Journal: Journal of Translational Medicine

Article Title: CEBPD-mediated SGPP2 upregulation via PERK/ER stress in endothelial cells disrupts S1P homeostasis and impairs angiogenesis in chronic endometritis

doi: 10.1186/s12967-025-07558-0

Figure Lengend Snippet: S1P exerts function via the S1PR1-STAT3/-VEGFA pathway. ( A ) qPCR detection of relative mRNA expression levels of S1PR1-5 in HUVECs. ( B ) Immunofluorescence staining of S1PR1 expression in HUVECs, (S1PR1: green, DAPI: blue). ( C ) Tube formation assay assessing the impact of S1PR1 inhibition on LPS + S1P-restored angiogenesis: HUVECs treated with S1PR1 inhibitor W146 (10 µM, S1PR1-i) or S1PR1-targeting siRNA (siS1PR1) during LPS + S1P stimulation. Branch node counts (ImageJ-quantified) reflect endothelial network formation capacity. ( D , E ) Wound healing assays evaluating migration under S1PR1 inhibition: Time-lapse imaging (0 h, 6 h, 12 h, 24 h) of HUVECs treated with S1PR1-i or siS1PR1 under LPS + S1P conditions; relative migration rates calculated from wound area reduction (ImageJ). ( F ) Transwell invasion assay quantifying invasive capacity: HUVECs (LPS + S1P + S1PR1-i or LPS + S1P + siS1PR1) seeded onto matrix gel-coated chimeric chambers (serum-free medium). After 24 h, invasive cells on the basolateral membrane surface were counted and quantified via ImageJ. ( G ) Western blot validation of STAT3, phosphorylated STAT3 (p-STAT3), VEGFA, and VEGFR2 expression in HUVECs treated with S1PR1-i under LPS + S1P conditions (vs. untreated controls); band intensities quantified via ImageJ. ( H ) Efficiency verification of siS1PR1 via Western blot (targeting S1PR1 knockdown) and ImageJ-based quantification. ( I ) Western blot analysis of STAT3, p-STAT3, VEGFA, and VEGFR2 in HUVECs treated with siS1PR1 under LPS + S1P conditions (vs. scrambled siRNA controls); band intensities quantified via ImageJ. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significant difference

Article Snippet: S1PR1 siRNA, CEBPD siRNA, SGPP2 siRNA, PERK siRNA, ATF4 siRNA and control siRNA were purchased from Sangon Biotech.

Techniques: Expressing, Immunofluorescence, Staining, Tube Formation Assay, Inhibition, Migration, Imaging, Transwell Invasion Assay, Membrane, Western Blot, Biomarker Discovery, Knockdown

Journal: Journal of Translational Medicine

Article Title: CEBPD-mediated SGPP2 upregulation via PERK/ER stress in endothelial cells disrupts S1P homeostasis and impairs angiogenesis in chronic endometritis

doi: 10.1186/s12967-025-07558-0

Figure Lengend Snippet: SGPP2 may be responsible for the decrease in S1P upon LPS stimulation. ( A ) Quantitative PCR (qPCR) analysis of S1P metabolic enzyme mRNA expression (SGPL1, SPHK1, SGPP1, SGPP2, SPNS2) in endometrial tissues from CE ( n = 10) and non-CE ( n = 10) patients, normalized to the reference gene B2M. ( B ) qPCR detected relative mRNA expression of S1P metabolic enzymes in HUVECs under LPS treatment compared to untreated control, normalized using B2M as a reference. ( C ) Venn diagram illustrating SGPP2 as the sole differentially expressed S1P metabolic enzyme identified in both human endometrial tissues (CE vs. non-CE) and HUVECs (LPS vs. CON). ( D ) Validation of SGPP2 changes in endometrial tissues from CE and non-CE patients via Western blot ( n = 6), quantified using ImageJ software. ( E ) Validation of SGPP2 changes in mouse endometrial tissues from the control and CE model groups via Western blot ( n = 6), quantified using ImageJ software. ( F , G ) Immunofluorescence staining of CD31 (red) and SGPP2 (green) in human and mouse endometrial tissues revealed that SGPP2 was primarily localized to vascular endothelial cells. ( H ) Western blot analysis of S1P metabolic enzymes (SGPL1, SPHK1, SGPP2, SPNS2) in HUVECs before and after LPS treatment, with band intensity quantification (ImageJ) to assess relative expression changes. ( I ) Immunofluorescence staining of SGPP2 (green ) in HUVECs, visualizing endogenous SGPP2 expression and subcellular localization. ( J ) ELISA detection of changes in S1P and extracellular S1P levels changes upon treatment with siRNA targeting SGPP2 (siSGPP2) versus control (siNC), and overexpression plasmid targeting SGPP2 (OE-SGPP2) versus control (OE-NC) in HUVECs. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01; ns, no significant difference

Article Snippet: S1PR1 siRNA, CEBPD siRNA, SGPP2 siRNA, PERK siRNA, ATF4 siRNA and control siRNA were purchased from Sangon Biotech.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control, Biomarker Discovery, Western Blot, Software, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Over Expression, Plasmid Preparation

Journal: Journal of Translational Medicine

Article Title: CEBPD-mediated SGPP2 upregulation via PERK/ER stress in endothelial cells disrupts S1P homeostasis and impairs angiogenesis in chronic endometritis

doi: 10.1186/s12967-025-07558-0

Figure Lengend Snippet: LPS-induced upregulation of SGPP2 mediated by transcription factor CEBPD. ( A ) Integrated bioinformatics analysis to identify potential upstream transcription factors (TFs) regulating SGPP2 expression. By combining RNA-seq data from LPS-treated HUVECs with the Human Transcription Factor Database (Human TFDB), Gene Transcription Regulation Database (GTRD), and hTFtarget database, candidate TFs were computationally predicted. ( B ) qPCR analysis of relative mRNA expression levels for 17 most probable candidate transcription factors in LPS-treated HUVECs, normalized to B2M as internal control. ( C ) Western blot was used to assess NF-κB, SMAD3, and CEBPD protein expression in HUVECs before and after LPS treatment. Band intensities were quantified by ImageJ software. ( D ) Immunofluorescence revealed significant nuclear translocation of CEBPD before and after LPS treatment (CEBPD: green, DAPI: blue). Images were captured at 20x magnification using high-content imaging. ( E ) Western blot validation of CEBPD and SGPP2 protein expression in HUVECs treated with small interfering RNA targeting CEBPD (siCEBPD) or non-targeting control siRNA (siNC). Protein band intensities were quantified using ImageJ software. ( F ) In silico prediction of CEBPD binding sites within the SGPP2 promoter using the JASPAR database, followed by chromatin immunoprecipitation (ChIP)-qPCR to experimentally validate direct binding of CEBPD to specific promoter regions. ( G ) Consensus binding motif of CEBPD (derived from JASPAR) and a schematic mechanism diagram illustrating the identified CEBPD binding sites within the SGPP2 promoter region. ( H ) Dual-luciferase reporter assay assessing the transcriptional regulation of SGPP2 by CEBPD. HUVECs were co-transfected with a CEBPD overexpression plasmid (CEBPD-pcDNA3.1) and reporter vectors containing either the wild-type (WT) SGPP2 promoter or a site-mutant (MT) SGPP2 promoter (with mutated CEBPD binding sites). Luciferase activity was measured to determine the functional impact of CEBPD on promoter activity. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significant difference

Article Snippet: S1PR1 siRNA, CEBPD siRNA, SGPP2 siRNA, PERK siRNA, ATF4 siRNA and control siRNA were purchased from Sangon Biotech.

Techniques: Expressing, RNA Sequencing, Control, Western Blot, Software, Immunofluorescence, Translocation Assay, Imaging, Biomarker Discovery, Small Interfering RNA, In Silico, Binding Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Derivative Assay, Luciferase, Reporter Assay, Transfection, Over Expression, Plasmid Preparation, Mutagenesis, Activity Assay, Functional Assay

Journal: Journal of Translational Medicine

Article Title: CEBPD-mediated SGPP2 upregulation via PERK/ER stress in endothelial cells disrupts S1P homeostasis and impairs angiogenesis in chronic endometritis

doi: 10.1186/s12967-025-07558-0

Figure Lengend Snippet: LPS-induced endoplasmic reticulum stress mediates CEBPD-SGPP2 upregulation via the PERK pathway. ( A ) Schematic model hypothesizing that LPS stimulation may induce ER stress in HUVECs, leading to upregulation of the CEBPD-SGPP2 axis. ( B ) qPCR analysis of ER stress-related molecular markers in HUVECs under LPS treatment (1 µg/mL, 24 h). Relative mRNA expression levels of key ER stress indicators were normalized to the reference gene B2M. ( C ) Western blot validation of PERK-eIF2α-ATF4 signaling pathway activation in HUVECs before and after LPS treatment. Protein expression levels of pathway components (PERK, eIF2α, p-eIF2α, ATF4) were quantified using ImageJ software to confirm the engagement of the canonical ER stress sensor pathway. ( D ) Western blot analysis of CEBPD and SGPP2 protein levels in HUVECs upon LPS stimulation, comparing cells transfected with siRNA targeting PERK (siPERK) versus siNC. Protein band intensities were quantified using ImageJ software. ( E ) Under LPS stimulation, changes in CEBPD and SGPP2 were verified by Western blot after treatment with ATF4-targeting siRNA (siATF4) compared to siNC. Protein band intensities were quantified using ImageJ software. All data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significant difference

Article Snippet: S1PR1 siRNA, CEBPD siRNA, SGPP2 siRNA, PERK siRNA, ATF4 siRNA and control siRNA were purchased from Sangon Biotech.

Techniques: Expressing, Western Blot, Biomarker Discovery, Activation Assay, Software, Transfection

Journal: Journal of Inflammation Research

Article Title: Identification and Validation of Lipid Metabolism-Related Biomarkers GPD1 and CEBPD in Metabolic Dysfunction-Associated Steatohepatitis

doi: 10.2147/JIR.S524204

Figure Lengend Snippet: Diagnostic Performance and Clinical Relevance of CEBPD and GPD1 in MASH Patients. ( A ) Comparison of risk distributions between patients with MASH and healthy controls in the test set. ( B ) ROC curves assessed the model’s diagnostic performance in the test set. ROC curves assessed the diagnostic performance of CEBPD ( C ) and GPD1 ( D ) in the train set. ( E ) Comparison of CEBPD and GPD1 expression levels between patients with MASH and HC in the test set. ROC curves evaluated the diagnostic performance of CEBPD ( F ) and GPD1 ( G ) in the test set. ( H and I ) The association between the expression level of CEBPD and GPD1 and the fibrosis stage. * p < 0.05, *** p < 0.001, **** p < 0.0001, ns not significant ( p ≥ 0.05).

Article Snippet: The GPD1 antibody was obtained from Proteintech, while the CEBPD antibody was sourced from Abmart.

Techniques: Diagnostic Assay, Comparison, Expressing

Journal: Journal of Inflammation Research

Article Title: Identification and Validation of Lipid Metabolism-Related Biomarkers GPD1 and CEBPD in Metabolic Dysfunction-Associated Steatohepatitis

doi: 10.2147/JIR.S524204

Figure Lengend Snippet: Immune Cell Profiling and Correlation with CEBPD and GPD1 Expression in MASH. ( A ) Heatmap showing correlations between 28 immune cell types in MerCorhort. ( B ) Heatmap showing the infiltrating enrichment of 28 immune cell types in MerCorhort. ( C ) Boxplot depicting differences in immune cell infiltration between patients with MASH and healthy controls. ( D ) Heatmap showing the correlation between CEBPD and GPD1 expression and immune cells in MASH. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns not significant ( p ≥ 0.05).

Article Snippet: The GPD1 antibody was obtained from Proteintech, while the CEBPD antibody was sourced from Abmart.

Techniques: Expressing

Journal: Journal of Inflammation Research

Article Title: Identification and Validation of Lipid Metabolism-Related Biomarkers GPD1 and CEBPD in Metabolic Dysfunction-Associated Steatohepatitis

doi: 10.2147/JIR.S524204

Figure Lengend Snippet: Validation of GPD1 and CEBPD in a MASH Animal Model. ( A ) IHC staining of normal and MASH liver tissues, n = 6, 400×, scale bar 100 μm. ( B ) AOD values for GPD1 in CON and MASH groups. ( C ) AOD values for CEBPD in CON and MASH groups. ( D ) WB analysis of normal and MASH liver tissues, n = 3. ( E ) Relative gray value measurements for GPD1 in CON and MASH groups. ( F ) Relative gray value measurements for CEBPD in CON and MASH groups. *** p ≤ 0.001.**** p ≤ 0.0001.

Article Snippet: The GPD1 antibody was obtained from Proteintech, while the CEBPD antibody was sourced from Abmart.

Techniques: Biomarker Discovery, Animal Model, Immunohistochemistry