cebpd sirna  (Ribobio co)

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: Cell reports

Article Title: HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

doi: 10.1016/j.celrep.2022.111136

Figure Lengend Snippet: Figure 5. HOTAIR-PRC2 regulates developmental pathways in differentiating gluteal preadipocytes (A) Venn diagram showing overlap between PRC2 target genes (from FUMA and CHEA) and shHOTAIR WGCNA DEGs (upregulated at all time points, light-green module). See also Figure S6. (B) qPCR confirmation of PCDH10 expression in shHOTAIR and shControl cells during adipogenesis (n = 3). Data were assessed using 2-way ANOVA; *p < 0.05. (C and D) ChIP to assess (C) SUZ12 and (D) H3K27me3 enrichment at the PCDH10 promoter in gluteal shHOTAIR and shControl cells on differentiation day 0 (n = 3). Mouse IgG antibody was used as a negative control. Data were assessed by Student’s t test; *p < 0.05. (E) Expression heatmap of shHOTAIR DEGs annotated to UniProtKB: Wnt signaling (shHOTAIR versus shControl; rlog normalized fold changes DESeq2). (F) HOTAIR1 gene expression in imGSAT cells expressing the 7TFP TOPflash luciferase vector following 72 h HOTAIR siRNA treatment (n = 3, paired t test). (G) TOPflash promoter activity in Control and siHOTAIR imGSAT cells (n = 5, paired t test). (H) Adipogenesis assessed by AdipoRed staining in control and siHOTAIR cells treated with CHIR99021 1 mM and 3 mM throughout adipogenic differentiation (n = 3, ANOVA). (I) Differential alternative splicing (DAS) events between shControl and shHOTAIR cells determined by SUPPA2:diffSplice. Alternative first (AF) or last (AL) exon, alternative 50 or 30 splice site (A5 and A3), mutually exclusive (MX) and skipped exons (SE), and retained introns (RI). (J) Number of genes that are differentially alternatively spliced at both the isoform (limma) and event (SUPPA2) level across each day of differentiation. (K) The proportion splice-in (PSI) of significantly expressed PPARG (GENCODE) isoforms in shControl and shHOTAIR cells across each day of differentiation. *p < 0.05 (shControl versus shHOTAIR). Data presented as means ± SEMs (B–D and F–H). See also Figure S7. n represents the number of experimental replicates.

Article Snippet: Lists of DAS genes were then uploaded to the gProfiler online g:GOSt tool (Raudvere et al., 2019) for overrepresentation analysis (Table S3D). siRNA rescue experiments Human siRNA oligo duplexes were used to transiently suppress CEBPD (Origene SR300761) or PTEN (Origene SR321496) from differentiation day4 (siRNA was added once on day4).

Techniques: Expressing, Negative Control, Gene Expression, Luciferase, Plasmid Preparation, Activity Assay, Control, Staining, Alternative Splicing