Journal: International Journal of Biological Sciences

Article Title: Orphan Nuclear Receptor NR4A1 Promotes Proliferation and Osteogenic Differentiation of Valvular Interstitial Cells through Activation of CCND2

doi: 10.7150/ijbs.122863

Figure Lengend Snippet: CCND2, a target gene regulated by NR4A1, is capable of promoting the osteogenic differentiation of VICs. (A) Venn diagram illustrating the overlap between differentially expressed genes and genes associated with cell cycle regulation. (B) Scatter plot showing the correlation between NR4A1 and CCND2 expression levels in single cells. (C) Violin plot illustrating CCND2 expression levels in calcified valves versus normal valves. (D-E) In the differential valve tissues, the levels of CCND2, as well as the calcification markers ALP and RUNX2, were assessed and quantified by WB analysis (n = 10, each group). (F) Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) assay showing the enrichment of the CCND2 promoter region by NR4A1 in VICs under CTR and OM conditions (n = 3, each group). (G) Schematic illustration of the CCND2 gene promoter region (2 kb upstream of the transcription start site, TSS). The locations of three putative NR4A1 binding sites (Binding site1, Binding site2, Binding site3), predicted by Jaspar, are indicated, along with their respective DNA sequences. P1, P2, and P3 represent different promoter fragment plasmids. (H) Luciferase reporter assay showing relative luciferase activity in VICs transfected with the empty vector control (PGL3-Basic, which serves as a negative control with no luciferase expression) or different truncated CCND2 promoter plasmids (PGL3-P1, PGL3-P2, PGL3-P3), either with empty vector (blue) or NR4A1-overexpressing vector (red) (n = 3, each group). (I-K) Ki-67 and EdU staining of VICs under osteogenic stimulation following CCND2 knockout. (L-O) WB experiments of CCND2, RUNX2, and ALP expression in VICs following CCND2 silencing (n = 3, each group). (P-T) Following the silencing of the CCND2 gene, representative images were obtained from Alizarin Red staining and ALP staining in VICs. (n = 3, each group). Scale bar: 50 μm. Data are means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001(ANOVA with Tukey's multiple comparisons test).

Article Snippet: The following are the sources of all antibodies and reagents used in this study: runt-related transcription factor 2 (RUNX2) (CST, 8486, dilution ratio of 1:1000 for WB), RUNX2 (Santa Cruz, sc-390715, dilution ratio of 1:100 for Immunofluorescence staining), alkaline phosphatase (ALP) (MAB29092, R&D, dilution ratio of 1:1000 for WB), GAPDH (Proteintech, 60004-1-Ig, dilution ratio of 1:10000 for WB), NR4A1(CST, 3960, dilution ratio of 1:1000 for WB, dilution ratio of 1:100 for Immunofluorescence staining and immunohistochemistry staining), NR4A1 (Proteintech, 12235-1-AP, ChIP), CCND2 (abclonal, A13284, dilution ratio of 1:1000 for WB), JMJD3 (Proteintech, 55354-1-AP, dilution ratio of 1:1000 for WB, dilution ratio of 1:100 for Immunofluorescence staining), H3K27me3 (Proteintech, 91403, dilution ratio of 1:1000 for WB), CD31 (Proteintech, 66065-2-Ig, dilution ratio of 1:100 for Immunofluorescence staining), Cy3-labeled anti-mouse IgG (GB21401, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining and Immunohistochemistry staining), Cy3-labeled anti-rabbit IgG (GB21403, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining), 488-labeled anti-mouse IgG (GB25301, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining), 488-labeled anti-rabbit IgG (GB25303, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining).

Techniques: Expressing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, ChIP-qPCR, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Control, Negative Control, Staining, Knock-Out

Journal: International Journal of Biological Sciences

Article Title: Orphan Nuclear Receptor NR4A1 Promotes Proliferation and Osteogenic Differentiation of Valvular Interstitial Cells through Activation of CCND2

doi: 10.7150/ijbs.122863

Figure Lengend Snippet: Silencing of CCND2 mitigates the osteogenic differentiation phenotype induced by NR4A1 overexpression in VICs. (A-E) Knockdown of CCND2 was able to inhibit the elevation of the levels of calcification markers ALP and RUNX2 induced by overexpression of NR4A1, as demonstrated by WB (n = 3, each group). (F-H) Visual representations of Ki-67 and EdU staining in VICs when NR4A1 was overexpressed and CCND2 was silenced (n = 3, each group). (I-M) Determination of calcified nodules and ALP activity in valvular interstitial cells with CCND2 gene silencing and NR4A1 overexpression (n = 3, per group). (N-R) Western blot analysis of calcification markers ALP and RUNX2 in VICs with NR4A1 silencing and CCND2 overexpression. (S-U) Visual representations of Ki-67 and EdU staining in VICs with NR4A1 silencing and CCND2 overexpression. (V-Z) Determination of calcified nodules and ALP activity in valvular interstitial cells with NR4A1 silencing and CCND2 overexpression. (n = 3, per group). Scale bar: 50 μm. Data are means ± SD. One-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

Article Snippet: The following are the sources of all antibodies and reagents used in this study: runt-related transcription factor 2 (RUNX2) (CST, 8486, dilution ratio of 1:1000 for WB), RUNX2 (Santa Cruz, sc-390715, dilution ratio of 1:100 for Immunofluorescence staining), alkaline phosphatase (ALP) (MAB29092, R&D, dilution ratio of 1:1000 for WB), GAPDH (Proteintech, 60004-1-Ig, dilution ratio of 1:10000 for WB), NR4A1(CST, 3960, dilution ratio of 1:1000 for WB, dilution ratio of 1:100 for Immunofluorescence staining and immunohistochemistry staining), NR4A1 (Proteintech, 12235-1-AP, ChIP), CCND2 (abclonal, A13284, dilution ratio of 1:1000 for WB), JMJD3 (Proteintech, 55354-1-AP, dilution ratio of 1:1000 for WB, dilution ratio of 1:100 for Immunofluorescence staining), H3K27me3 (Proteintech, 91403, dilution ratio of 1:1000 for WB), CD31 (Proteintech, 66065-2-Ig, dilution ratio of 1:100 for Immunofluorescence staining), Cy3-labeled anti-mouse IgG (GB21401, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining and Immunohistochemistry staining), Cy3-labeled anti-rabbit IgG (GB21403, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining), 488-labeled anti-mouse IgG (GB25301, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining), 488-labeled anti-rabbit IgG (GB25303, Servicebio, dilution ratio of 1:200 for Immunofluorescence staining).

Techniques: Over Expression, Knockdown, Staining, Activity Assay, Western Blot