Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: (A) Detection of endogenous HIF-1α or HIF-2α in whole cell extracts by immunoblotting after the indicated period of hypoxia exposure. Alpha-tubulin levels for each immunoblot are also shown. (B) Same as (A) except after low glucose exposure. (C) Cellular acetate levels generated after the indicated period of hypoxia exposure ( n = 3 biological replicates/time-point; single measurement/replicate; mean/SD). (D) Same as (C) except after low glucose exposure. (E) Acetylation of ectopic HA-tagged HIF-2α after pulldown (PD) and detection by immunoblotting (IB) after the indicated period of hypoxia exposure with pharmacological inhibition of Sirt1 by sirtinol and nicotinamide (NAM). Levels of ectopic HIF-2α and alpha-tubulin levels from whole cell extracts prepared under the same conditions are also shown. (F) Same as (E) except after low glucose exposure. (G) Detection of endogenous CBP/HIF-2α or p300/HIF-2α complexes by immunoblotting (IB) after early (4 hr) or late (16 hr) hypoxia exposure. (H) Same as (G) except after early (2 hr) or late (24 hr) low glucose exposure. All experiments performed with HT1080 whole cell extracts.

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Western Blot, Generated, Inhibition

Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: (A) Acetylation of endogenous HIF-2α after immunoprecipitation (IP) and detection by immunoblotting (IB) after early (4 hr) hypoxia exposure with pharmacological inhibition of Sirt1 by sirtinol and nicotinamide (NAM). (B) Same as (A) except after late (24 hr) low glucose exposure. (C) Detection of endogenous CBP/HIF-2α or p300/HIF-2α complexes by immunoblotting (IB) after early (4 hr) hypoxia exposure. (D) Same as (C) except after late (24 hr) low glucose exposure. (E) Detection of ACSS2 or ACLY in HT1080 subcellular fractions by immunoblotting (IB) after early (4 hr) or late (16 hr) hypoxia exposure. (F) Same as (E) except after early (2 hr) or late (24 hr) low glucose exposure. Experiments in (A)-(D) performed with HT1080 whole cell extracts.

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Immunoprecipitation, Western Blot, Inhibition

Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: (A) Acetylation of ectopic HA-tagged wild-type HIF-2α after pulldown (PD) and detection by immunoblotting (IB) after (4 hr) incubation with vehicle (Veh), or the short chain fatty acids acetate (Ac), propionate (Pr), and butyrate (Bu) with pharmacological inhibition of Sirt1 by sirtinol and nicotinamide (NAM). (B) Acetylation of endogenous HIF-2α after immunoprecipitation (IP) and detection by immunoblotting (IB) after (4 hr) incubation with acetate and following control, ACSS1, ACSS2, ACLY, CBP or p300 knockdown. (C) Endogenous CBP/HIF-2α or p300/HIF-2α complexes after (4 hr) treatment with vehicle (Veh), acetate (Ac), propionate (Pr), or butyrate (Bu). (D) Endogenous CBP/HIF-2α or p300/HIF-2α complexes detected by immunoblotting (IB) after (4 hr) acetate exposure following ACSS2 knockdown. (E) Detection of ACSS2 or ACLY in HT1080 subcellular fractions by immunoblotting (IB) after (4 hr) acetate exposure. Experiments in (A)-(D) performed with HT1080 whole cell extracts.

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Western Blot, Incubation, Inhibition, Immunoprecipitation, Control, Knockdown

Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: Semi-quantitative RTPCR measurement of HIF-1 selective (PGK1), HIF-2 selective (MMP9, GLUT1), or HIF-1/HIF-2 co-regulated (VEGFa, PAI1) target genes following HIF-1α, HIF-2α, ACLY, ACSS2, p300, CBP, or SIRT1 knockdown and after (A) early (4 hr) hypoxia exposure, or (B) after incubation under late (24 hr) low glucose conditions. Comparison by one-tailed t-test between control knockdown/treatment and specified knockdown/treatment with significant reductions compared to control indicated (single pool of triplicate biological replicates/manipulation; triplicate measurements/pool; mean/SD; *, P<0.05; **, P<0.10).

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Reverse Transcription Polymerase Chain Reaction, Knockdown, Incubation, Comparison, One-tailed Test, Control

Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: HIF target genes induced in (A) HT1080 cells expressing no ectopic HIF (control), oxygen-insensitive (PPN) HIF-1α, or PPN HIF-2α following control or ACSS2 knockdown, and treated with vehicle or acetate, or in (B) HT1080 cells expressing PPN HIF-2α following control, p300, CBP, or ACSS2 knockdown, and treated with vehicle or acetate. Comparison by one-tailed t-test between vehicle (empty bars) and acetate treatment (filled bars) with significant increases compared to vehicle treatment indicated (single pool of triplicate biological replicates/manipulation; triplicate measurements/pool; mean/SD; *, P<0.05; **, P<0.10).

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Expressing, Control, Knockdown, Comparison, One-tailed Test

Journal: PLoS ONE

Article Title: The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

doi: 10.1371/journal.pone.0116515

Figure Lengend Snippet: Endogenous acetate generated in response to hypoxia or glucose deprivation, or exogenous acetate originating from neighboring cells or from gastrointestinal uptake, stimulates ACSS2-dependent acetyl CoA production in the cytosol, but also directs production of an acetyl CoA pool that is localized in the nucleus upon ACSS2 nuclear translocation. In the cytosol, ACSS2 contributes to lipid synthesis, likely for cell growth and proliferation. In the nucleus, the acetyltransferase/coactivator CBP uses this specific ACSS2-derived acetyl CoA pool for HIF-2α acetylation and CBP/HIF-2α complex formation, which augments HIF-2 signaling. CBP is only bound to HIF-2α while it is undergoing the acetylation reaction, which occurs as long as ACSS2-generated acetyl CoA remains available. When acetylation of HIF-2α is complete, CBP is released. SIRT1 then deacetylates HIF-2α, restoring HIF-2α to a CBP substrate. After transformation or as a tumor forms, acetate may be produced constitutively, which further augments tumor growth. In the absence of ACSS2-generated acetyl CoA, HIF-2α complexes with p300 during hypoxia, but not during glucose deprivation. The p300/HIF-2α complex, however, is inefficient at inducing HIF-2 signaling compared to CBP/HIF-2α.

Article Snippet: After washing, immunoprecipitated materials were eluted and immunoblotted with anti-human p300 (1:500 dilution; Cat. No. sc-584, Santa Cruz Biotechnology), anti-human CBP (1:500 dilution; Cat. No. 4772, Cell Signaling Technology), or anti-human HIF-2α mouse monoclonal (1:1,000 dilution; Cat. No. NB100-132, Novus Biologicals) antibodies.

Techniques: Generated, Translocation Assay, Derivative Assay, Transformation Assay, Produced

Journal: Bioengineered

Article Title: Knockdown of growth factor receptor bound protein 7 suppresses angiogenesis by inhibiting the secretion of vascular endothelial growth factor A in ovarian cancer cells

doi: 10.1080/21655979.2021.2005225

Figure Lengend Snippet: Knockdown of GRB7 in SKOV-3 cells decreases the expression of proteins associated with angiogenesis. HUVECs were seeded in the lower chamber of a transwell insert, and SKOV-3 cells transfected with negative control siRNA (NC) or siRNAs targeting GRB7 (siGRB7-1 or siGRB7-2) were seeded in the upper chamber of the transwell insert. These conditions are designated as HUVECs-NC, HUVECs-siGRB7-1, and HUVECs-siGRB7-2. After co-culture for 24 h, HUVECs were harvested for western blotting to measure the expression of VEGFR2, p-MEK1, p-ERK1/2, NOTCH1, and DLL4

Article Snippet: All primary antibodies were purchased from Proteintech Group, Inc., and their catalog numbers and dilutions were as follows: anti-GRB7 (Catalog number 10,045-1-Ig; 1:1000), anti-vascular endothelial growth factor A (VEGFA) (19,003-1-AP, 1:2000), anti-vascular endothelial growth factor receptor-2 (VEGFR2) (26,415-1-AP, 1:800), anti-mitogen-activated protein kinase kinase 1 (MAP2K1/MEK1) (28,930-1-AP, 1:3000), anti-extracellular signal-regulated kinases 1 and 2 (ERK1/2) (28,733-1-AP, 1:4000), anti-notch receptor 1 (NOTCH1) (20,687-1-AP, 1:1000), anti-delta-like canonical Notch ligand 4 (DLL4) (21,584-1-AP, 1:600), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (10,494-1-AP, 1:6000).

Techniques: Expressing, Transfection, Negative Control, Co-Culture Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: Summary of PCR/qPCR primer pairs

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: Validation of microarray data by qPCR and conventional PCR. a qPCR CLDN1, b qPCR ICAM1, c qPCR MMP9, d conventional PCR. MMP9 was significantly up-regulated in TNF-α-treated (100 ng/ml) M13SV1-Cre cells, whereas significantly elevated CLDN1 and ICAM1 expression levels were detected in TNF-α-treated (100 ng/ml) MDA-MB-435-pFDR1 cells. Likewise, increased CCL2 and VEGFC expression levels were detected in TNF-α-treated (100 ng/ml) MDA-MB-435-pFDR1 cells. Data are presented as the mean ± SD of at least three independent experiments ( a ) or the representative data of three independent experiments ( b ). Statistical analysis: unpaired, two-tailed Student’s t -test: * = p < 0.05; ** = p < 0.01

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Microarray, Expressing, Two Tailed Test

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: TNF-α-induced cell fusion could be blocked by the inhibition of MMP9. a The knockdown of CLDN1 expression with specific siRNA was not correlated with a decreased TNF-α-induced (100 ng/ml) fusion rate. b siRNA-mediated CLDN1 expression levels of M13SV1-Cre cells were stably down-regulated over 72 h. c Inhibition of ICAM1 activity using a blocking antibody did not impair the TNF-α-induced fusion between M13SV1-Cre and MDA-MB-435-pFDR1 cells. d The TNF-α-induced fusion of M13SV1-Cre and MDA-MB-435-pFDR1 cells was impaired by the MMP9 inhibitor SB-3CT in a dose-dependent manner. The mean ± SD of at least three independent experiments is shown. Statistical analysis: ANOVA F-test and Scheffé post-hoc test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Inhibition, Expressing, Stable Transfection, Activity Assay, Blocking Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: TNF-α-induced MMP9 expression in M13SV1-Cre cells was blocked by minocycline. M13SV1-Cre and MDA-MB-435-pFDR1 cells were treated for 3 days with 100 ng/ml TNF-α, 10 μg/ml minocycline and a combination of both. The expression levels of a ) CLDN1, b ) ICAM1 and c ) MMP9 were determined by qPCR, Western blot analysis and zymography (only MMP9). Significantly decreased MMP9 mRNA levels were detected in TNF-α and minocycline-treated M13SV1-Cre cells as compared with TNF-α-treated cells and were validated by zymography. d ) Co-immunoprecipitation data show that MMP9 binds to ICAM1. ICAM1 expression and co-immunoprecipitated MMP9 were markedly decreased in the lysates of TNF-α + minocycline-treated M13SV1-Cre cells compared with the lysates from TNF-α-stimulated cells. e ) No differences in sICAM1 levels were detected in M13SV1-Cre and MDA-MB-435-pFDR1 cells that were treated with TNF-α, minocycline and a combination of both. Shown are the mean ± SD or representative Western blot data for at least three independent experiments. Statistical analysis: ANOVA F-test and Scheffé post-hoc test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Expressing, Western Blot, Zymography, Immunoprecipitation

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: The co-administration of minocycline, SB-3CT and the ICAM1 blocking antibody, yielded different results. No additive inhibitory effect of minocycline and SB-3CT regarding the TNF-α-induced fusion of M13SV1-Cre and MDA-MB-435-pFDR1 cells was observed. In contrast, the incubation of cells in the presence of minocycline, ICAM1 blocking antibody and TNF-α yielded a significantly increased fusion rate. The data shown are the mean ± SD of at least three independent experiments. Statistical analysis: ANOVA F-test and Scheffé post-hoc test: * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Blocking Assay, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: Up-regulated genes (FC ≥ 2) in TNF-α treated cells under normoxic and hypoxic conditions

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Sequencing, Binding Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells

doi: 10.1186/s12964-018-0226-1

Figure Lengend Snippet: Validation of qPCR and conventional RT-PCR data by Western blot and zymography. a Western blot analysis: CCL2 protein expression was only detected in TNF-α-treated (100 ng/ml) MDA-MB-435-pFDR1 cells, whereas CLDN1 expression was absent from these cells but was clearly detectable in M13SV1-Cre cells. Increased ICAM1 expression was observed in TNF-α-treated cells, whereas increased MMP9 expression was only found in TNF-α-stimulated M13SV1-Cre cells. Two discrete bands were detected for MMP9 in Western blot analysis; the upper band represents inactive MMP9 and the lower band represents active MMP9. ICAM1 Western blot data were rearranged because ICAM1 samples were originally loaded in a different order. b Zymography: markedly increased MMP9 expression levels and activity were detected in TNF-α-treated M13SV1-Cre cells, whereas even in TNF-α-stimulated MDA-MB-435 cells slightly enhanced MMP9 expression levels could be identified. The data shown are representative of at least three independent experiments

Article Snippet: The samples were incubated with anti-ICAM1/CD54 (5 μg/ml; mouse monoclonal, clone 11C81; Bio-Techne GmbH, Wiesbaden-Nordenstadt, Germany) or IgG1 (5 μg/ml; Beckman Coulter, Krefeld, Germany) overnight at 4 °C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Zymography, Expressing, Activity Assay

Journal: BMC Pregnancy and Childbirth

Article Title: Comprehensive identification of immune-related biomarkers and therapeutic targets in preeclampsia: integrative bioinformatics and experimental validation

doi: 10.1186/s12884-025-08169-9

Figure Lengend Snippet: Hub gene identification and diagnostic signature construction for PE. A Training error trajectory of RF algorithmic optimization; B Scatter plot of MeanDecreaseAccuracy and MeanDecreaseGini for IRDEGs; C , D LASSO regression modeing: diagnostic parameter optimization curve ( C ) and coefficient development trajectory. E ROC curve analysis of the diagnostic model constructed with four key genes (FLT1, PIK3CB, KLRD1, and APLN) using RF algorithm in the training dataset GSE75010 (AUC = 0.9463); F Validation of the four-gene diagnostic model performance in the independent test dataset GSE44711 , achieving an AUC of 0.9844, which demonstrates robust predictive capability. PE, Preeclampsia; IRDEGs, Immune-Related Differentially Expressed Genes; LASSO, Least Absolute Shrinkage and Selection Operator; RF, Random Forest

Article Snippet: Sections were incubated overnight at 4 °C with antibodies targeting FLT1 (Proteintech, 13,687–1-AP, 1:100), PIK3CB (Proteintech, 20,584–1-AP, 1:100), KLRD1 (Affinity, DF6773, 1:100), and APLN (Affinity, DF13350, 1:100).

Techniques: Diagnostic Assay, Construct, Biomarker Discovery, Selection

Journal: BMC Pregnancy and Childbirth

Article Title: Comprehensive identification of immune-related biomarkers and therapeutic targets in preeclampsia: integrative bioinformatics and experimental validation

doi: 10.1186/s12884-025-08169-9

Figure Lengend Snippet: Immune cell landscape analysis of GSE75010 expression data. A Correlation matrix visualizing relationships between infiltrating immune cell populations in GSE75010 dataset; B Violin plots of differential immune cell infiltration patterns between PE and control specimens. Control group indicated in red; preeclampsia group in blue; C-F Bubble plots illustrating relationships between immune cell abundance and expression level of FLT1 ( C ), APLN ( D ), PIK3CB ( E ), and KLRD1 ( F ). Horizontal axis displays correlation coefficients, vertical axis shows immune cell subtypes, and bubble color intensity represents statistical significance values

Article Snippet: Sections were incubated overnight at 4 °C with antibodies targeting FLT1 (Proteintech, 13,687–1-AP, 1:100), PIK3CB (Proteintech, 20,584–1-AP, 1:100), KLRD1 (Affinity, DF6773, 1:100), and APLN (Affinity, DF13350, 1:100).

Techniques: Expressing, Control

Journal: BMC Pregnancy and Childbirth

Article Title: Comprehensive identification of immune-related biomarkers and therapeutic targets in preeclampsia: integrative bioinformatics and experimental validation

doi: 10.1186/s12884-025-08169-9

Figure Lengend Snippet: Interaction network analysis of hub genes. A mRNA-miRNA regulatory network of hub genes. mRNAs represented as Orange polygons represent, miRNAs displayed as blue ellipses; B mRNA-TF regulatory network of hub genes. mRNAs visualized as orange diamonds; regulatory TFs shown as blue diamonds; C Pharmaceutical-gene interaction network for FLT1, PIK3CB, KLRD1, and APLN. Experimental drugs depicted with blue connections; approved medications shown with red connections. Node dimensions correspond to connectivity degree, and connection thickness represents the interaction strength. TF: Transcription factors

Article Snippet: Sections were incubated overnight at 4 °C with antibodies targeting FLT1 (Proteintech, 13,687–1-AP, 1:100), PIK3CB (Proteintech, 20,584–1-AP, 1:100), KLRD1 (Affinity, DF6773, 1:100), and APLN (Affinity, DF13350, 1:100).

Techniques: Medications

Journal: BMC Pregnancy and Childbirth

Article Title: Comprehensive identification of immune-related biomarkers and therapeutic targets in preeclampsia: integrative bioinformatics and experimental validation

doi: 10.1186/s12884-025-08169-9

Figure Lengend Snippet: Expression of hub IRDEGs in preeclamptic placental tissues and hypoxia-induced HTR-8/SVneo cells. A Representative histological immunostaining of FLT1, PIK3CB, KLRD1, and APLN protein in control and preeclamptic placental tissues. Scale indicator = 50 μm. Brown staining indicates positive protein expression. B Correlation heatmap illustrating relationships between hub IRDEG expression levels and clinical parameters in preeclamptic patients. Color intensity represents correlation coefficient strength (r values), with red indicating positive correlation and blue indicating negative correlation. All marked correlations reached statistical significance ( p < 0.05). C Relative mRNA expression of hub IRDEGs in HTR-8/SVneo trophoblast cells under normoxic (control) and hypoxic (model: 1% O₂, 5% CO₂, 94% N₂, 48 h) conditions, quantified by qRT-PCR. D Representative Western blot analysis (left) and densitometric quantification (right) of FLT1, PIK3CB, KLRD1, and APLN protein expression in HTR-8/SVneo cells under normoxic and hypoxic conditions. GAPDH served as loading control. Data are presented as mean ± SD from three independent experiments performed in triplicate. * p < 0.05 compared to normoxic control or non-preeclamptic controls. SD, standard deviation

Article Snippet: Sections were incubated overnight at 4 °C with antibodies targeting FLT1 (Proteintech, 13,687–1-AP, 1:100), PIK3CB (Proteintech, 20,584–1-AP, 1:100), KLRD1 (Affinity, DF6773, 1:100), and APLN (Affinity, DF13350, 1:100).

Techniques: Expressing, Immunostaining, Control, Staining, Quantitative RT-PCR, Western Blot, Standard Deviation