Journal: Mechanisms of ageing and development

Article Title: Long-term caloric restriction ameliorates T cell immunosenescence in mice.

doi: 10.1016/j.mad.2022.111710

Figure Lengend Snippet: Fig. 7. Effects of caloric restriction on expression of T cell exhaustion-associated transcription factors in the spleen. Spleen cells were isolated from groups of mice fed and sacrificed according to the protocols described in Fig. 1. Cells were stained with antibodies against CD4, CD8, NR4A1, and TOX and the proportions of NR4A1+ (A) and TOX+ (B) T cells were analyzed by flow cytometry. Data are the means ± SE of n = 7 (Young, CR, and sCR) or n = 6 (CON) mice per group. * P < 0.05 vs CON by ANOVA followed by Dunnett’s test.

Article Snippet: For the detection of intracellular NR4A1 and TOX, splenocytes were incubated with FITC-anti-CD4 or APC-anti-CD8 mAbs, fixed and permeabilized as described above, and incubated with PE-antiNR4A1 (REA704; Miltenyi Biotec, San Diego, CA, USA) or PE-antiTOX (TXRX10, Invitrogen) mAbs.

Techniques: Expressing, Isolation, Staining, Flow Cytometry

Journal: Cell Reports

Article Title: NR4A Nuclear Receptors Target Poly-ADP-Ribosylated DNA-PKcs Protein to Promote DNA Repair

doi: 10.1016/j.celrep.2019.01.083

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-NR4A1 , BosterBio , Cat# PB9766.

Techniques: Recombinant, SYBR Green Assay, Fractionation, Luciferase, Stable Transfection, Expressing, Plasmid Preparation, Software

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: METTL3 inhibits NR4A1 expression via m 6 A-dependent manner. A , B Direct RNA-seq analysis of the m 6 A abundance and sites in shR-NC and shR-METTL3 HeLa cells. C Volcano plot displaying the differentially expressed genes base on direct RNA-seq. D Venn diagram showed the candidate target gene from TCGA, GSE39001 and Direct RNA-seq. E , F RT-qPCR and western blot assays of the mRNA ( E ) and protein ( F ) expression of NR4A1 in CC cells with overexpression and knockdown of METTL3. G m 6 A RIP-qPCR analysis of NR4A1 mRNA in control and METTL3-silenced HeLa cells. H, The stability of NR4A1 mRNA in control and METTL3-silenced HeLa cells. Transcription was inhibited by act-D at 5 µg/ml during indicated times. I Left: Cells were incubated with 100 µg/ml CHX at the indicated times, the protein stability of NR4A1 was detected by western blot assay. Right: Quantification of protein stability. J Co-IP assay for the interactions between METTL3 and NR4A1. K Schematic depiction of mutated (GG A CA to GG C CA) m 6 A site in CDS region in NR4A1. L RT-qPCR analysis showed the expression of EGFP from cells transfected with the indicated plasmids, and normalized to the corresponding β-actin (left) and RFP (right) values. All experiments were performed at least 3 independent times, and data are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Western Blot, Over Expression, Incubation, Co-Immunoprecipitation Assay, Transfection

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: NR4A1 suppresses CC cells progression. A The mRNA expression of NR4A1 in CC were analyzed by GENT2 and GSE39001 datasets. For violin polts, midlines indicate the median, upper and lower lines indicate the first and third quartiles. B Representative images of IHC staining showed the protein levels of NR4A1 in CC tissues and paired adjacent nontumor tissues (n = 8, left) and quantitatively analyzed (right). C MTT assay showed the proliferative ability of CC cells. D The effect of NR4A1 on cell migration and invasion in CC cells. E The proliferative ability was investigated by colony formation. F Western blot assay of N-cadherin, Vimentin and E-cadherin protein levels in overexpression and knockdown of NR4A1 CC cells. All experiments were performed at least 3 independent times, and data are presented as means ± SD expect where otherwise specified. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Expressing, Immunohistochemistry, MTT Assay, Migration, Western Blot, Over Expression

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: YTHDF2 sufficient to mediate the degradation of NR4A1 mRNA via m 6 A-dependent pathway. A Left: Overlap of YTHDF target genes identified by published RIP–seq and PAR-CLIP-seq in HeLa cells. Right: Venn diagram showed the candidate targets (PAR-CLIP + RIP targets) among all YTHDF paralogs. B The levels of YTHDF paralogs in TCGA-CC cohorts. Midlines indicate the median, upper and lines indicate the first and third quartiles. C The NR4A1 mRNA expression upon silencing YTHDF paralogs were analyzed by GSE134380 dataset. D-E, NR4A1 mRNA ( D ) and protein ( E ) levels were measured by RT-qPCR and western blot assay in HeLa cells. F Protein levels of NR4A1 in CC cells with overexpression wild-type (WT) or m 6 A recognition defective YTHDF2 (W432A and W486A) plasmids. G RIP-qPCR assay of the NR4A1 mRNA enrichment by YTHDF2 in overexpression YTHDF2 and control HeLa cells. H The stability of NR4A1 mRNA and non-methylated mRNA (PPP1R3C) upon the silencing of YTHDF2 were determined by RT-qPCR after treatment with act-D during indicated time points. I Left: The protein stability of NR4A1 was examined by western blot assay after treatment with CHX during indicated time points. Right: Quantification of protein stability. J The interaction between YTHDF2 protein and NR4A1 protein was measured via Co-IP assay. K The relative EGFP levels of co-transfected with the indicated plasmids, and normalized to the level of β-actin (left) and RFP (right) mRNA. All experiments were performed at least 3 independent times, and data are presented as means ± SD expect where otherwise specified. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Methylation, Co-Immunoprecipitation Assay, Transfection

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: YTHDF2 recruits DDX6 to promote NR4A1 mRNA decay. A , B KEGG and GO pathway enrichment analysis of the high-confidence interacting proteins with YTHDF2. C Overlap of RNA degradation related genes and cytoplasmic mRNA processing body related genes as identified by A and B . D The interaction of YTHDF2 protein and DDX6 protein via Co-IP assay. E Confocal microscopy images indicated that YTHDF2 (red) were colocalized with P-bodies marker DDX6 (green). F-G, The NR4A1 mRNA ( F ) and protein ( G ) were determined by RT-qPCR and western blot assays upon silencing DDX6 in HeLa cells. H The mRNA stability of NR4A1 mRNA in HeLa cells after treatment with act-D during indicated time points in shR-DDX6 and shR-NC HeLa cells. I RIP-qPCR was used to determine the enrichment of NR4A1 in shR-NC and shR-YTHDF2 HeLa cells by using a DDX6-antibody. J The effect of YTHDF2 on DDX6 protein expression via western blot assay. K, RIP-qPCR was performed to measure the enrichment of NR4A1 in DDX6 knockdown and control cells using a YTHDF2-antibody. All experiments were performed at least 3 independent times, and data are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Co-Immunoprecipitation Assay, Confocal Microscopy, Marker, Quantitative RT-PCR, Western Blot, Expressing

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: NR4A1 attenuates malignant progression that induced by YTHDF2 in CC cells. A GEO database (GSE7410 and GSE63514) and GENT2 database of YTHDF2 mRNA in CC cohorts. The three lines inside the violin plots indicate the first quartile, median and third quartile. B Representative images of IHC staining for YTHDF2 protein from CC tissues and matched adjacent non-tumor tissues (n = 8, left) and quantitatively analyzed (right). C Transwell migration and invasion assays in CC cells expressing the specified plasmids. D , E The MTT (D) and colony formation assays ( E ) showed the cell proliferative ability with transfected the indicated plasmids in CC cells. F Western blot assay showed that the expression of N-cadherin, Vimentin and E- cadherin with transfected the indicated plasmids in CC cells. All experiments were performed at least 3 independent times, and data are presented as means ± SD except where otherwise specified. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Immunohistochemistry, Migration, Expressing, Transfection, Western Blot

Journal: Cell & Bioscience

Article Title: RNA N 6 -methyladenosine modification mediates downregulation of NR4A1 to facilitate malignancy of cervical cancer

doi: 10.1186/s13578-022-00937-w

Figure Lengend Snippet: YTHDF2-NR4A1 axis meditates the transcription repression of AKT1 by recruiting LHC complex. A YTHDF2 was positively correlated with AKT1 in GEPIA2 database. B The mRNA levels of AKT1 were detected by RT-qPCR with overexpression and knockdown of YTHDF2. C Western blot assay was used to detect the alteration of AKT and AKT phosphorylation after overexpression and knockdown of YTHDF2 in CC cells. D RIP assay of the enrichment of AKT1 transcripts by YTHDF2. E RNA remaining for AKT1 in HeLa cells transfected with control and YTHDF2 knockdown were determined by RT-qPCR after treated with act-D at indicated times. F GSEA identified a significant association between NR4A1 and AKT pathway. G Western blot showed the protein levels of AKT1 and p-AKT1 after overexpression and knockdown of NR4A1 in CC cells. H Co-IP assay was performed to analyze the interaction between NR4A1 protein and SP1 protein. I – K The analysis of SP1-bound site of AKT1 promoter. L The EGFP reporter assay was preformed to measure the EGFP levels by co-transfected with the indicated plasmids. β-actin (left) and RFP (right) were used for normalization. M Binding of NR4A1 to LSD1, CoREST and HDAC1 in HeLa cells as shown by Co-IP assay. N ChIP assay of AKT1 promoter binds of NR4A1 and components of LHC complex. O The mRNA and protein expression were measured by RT-qPCR (left) and western blot assay (right) after transfected of shR-LSD1, shR-CoREST and shR-HDAC1, respectively, upon a NR4A1-overexpression background. P All findings in this study are presented as a schematic diagram. All experiments were performed at least 3 independent times, and data are presented as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: The primary antibodies used in this study were anti-β-actin (immunoway), anti-NR4A1 (immunoway), anti-METTL3 (abcam), anti-YTHDF1(Saier Biotechnology), anti-YTHDF2 (abcam), anti-YTHDF3(Saier Biotechnology), anti-AKT1 (Saier Biotechnology), anti-p-AKT1(S473) (Wanlei Biotechnology), anti-DDX6 (Saier Biotechnology), anti-LSD1 (Saier Biotechnology), anti-CoREST (Saier Biotechnology), anti-HDAC1 (Saier Biotechnology), anti-EGFP (Saier Biotechnology), anti-Vimentin (abcam), anti-N-Cadherin (abcam), anti-SP1 (abcam), anti-E- Cadherin (abcam) and anti-Flag (MBL). β-actin was the internal reference.

Techniques: Quantitative RT-PCR, Over Expression, Western Blot, Transfection, Co-Immunoprecipitation Assay, Reporter Assay, Binding Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Deciphering oligomeric proanthocyanidins’ dual osteoprotective mechanisms at single-cell resolution: NR4A1 -mediated PTGS2 suppression and β-catenin- Runx2 activation

doi: 10.3389/fimmu.2025.1679987

Figure Lengend Snippet: Single-cell atlas of MSC subtypes in osteopenia. (A) Circular plot illustrated the clustering of three MSC subtypes in osteopenia, with contour curves depicting the distribution of each subtype. The outer, middle, and inner axes represented the log-scaled clusters, groups, and cell cycle phases of each subtype, respectively. (B) The bubble plot displayed the mean expression levels of the top 5 DEGs in each MSC subtype. The bubble size corresponded to the percentage of gene expression, while the color represented normalized data. (C) UMAP plots illustrated the distribution of signature genes across three MSC subtypes. Contour density lines were overlaid to highlight regions with higher gene expression intensity. (D) The word cloud graphs presented the activity of different pathways in each MSC subtype. (E) The heatmap demonstrated the enrichment analysis results of the top 5 GO-BP terms for the three types of cells. (F) The GSEA enrichment analysis revealed the GO-BP terms related to the DEGs in C2 NR4A1 + MSCs. *** P < 0.001. (G) The heatmap revealed the metabolism-related pathways enriched in different MSC subtypes and C4 NR4A1 + MSCs.

Article Snippet: Immunoprecipitation was performed using anti- NR4A1 antibody (Cell Signaling Technology (CST), Danvers, MA, USA, Cat. No. 13851) or normal IgG as a negative control.

Techniques: Expressing, Gene Expression, Activity Assay

Journal: Frontiers in Immunology

Article Title: Deciphering oligomeric proanthocyanidins’ dual osteoprotective mechanisms at single-cell resolution: NR4A1 -mediated PTGS2 suppression and β-catenin- Runx2 activation

doi: 10.3389/fimmu.2025.1679987

Figure Lengend Snippet: Cell–cell communication atlas. (A) The circle plot displayed the number of interactions (left) and interaction strength (right) between C2 NR4A1 +MSCs, as sources and targets, and other cells. (B) The heatmap described the relative strength of various signaling pathways in the outgoing and incoming signaling patterns of three MSC subtypes and three other cell types. (C) The heatmap quantified the communication probabilities between three MSC subtypes and three other cell types within the FGF signaling network. (D) The heatmap depicted the centrality scores of the FGF signaling pathway network. (E) The violin plot visualized the expression levels of key ligand and receptor in the FGF signaling pathway across three MSC subtypes and three other cell types. (F) The hierarchical plot depicted the interactions between three MSC subtypes and three other cell types within the FGF signaling pathway network. (G) The circle plot described the interactions between three MSC subtypes and three other cell types within the FGF7–FGFR1 signaling network.

Article Snippet: Immunoprecipitation was performed using anti- NR4A1 antibody (Cell Signaling Technology (CST), Danvers, MA, USA, Cat. No. 13851) or normal IgG as a negative control.

Techniques: Protein-Protein interactions, Expressing

Journal: Frontiers in Immunology

Article Title: Deciphering oligomeric proanthocyanidins’ dual osteoprotective mechanisms at single-cell resolution: NR4A1 -mediated PTGS2 suppression and β-catenin- Runx2 activation

doi: 10.3389/fimmu.2025.1679987

Figure Lengend Snippet: Transcriptional regulatory networks and OPC response characteristics in MSC subtypes. (A) The UMAP plot on the left visualized the clustering distribution of three MSC subtypes based on TF activation levels, while the UMAP plots on the right highlighted each MSC subtype’s distribution separately. (B) The heatmap displayed the two regulatory modules determined based on pySCENIC regulatory rules and AUCell similarity scores. (C) Bar plots provided the AUC scores of TFs across different MSC subtypes within the two regulatory modules. (D) UMAP plots depicted the differential expression distribution of TFs within the two regulatory modules. (E) Scatter plots provided the ranking of regulon activity score for different MSC subtypes within the two regulatory modules. (F) The heatmap demonstrated the top 5 TFs of each MSC subtype. (G) The heatmap displayed the expression of 10 target genes of OPC across different MSC subtypes. (H, I) Bar plots and UMAP plots visualized the expression levels and distribution of two target genes of OPC in C2 NR4A1 + MSCs, respectively.

Article Snippet: Immunoprecipitation was performed using anti- NR4A1 antibody (Cell Signaling Technology (CST), Danvers, MA, USA, Cat. No. 13851) or normal IgG as a negative control.

Techniques: Activation Assay, Quantitative Proteomics, Activity Assay, Expressing

Journal: Frontiers in Immunology

Article Title: Deciphering oligomeric proanthocyanidins’ dual osteoprotective mechanisms at single-cell resolution: NR4A1 -mediated PTGS2 suppression and β-catenin- Runx2 activation

doi: 10.3389/fimmu.2025.1679987

Figure Lengend Snippet: OPC suppresses PTGS2 expression in MSCs via inhibition of NR4A1 transcriptional activity. (A) The CCK-8 assay showing the effects of various concentrations of OPC (1–40 μM) on MSC viability. (B) Representative images of crystal violet-stained colonies formed by MSCs treated with increasing concentrations of OPC. (C, D) Western blot and quantification showing reduced protein levels of PTGS2 and NR4A1 upon OPC treatment. (E) RT-qPCR results confirming the downregulation of PTGS2 and NR4A1 mRNA in OPC-treated MSCs. (F) Representative images of wound healing assays demonstrating enhanced migratory ability of MSCs upon OPC exposure. (G) Quantification of colony number showing a peak at 20 μM OPC, followed by a decline at 40 μM. (H) Schematic representation of the PTGS2 promoter indicating three NR4A1 -binding sites (TFBS1–3) and their mutated sequences. (I) Western blot confirming NR4A1 overexpression in MSCs. (J) RT-qPCR showing increased PTGS2 transcription upon NR4A1 overexpression ( P < 0.01). (K) ChIP-qPCR demonstrating NR4A1 binding to the PTGS2 promoter, which is reduced by OPC or anti- NR4A1 treatment. Data are presented as mean ± SEM. ** P < 0.01, *** P < 0.001 versus control; ## P < 0.01 versus the OP group.

Article Snippet: Immunoprecipitation was performed using anti- NR4A1 antibody (Cell Signaling Technology (CST), Danvers, MA, USA, Cat. No. 13851) or normal IgG as a negative control.

Techniques: Expressing, Inhibition, Activity Assay, CCK-8 Assay, Staining, Western Blot, Quantitative RT-PCR, Binding Assay, Over Expression, ChIP-qPCR, Control

Journal: Frontiers in Immunology

Article Title: Deciphering oligomeric proanthocyanidins’ dual osteoprotective mechanisms at single-cell resolution: NR4A1 -mediated PTGS2 suppression and β-catenin- Runx2 activation

doi: 10.3389/fimmu.2025.1679987

Figure Lengend Snippet: OPC attenuates OVX-induced bone loss via a β-catenin-dependent NR4A1 – Runx2 signaling axis. (A–C) Micro-CT analysis showing bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) in femurs of sham, OVX, and OPC-treated mice. (D) Quantification of TRAP + osteoclast numbers. (E, F) Serum CTX and PINP levels assessed by ELISA. (G) Western blot of PTGS2 , NR4A1 , RANKL, OPG, Runx2 , and OCN in femurs. (H) RT-qPCR analysis of corresponding gene expression. (I) Co-immunoprecipitation showing interaction between Flag- NR4A1 and His- Runx2 in β-catenin fl/fl and Osx-Cre-β-catenin fl/fl MSCs. (J) Protein expression of target genes in femurs from β-catenin-deficient mice treated with OPC. (K, L) ELISA analysis of serum PINP and CTX levels in β-catenin fl/fl versus Osx-Cre-β-catenin fl/fl mice. Data are shown as mean ± SEM. * P < 0.05, ** P < 0.01 versus indicated groups; ## P < 0.01 versus OVX + vehicle.

Article Snippet: Immunoprecipitation was performed using anti- NR4A1 antibody (Cell Signaling Technology (CST), Danvers, MA, USA, Cat. No. 13851) or normal IgG as a negative control.

Techniques: Micro-CT, Enzyme-linked Immunosorbent Assay, Western Blot, Quantitative RT-PCR, Gene Expression, Immunoprecipitation, Expressing

Journal: Advanced Science

Article Title: Stress‐Induced Activation of Prolactin‐NR4A1‐Midkine Axis Exacerbates Skin Inflammation

doi: 10.1002/advs.202509679

Figure Lengend Snippet: Stress modulates fibroblasts via PRL‐NR4A1 signaling. A) Cell divergence estimation (upper panel) and Nr4a1 expression (lower panel) by Dynamo. B) RNA velocity (Dynamo) estimation of fibroblast fate decision in the four study groups. C) Prediction of the fibroblast fate redirection after in silico knockout of Nr4a1 . D) Representative immunofluorescent labeling of NR4A1 in human psoriasis vs healthy controls. E) Representative immunofluorescent labeling of Nr4a1 in the four mouse groups. F) Representative cell immunofluorescent labeling of p‐NR4A1 and pan‐NR4A1 in human fibroblasts after PRL treatment. G) Western blot of the p‐NR4A1 expression levels in nuclear and cytosolic fractions from human fibroblasts treated with PRL at different time points. H) Western blot of the p‐NR4A1 expression levels in human fibroblasts treated with PRL at different time points. I, J) Western blot analysis (I) and QRT‐PCR (J) of MDK expression after NR4A1 knockdown in human primary fibroblasts ( n = 3). K) Psoriasis‐related inflammatory gene expression in keratinocytes treated with conditioned medium (CM) from fibroblasts with or without siNR4A1 transfection ( n = 3). The hashtag indicates the P value before PRL stimulation, and the asterisk indicates the P value after PRL stimulation. L,M) Volcano plots (left) and bar plots (right) showing gene expression differences and enriched pathways in fibroblasts with or without siNR4A1 transfection, before (L) and after PRL stimulation (M) ( n = 3). All results are shown as the means ± SD. * p < 0.05, ** p < 0.01, ### p < 0.001, **** p < 0.0001, ns, not significant (J and K: two‐way ANOVA). Scale bar, 50 µm. CM: conditioned medium; Con: Control; CRS: chronic restraint stress; IMQ: imiquimod; MDK: midkine; p‐NR4A1: phosphorylated forms of NR4A1; PRL: prolactin; Veh: vehicle.

Article Snippet: The membrane was subsequently blocked with 5% BSA in PBS and probed with primary antibodies: PRLR (1:1000, Abcam), NR4A1 (1:1000, Proteintech), p‐NR4A1 (1:1000, Cell Signaling Technology), MDK (1:1000, Santa Cruz Biotechnology).

Techniques: Expressing, In Silico, Knock-Out, Labeling, Western Blot, Quantitative RT-PCR, Knockdown, Gene Expression, Transfection, Control

Journal: Advanced Science

Article Title: Stress‐Induced Activation of Prolactin‐NR4A1‐Midkine Axis Exacerbates Skin Inflammation

doi: 10.1002/advs.202509679

Figure Lengend Snippet: Overexpression of NR4A1 primes fibroblasts toward a hyperinflammatory phenotype. A) Prediction of the fibroblast fate redirection after in silico overexpression of Nr4a1. B) Western blot analysis of MDK expression in NR4A1 ‐overexpressing fibroblasts. C) Relative gene expression levels of inflammatory profiles in NR4A1 ‐overexpressing fibroblasts ( n = 8–9). D) PCA data of primary human fibroblasts with and without NR4A1 overexpression. E) Volcano plot of DEGs in fibroblasts with or without NR4A1 overexpression ( n = 3). F) Enriched pathways in NR4A1‐overexpressing fibroblasts. Count indicates the number of genes enriched in corresponding pathways. G) Description of the NR4A1‐binding sites in the MDK (up) or PRLR (down) promoter (left panel). Primary fibroblasts transfected with lentivirus encoding e‐GFP or Flag‐n‐ NR4A1 for ChIP assay with antibodies against NR4A1 (right panel) ( n = 3). H) Effects of NR4A1 overexpression on the wild‐type or sequence variant promoter activity of the MDK or PRLR gene in transfected fibroblasts ( n = 3). All results are shown as the means±SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns, not significant. (C and G: unpaired Student's t test; H: two‐way ANOVA). IMQ: imiquimod; MDK: midkine; OE: overexpression; PCA: principal components analysis; p‐NR4A1: phosphorylated forms of NR4A1; PRL: prolactin; sv: sequence variant; Veh: vehicle.

Article Snippet: The membrane was subsequently blocked with 5% BSA in PBS and probed with primary antibodies: PRLR (1:1000, Abcam), NR4A1 (1:1000, Proteintech), p‐NR4A1 (1:1000, Cell Signaling Technology), MDK (1:1000, Santa Cruz Biotechnology).

Techniques: Over Expression, In Silico, Western Blot, Expressing, Gene Expression, Binding Assay, Transfection, Sequencing, Variant Assay, Activity Assay