL-6-treated chondrocytes increase matrix degradation inhibited by BMAL-1. (A) MCCs transiently transfected with control plasmid or bmal1 plasmid were cultured in the presence of IL-6. Western blotting showed that IL-6-induced increases in P-ERK, MMP3, MMP9, and MMP13 were abrogated in BMAL-1-overexpressing cells, and IL-6-induced decreases in COL2 were reversed by the bmal1 plasmid. (B) Alcian blue staining results for MCCs transfected with 20 nM con plasmid or 20 nM bmal1 plasmid and stimulated with 20 ng/ml IL-6 for 12 h. (C) BMAL-1 overexpression enhanced MCC proliferation in medium containing 20 ng/ml IL-6, as determined by CCK-8 assays. All experiments were performed in triplicate, and the results are expressed as the mean ± SD. The P value is written in the figure corner. Scale bar: 50 mm.*P < 0.05.
Fig 2: BMAL-1 regulates TMJ-OA in vitro via the MAPK/ERK signaling pathway. (A) MCCs transiently transfected with control siRNA (con-siRNA) or bmal1-siRNAs were cultured in the presence of IL-6. Western blots of MCC lysates showed increased P-ERK, MMP3, MMP9, and MMP13 levels and inhibited COL2 expression in cells transfected with bmal1-siRNAs and treated with or without IL-6. (B) Alcian blue staining results for MCCs transfected with 50 nM con-siRNA or 50 nM bmal1-siRNAs in normal medium or stimulated with 20 ng/ml IL-6 for 12 h. (C) BMAL-1 knockdown inhibited MCC proliferation in normal medium, as determined by CCK-8 assays. All experiments were performed in triplicate, and the results are expressed as the mean ± SD. The P value is written in the figure corner. Scale bar: 50 μm.
Fig 3: BMAL-1 overexpression reverses TMJ-OA in rats. Ad-NC plasmid and Ad-bmal1 plasmid were injected IA into the TMJs of mice once per week for 4, 6, and 8 weeks. (A) Compared with that in rats transfected with Ad-NC-plasmid, the P-ERK, MMP3/13 and ADAMTS5 upregulation was significantly delayed in bmal1 overexpression rats. (B) qRT-PCR results indicated decreased bmal1 mRNA levels in vivo after Ad-bmal1 plasmid transfection. (C) Western blotting assay images show that CRD-induced P-ERK upregulation in cartilage was markedly abrogated in bmal1 overexpression rats. (D) The CRD-induced MMP3 mRNA upregulation in cartilage was markedly abrogated in bmal1 overexpression rats. (E) The CRD-induced MMP13 mRNA upregulation in cartilage was markedly abrogated in bmal1 overexpression rats. (F) The CRD-induced ADAMTS5 mRNA upregulation in cartilage was markedly abrogated in bmal1 overexpression rats. (G) The CRD-induced COL2 mRNA downregulation in cartilage was markedly abrogated in bmal1 overexpression rats. All experiments were performed in triplicate, and the results are expressed as the mean ± SD. *P < 0.05; **P < 0.01, ***P < 0.005. NS, not significant.
Fig 4: Expression analysis of BMAL1 and CD34. (A) ANOVA test was used for comparisons the expression of BMAL1 in glioma and normal tissues with different pathological grades. (B) A set of representative graphs displaying the number of microvessels in gliomas by labeling CD34. (C) Two-sample t-test was used for comparisons between the two groups.*p < 0.05, **p < 0.01. ***p < 0.001.
Fig 5: Circadian rhythm of NRF2 gene in the kidney. (A) Total NRF2 protein expression levels by western blot analysis. A strong circadian rhythm of total NRF2 protein expression was revealed in normal kidney. The peak of NRF2 protein expression was between ZT0 and ZT4, with a trough between ZT12 and ZT16. (B) Nuclear NRF2 protein expression levels by western blot analysis. The nuclear NRF2 protein expression in normal kidney tissue also exhibited a circadian rhythm, with an amplitude and peak phase that mirrored total NRF2 protein expression. NRF2 densitometry (mean ± SD; n=5) was normalized to β-actin or lamin B. One-way ANOVA for the effect of time, *P<0.05. (C) NRF2 protein expression levels by immunostaining. Positive expression in the cytoplasm and/or nucleus was stained brown (original magnification, x200). The nuclear NRF2 protein expression in renal tubular epithelial cells displayed clear diurnal variability. Compared with ZT0, the expression of nuclear NRF2 protein at ZT12 group was weaker. Data presented as mean ± SD, n=5. *P<0.05. (D) The CLOCK, BMAL1 and NRF2 mRNA expression by quantitative PCR. CLOCK and BMAL1 mRNA expression displayed a strong endogenous circadian rhythm, corresponding to that of NRF2 mRNA in normal kidney. Data (mean ± SD; n=5) were normalized to GAPDH. One-way ANOVA for the effect of time, *P<0.05. NRF2, nuclear factor erythroid 2-related factor 2.
Supplier Page from Abcam for Anti-BMAL1 antibody