Journal: Cell Death Discovery

Article Title: KDM4B modulates ERα signaling pathway to participate in vascular smooth muscle cell calcification

doi: 10.1038/s41420-025-02765-6

Figure Lengend Snippet: A Confocal images showing the distribution of KDM4B and ERα in HASMCs after 48-h treatments in the presence of β-GP, either without or with 100 nM E2. This 48-h time point represents an early stage of calcification and was specifically chosen to examine KDM4B’s subcellular localization rather than changes in its expression levels at this stage. Cells were stained for ERα (green) and KDM4B (red), with nuclei counterstained by DAPI (blue) to indicate nuclear morphology. Scale bar = 50 μm. B KDM4B directly binds GST-ERαAF1 and GST-ERαAF2 in vitro. The GST-ERαAF1 and GST-ERαAF2 proteins were expressed in prokaryotic cells and purified using GST beads and then incubated with the lysate of HEK-293 T cells expressing HA-KDM4B. After being washed with cold PBS, the eluted complexes were subjected to western blotting and detected with specific antibodies. C HASMC cells were co-transfected with PcDNA3.1/HA-KDM4B and ERα plasmids and treated with or without estrogen. Whole-cell extracts were immunoprecipitated with the anti-HA antibody or IgG after transfection for two days. Input means 5% of the entire extract for each column. D , E Co-IP experiments show endogenous KDM4B and ERα associated with each other in HASMC cells. Reciprocal Co-IP and immunoblotting were performed with antibodies as indicated. A 5% fraction of the input cell lysate before immunoprecipitation was loaded as a control. F , G Co-IP experiments show the interaction between endogenous KDM4B and ERα in MOVAS cells. H HEK-293 cells were co-transfected with PcDNA3.1/HA-KDM4B and ERα plasmids and treated with or without estrogen (E2, 100 nM). Whole-cell extracts were immunoprecipitated with the anti-HA antibody or IgG after transfection for two days. Input means 5% of the whole extract for each column.

Article Snippet: Cell lines were co-transfected with the listed constructs according to the manufacturer’s instructions using jetPRIME TM DNA Transfection Reagent (Polyplus transfection).

Techniques: Expressing, Staining, In Vitro, Purification, Incubation, Western Blot, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Control

Journal: Cell Death Discovery

Article Title: KDM4B modulates ERα signaling pathway to participate in vascular smooth muscle cell calcification

doi: 10.1038/s41420-025-02765-6

Figure Lengend Snippet: A Western blotting of HASMCs overexpressing KDM4B following transfection with the HA-KDM4B. B , E Alizarin red staining of HASMCs. C , F Quantification of calcium deposition in HASMCs. D , G Western blotting analysis for HA-KDM4B, Runx2, BMP2, RANKL, and β-actin (used as a loading control). The KDM4Bwt or KDM4Bmut were overexpressed in HASMCs treated with β-GP and used as the control condition for each parameter. H Western blotting of MOVAS cells overexpressing KDM4B following transfection with the HA-KDM4B. I , L Alizarin red staining of MOVAS cells. J Quantification of calcium deposition in MOVAS. K Western blotting analysis and quantification of protein expression for HA-KDM4B, Runx2, RANKL, and β-actin (used as a loading control). The KDM4Bwt or KDM4Bmut were overexpressed in MOVAS cells treated with β-GP and used as the control condition for each parameter. Data were collected from 3 replicates and expressed as the mean ± SD. * p < 0.05, ** p < 0.01, and ns stands for non-significant.

Article Snippet: Cell lines were co-transfected with the listed constructs according to the manufacturer’s instructions using jetPRIME TM DNA Transfection Reagent (Polyplus transfection).

Techniques: Western Blot, Transfection, Staining, Control, Expressing