Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Quantification of human MTTP activity at the plateau phase (170-210 min) non-steatosis (n=11) and steatosis samples (n=12). Each patient is represented by a group of approximately 5 data points (dots). (B-C) Representative western blot showing reduced CEPT1 and MTTP protein levels in human liver tissue from non-steatosis (n=11) and steatosis samples (n=12). GAPDH serves as a loading control. (D) Quantification of hepatic CEPT1 (C) and MTTP (D) protein levels. (E-F) Representative immunofluorescence images of human liver sections, comparing non-steatosis and steatosis conditions: (E) H&E staining with DAPI (blue) and MTTP (green); white arrows highlight regions of positive MTTP staining; (F) H&E staining alongside DAPI (blue), CD31 (red), and CEPT1 (green), white arrows indicate areas of CD31–CEPT1 colocalization, shown in yellow; (G) Quantification of MTTP integrated density, showing a significant decrease in steatosis (*p<0.05). (H) Quantification of CEPT1/CD31 co-localization, which is significantly reduced in steatotic samples (**p<0.01). Data represent mean ± SEM. Statistical significance was determined by Unpaired t-test.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activity Assay, Western Blot, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Schematic illustrating the proposed impact of endothelial CEPT1 (EC CEPT1) on hepatic MTTP and PPARα regulation. EC CEPT1 influences hepatocyte function, potentially impacting VLDL assembly and fatty acid metabolism. Fenofibrate (FEN), a PPARα activator, is included to indicate pharmacological activation of PPARα in this pathway (B) Experimental setup illustrating co-culture of HUVECs with HepG2 cells ± FEN treatment (50 µM) for 48h. HUVECs were transfected with si CEPT1 +. (C-E) Relative mRNA expression of CEPT1 (C), PPARα (D), and MTTP (E) in HUVECs transfected with siRNA targeting Cept1 compared to untransfected HUVECs (control), normalized to controls (n=3). Relative mRNA expression levels of MTTP (F) and PPARα (G) in HepG2 cells co-cultured with HUVECs ± siRNA targeting CEPT1 and ±FEN treatment (50µM) for 48h, normalized to controls (n=3). (H) MTTP activity curve (pmol transferred/µg protein) over time for HepG2 cells from control and co-culture with HUVEC si CEPT1 + cells ±FEN treatment (50µM for 48h). Plateau indicates maximal MTTP transfer capacity, as determined by endpoint assay guidelines. (I) Quantification of MTTP activity at the plateau phase (195-240 min) for HepG2 cells from control and co-culture with HUVEC si CEPT1 + cells ±FEN treatment. Data were analyzed by Mann-Whitney U test; n=4. (J) Experimental setup illustrating co-culture of HepG2 with si PPARα + transfected HUVECs vs control (HUVECs si PPARα -) for 48h. (K) Relative mRNA expression of MTTP in HepG2 co-culture with HUVECs si PPARα + vs positive control (HUVECs si GFP + ) and negative control (un-transfected HUVECs) (n=9). Data were analyzed by t-test or one-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activation Assay, Co-Culture Assay, Transfection, Expressing, Control, Cell Culture, Activity Assay, End Point Assay, MANN-WHITNEY, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Schematic representation of experimental design. Tamoxifen was administered intraperitoneally to induce Cre recombinase activity in Cept1 fl/fl Apoe +/+ and Cep1 fl/fl Apoe -/- mice. After tamoxifen treatment, mice were fed a 42% high-fat diet for 12 weeks. Serum samples were collected weekly for further analysis. (B) Body weight measurements (in grams) of mice over 12-week period. (C-E) Serum lipid profile after 12 weeks on a high-fat diet. Mean serum levels of total cholesterol (mg/dL) (C), triglycerides (mg/dL) (D), and free fatty acids (ng/dL) (E) in Cept1 fl/fl Apoe +/+ and Cep1 fl/fl Apoe -/- mice compared to controls. All data are presented as mean ± SEM. Statistical significance was determined by Unpaired t-test. *p<0.05, **p<0.01, ****p<0.0001, and n ≥ 6 for all groups.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: (A) Representative H&E-stained liver sections from Cept1 fl/fl Cre + Apoe -/- , Cept1 fl/fl Cre - Apoe -/- , Cept1 fl/fl Cre + Apoe +/+ and Cept1 fl/fl Cre - Apoe +/+ mice. Scale bar = 100 μm. (B) Quantification of hepatic lipid accumulation as measured by ORO intensity in liver sections across all experimental groups. Data analyzed by one-way ANOVA with Tukey’s post-hoc test; n=4-6 per group; **p<0.01. (C) Representative Western blots of MTTP, PPARα, CEPT1, and GAPDH in liver lysates of Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ . Molecular weights (kDa) are indicated. Quantification of hepatic Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ MTTP (D), PPARα (E), CEPT1 (F) protein levels normalized to GAPDH in all mice groups; n=3. Hepatic Mttp (G) and Cept1 (H) gene expression in Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ . (I) Representative Western blots of MTTP, PPARα, CEPT1, and GAPDH in liver lysates of Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- . Molecular weights (kDa) are indicated. Quantification of hepatic Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe -+/+ MTTP (J), PPARα (K), CEPT1 (L) protein levels normalized to GAPDH in all mice groups; n=3. Hepatic Mttp (M) and Cept1 (N) gene expression in Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- . Data were analyzed by t-test. (O) Liver MTTP activity curve (pmol transfered/μg protein) over time in Cept1 fl/fl Cre + Apoe -/- , and Cept1 fl/fl Cre + Apoe +/+ mice compared to controls. Plateau represents the maximal transfer capacity achieved under these experimental conditions, as per manufacturer’s guidelines for endpoint assays. (P-Q) MTTP activity at the plateau phase (180-240 min) in Cept1 fl/fl Cre + Apoe +/+ (P), and Cept1 fl/fl Cre + Apoe -/- (Q) mice compared to controls. Data were analyzed by Mann-Whitney U test; n=4. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Staining, Western Blot, Gene Expression, Activity Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Endothelial CEPT1 Regulates Hepatic MTTP-Mediated Lipid Metabolism and Impacts Aortic Atherosclerosis

doi: 10.1101/2025.09.21.677657

Figure Lengend Snippet: Representative images of ORO stained en face preparations of the aortas in Cept1 fl/fl Cre - Apoe +/+ and Cept1 fl/fl Cre + Apoe +/+ (A). Scale bar=5mm. Quantification of plaque area in the total aorta (B; n ≥ 5), aortic arch (C; n ≥ 4), thoracic aorta (D; n ≥ 5), and aortoiliac segment (E; n ≥ 4) of Cept1 fl/fl Cre - Apoe +/+ , and Cept1 fl/fl Cre + Apoe +/+ mice. Representative images of ORO stained en face preparations of the aortas in Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- (F). Scale bar=5mm. Quantification of plaque area in the total aorta (G; n ≥ 5), aortic arch (H; n ≥ 4), thoracic aorta (I; n ≥ 5), and aortoiliac segment (J; n ≥ 4) of Cept1 fl/fl Cre - Apoe -/- , and Cept1 fl/fl Cre + Apoe -/- mice. (K) Representative images of ORO-stained aortic root sections from Cept1 fl/fl Cre + Apoe +/+ and Cept1 fl/fl Cre + Apoe -/- mice compared to controls. Scale bar=100µm. (L-M) Quantification of lipid deposition in the aortic root sections; n ≥5 Cept1 fl/fl Cre + Apoe +/+ (L) and Cept1 fl/fl Cre + Apoe -/- (M) compared to their corresponding controls. (N) Representative immunofluorescence staining for CD68 (green) and DAPI (blue) in the aortic root of Cept1 fl/fl Cre - Apoe -/- and Cept1 fl/fl Cre + Apoe -/- mice. Scale bar=100μm. (O) Quantifying means CD68 intensity (arbitrary units, a.u.) (n=5). t-tests determined statistical significance. *p<0.05, **p<0.01.

Article Snippet: Sections were incubated overnight at 4°C with the following primary antibody anti-CD68 (1:200, Bio-Rad, MCA17576A), CD31 (1:250, Santa Cruz, sc-376764), MTTP (1:150, Thermo Fischer, PA5-76049) and CEPT1 (1:250, Bioss, bs-12284R-Cy3).

Techniques: Staining, Immunofluorescence

Journal: PLoS ONE

Article Title: The E2F2 Transcription Factor Sustains Hepatic Glycerophospholipid Homeostasis in Mice

doi: 10.1371/journal.pone.0112620

Figure Lengend Snippet: Partial hepatectomy was performed on E2F2 +/+ (wild-type, WT) and E2F2 -/- mice, and were sacrificed 48 hours later. Quiescent (0-h, solid bars) and regenerating (48-h, open bars) livers were harvested and total RNA from each mouse was examined in triplicate by quantitative real-time PCR for the following genes: Pemt, phosphatidylethanolamine N-methyltransferase; Chpt1, choline phosphotransferase 1; Cept1, choline/ethanolamine phosphotransferase 1; Pebp1, phosphatidylethanolamine binding protein 1; Sptlc2, serine palmitoyltransferase, long chain base subunit 2; Fads2, fatty acid desaturase 2; Scd1, stearoyl-CoA desaturase 1. Relative transcript abundance was calculated for each mouse using a normalization factor computed by Genorm software for pyruvate carboxylase, transferrin receptor and vascular endothelial zinc finger containing factor 1 mRNAs. Values are referred to 0-h WT mice samples (100%). Results are presented as means ± SD (n = 8 mice per group), except for Scd1 transcript levels, which were estimated in 3 pools of eight mice each from each genotype (n = 3). Statistical differences between regenerating and quiescent liver of a genotype are denoted by ** P ≤0.01, *** P ≤0.001, and between the same tissue condition of different genotypes are denoted by # P ≤0.05, ## P ≤0.01, ### P ≤0.001 (unpaired, 2-tailed Student's t -test).

Article Snippet: References of TaqMan assays were the following: Mm00839436_m1 for phosphatidylethanolamine N-methyltransferase (Pemt, GI:33667035); Mm00772290_m1 for stearoyl-CoA desaturase 1 (Scd1, GI:118130513); Mm01208299_g1 for fatty acid desaturase 2 (Fads2, GI:9790070); Mm00522694_m1 for choline phosphotransferase 1 (Chpt1, GI:38604066); Mm01200029_g1 for choline/ethanolamine phosphotransferase 1 (Cept1, GI:142365741); Mm02601848_g1 for phosphatidylethanolamine binding protein 1 (Pebp1, GI:84794551); and Mm00448878_m1 for serine palmitoyltransferase, long chain base subunit 2 (Sptlc2, GI:142366849).

Techniques: Real-time Polymerase Chain Reaction, Binding Assay, Software

Journal: Journal of Lipid Research

Article Title: Diabetes adversely affects phospholipid profiles in human carotid artery endarterectomy plaques [S]

doi: 10.1194/jlr.M081026

Figure Lengend Snippet: CEA plaques from diabetic subjects have higher CEPT1 expression. A: The extracranial common carotid artery (CCA) bifurcation is prone to atherosclerosis. Within the plaque there are segments of MAX disease at the level of the carotid bifurcation and segments of MIN disease at the distal edge of the plaque within the lumen of the internal carotid artery (ICA). ECA, external carotid artery. B: Pentachrome staining of CEA plaque demonstrates that MIN diseased segments retain an intima layer surrounded by a relatively intact internal elastic lamina and partial media layer. The MAX segment has a grossly abnormal thick intima layer with areas of necrosis, intra-plaque hemorrhage, non-intact internal elastic lamina layer, and a thinner medial segment. Black, elastic fibers; yellow, collagen fibers; blue, mucin; bright red, fibrin. C: Quantified Western blot analysis demonstrates higher CEPT1 protein expression in CEA plaques of diabetic subjects (n = 11) compared with nondiabetic (n = 7) subjects. D: Immunofluorescent staining demonstrates more CEPT1-DAPI-positive cells in the superficial intima of MAX segments. Isolectin staining for ECs and smooth muscles, DAPI staining for nuclei, and ^ indicates vessel lumen. E: Quantitative analysis of CEPT1-positive cells in five random of 20× images demonstrates higher CEPT1 content in MAX and MIN diseased segments of diabetic subjects, and also higher content in MAX segments of diabetic (n = 8) and nondiabetic (n = 17) subjects. F: Relative expression of cept1 was higher in diabetic MAX and MIN diseased segments. Error bars = SEM, * P < 0.05 and *** P < 0.001.

Article Snippet: CEA sections were then stained with Pentachrome, as previously described , or with anti-CEPT1 antibody (1:50; Proteintech, Rosemont, IL), followed by labeling with Alexa Fluor 594-conjugated secondary antibody (1:250; Invitrogen, Waltham, MA).

Techniques: Expressing, Staining, Western Blot, Muscles

Journal: Journal of Lipid Research

Article Title: Diabetes adversely affects phospholipid profiles in human carotid artery endarterectomy plaques [S]

doi: 10.1194/jlr.M081026

Figure Lengend Snippet: CEPT1-derived pPEs contribute to AA production. LDL particles internalized by vascular macrophages and smooth muscle cells are oxidized (ox-LDL). The ox-LDL is hydrolyzed in the cellular lysosomes and peroxisomes to generate triglycerides (TGs) and free fatty acids. Through the Kennedy pathway, free fatty acids are utilized in the de novo biosynthesis of pPEs. Hydrolysis of pPEs by cPLA 2 leads to production of AA, which may be released into the extracellular space to alter plaque physiology and progression. pPEs can also be converted to PSs in the ER via PS synthases.

Article Snippet: CEA sections were then stained with Pentachrome, as previously described , or with anti-CEPT1 antibody (1:50; Proteintech, Rosemont, IL), followed by labeling with Alexa Fluor 594-conjugated secondary antibody (1:250; Invitrogen, Waltham, MA).

Techniques: Derivative Assay

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A) Min and Max diseased peripheral arterial segments were procured from the lower extremities of patients with severe PAD, and with or without T2D. Peripheral arterial segments were also harvested from health organ donors as controls. B-E) Western blot of CEPT1 in healthy, non-T2D Min, T2D Min, non-T2D Max, and T2D Max-diseased arterial segments was quantified relative to GAPDH (n=3-8). F-J) Immunostaining of arterial segments for CEPT1, ACOX1, VEGF2R, p-Akt, and p-eNOS that colocalized with CD31. Colocalization between two channels (fractions of CEPT1, ACOX1, VEGF2R, p-Akt, and p-eNOS colocalized with CD31) were expressed by Manders coefficients. Data are presented as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. P values were from 1-way ANOVA and unpaired Student t -test.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Western Blot, Immunostaining

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A) Experimental design of aortic cell isolation for scRNA-seq analysis. B) t-SNE of EC and stromal cells separated by genotypes. C) Pie-chart representing the relative abundance of each cluster. D) Violin plot of Cept1 gene expression in EC sub-cluster. E) Pathway analysis of Differential Expressed Gene (DEG) between Cept1 overexpression and control in EC1 sub-cluster (FDR adjusted p value of <0.05, log (FC) > 0.1 and pct.1 >0.6). F-H) Module score from significantly upregulated pathways.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Cell Isolation, Gene Expression, Over Expression, Control

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A) Graphical representation of the experiment design. B) Representative Doppler perfusion of HLI of Cept1 fl/fl Cre − and Cept1 fl/fl Cre + mice (n=8 mice) treated with STZ. C-D) Quantitative graphical representation of Doppler perfusion of the hind-paw and gastrocnemius muscle. E-F) Limb and ischemic damage severity were evaluated on days 3, 7, 14, and 21. G) Representative images showing HE and Isolectin B4 (IB4) staining in the gastrocnemius muscle at 21 days after artery ligation (n=8 mice). Scale bar =50 μm. H-I) Quantification of relative microvessel density and relative muscle fiber area (n=8 mice). Data are presented as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. p values were from unpaired Student t -test.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Staining, Ligation

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A-B) Cell proliferation of the EC isolated from the liver and lung of Cept1 fl/fl Cre − and Cept1 fl/fl Cre + (n=3). C) Migration of cells was assessed using scratch assay from EC from the liver and lung of Cept1 fl/fl Cre − and Cept1 fl/fl Cre + (n=3 per condition). D-F) Quantitative representations of percent change in migration at different time points (t= 0, 3, 6, and 16h; n=3). F-H) Aortic ring assay to evaluate the growth of sprouts from the aortic rings of Cept1 fl/fl Cre − and Cept1 fl/fl Cre + were represented and quantitively analyzed at day 6 (n=3). I-K) Quantitative analysis of percent of tube formation of cells (n=3). L) Efficiency of Cept1 lentiviral transduction was confirmed using RT-PCR (n=3). M) Proliferation of HUVEC treated with Cept1 lentiviral particles (n=3). N) Representative image of the change of cell migration from HUVEC treated with Cept1 lentiviral particle and its control (t=0, 6, and 16h). O) Quantitative analysis of percent change of migration of cells (n=3). P-R) Representative image of the tube formation from HUVEC treated with Cept1 lentiviral particle and its control. Quantitative analysis of percent of tube formation of cells (n=3). Data are presented as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. p values were from unpaired Student t -test.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Isolation, Migration, Wound Healing Assay, Aortic Ring Assay, Transduction, Reverse Transcription Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A) Western blotting demonstrates that CEPT1 content is increased in ECs isolated from Cept1 fl/fl Cre + mice. Representative blots of VEGF2R, PPAR α , ACOX1, p-Akt, t-Akt and p-eNOS, t-eNOS are also provided. B) Quantitative representation of protein content relative to GAPDH as a loading control (n=3). C) RT-PCR analysis demonstrates Cept1 is overexpressed in ECs isolated from Cept1 fl/fl Cre + mice (n=3). Gene expression of Pparα, Acox1 , Vegfa, and Vegf2r is also provided (n=3). D) Western blotting demonstrates that CEPT1 content is increased in HUVECs transduced with Cept1 lentiviral particles (MOI 5). Representative blots of VEGF2R, PPAR α , ACOX1, p-AKT, t-AKT and p-eNOS, t-eNOS are also provided. E) Quantitative representation of protein content relative to GAPDH as a loading control (n=3). F) RT-PCR analysis demonstrates Cept1 is overexpressed in transduced ECs (n=3). Gene expression of Pparα, Acox1 , Vegfa, and Vegf2r is also provided (n=3). Data are presented as mean ± SEM. * p<0.05; ** p<0.01; *** p<0.001. p values were from 1-way ANOVA and unpaired Student t -test.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Western Blot, Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Transduction

Journal: bioRxiv

Article Title: Endothelial Cept1 Promotes Post-Ischemic Angiogenesis in a Pparα -Dependent Fashion

doi: 10.1101/2025.03.11.642511

Figure Lengend Snippet: A-B) HUVECs were transfected with siRNA targeting Pparα or siRNA control (si-Ct) for 72 h, and the expression of Pparα was confirmed through RT-PCR. Relative gene expression of Cept1 and Pparα was performed using RT-PCR (n=3). C-D) Representative cell migration assay images at t= 0, 6, and 16h were taken from HUVEC Cept1 cDNA, Cept1 cDNA + si Pparα , and si Pparα . A quantitative graphical representation of cell migration was shown (n=3). E-F) Representative images and quantitative analysis of tubule formation of HUVEC with different conditions after 6h of incubation of Matrigel (n=3). G-H) EC from Cept1 fl/fl Cre + were treated with GW6471 (10uM), ZM323881 (80nM), LY294002 (30uM), and L- NAME (0.5uM) and cell migration at t = 0, 6, and 16h was assessed and quantified, relative to EC from Cept1 fl/fl Cre + and control (n=3). I-J) HUVEC were treated with GW6471 (10uM), ZM323881 (80nM), LY294002 (30uM), and L- NAME (0.5uM) and cell migration at t = 0, 6, and 16h was assessed and quantified, relative to control (n=3). Data are presented as mean ± SEM. * p<0.05; ** p<0.01. P values were from 1-way ANOVA and unpaired Student t -test.

Article Snippet: Proteins for western blot and immunostaining were detected with rabbit anti-CEPT1 (bs-12284R; Bioss USA), mouse anti-PPARα (66826-1-1g; Proteintech), rabbit anti-ACOX1 (10957-1-AP; Proteintech), rabbit anti-VEGF-A (bs-1665R; Bioss USA), rabbit anti-VEGF2r (#2472; Cell Signaling), rabbit anti-eNOS (PA1-037; Thermo Fisher), rabbit anti-p-eNOS (MA5-14957; Thermo Fisher), rabbit anti-Akt (MA5-14916; Thermo Fisher), rabbit anti-p-Akt (#9271; Cell Signaling), and mouse anti-CD31 (sc-376764; Santa Cruz Biotechnology, Inc).

Techniques: Transfection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Cell Migration Assay, Migration, Incubation