Journal: Development (Cambridge, England)

Article Title: Developmental vascular pruning in neonatal mouse retinas is programmed by the astrocytic oxygen-sensing mechanism

doi: 10.1242/dev.175117

Figure Lengend Snippet: PHD2 deficiency promotes HIF-α accumulation and VEGF-A expression in retinal astrocytes. (A) Quantitative RT-PCR (qPCR) analysis for PHD2, VEGF-A and EPO. Total RNA used for qPCR was isolated from primary retinal astrocytes cultured from neonatal mice (P3). All data points were normalized against β-actin values. (B) ELISA for VEGF-A and EPO. Conditioned media from retinal astrocyte cultures were collected and assayed with VEGF and EPO ELISA kits. (C) Western blotting of nuclear extracts isolated from cultured retinal astrocytes. For quantification, PHD2, HIF-1α and HIF-2α signals were normalized to that of α-actin. n=5 mice/group. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: For ELISA, conditioned media were collected from retinal astrocyte cultures, and used for VEGF-A and EPO ELISA kits, respectively (R&D Systems, now part of Thermo Fisher; catalog numbers MMV00 for VEGF kit, and MEP00B for EPO kit).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Molecular Therapy

Article Title: Erythropoietin Gene-enhanced Marrow Mesenchymal Stromal Cells Decrease Cisplatin-induced Kidney Injury and Improve Survival of Allogeneic Mice

doi: 10.1038/mt.2011.162

Figure Lengend Snippet: Effect of conditioned media (CM) from mesenchymal stromal cells (MSCs) and from Epo-MSCs on P-Akt expression in kidney cells. MM55.K mouse kidney epithelial cells were exposed to cisplatin, with/without MSCs CM or Epo-MSCs CM (a) at increasing concentrations or (b) at highest concentrations with/without a neutralizing anti-IGF-1 antibody (IGF1 Ab), and/or anti-VEGF antibody (VEGF Ab), or anti-Epo antibody (Epo Ab), as detailed in the Materials and Methods. Cells were collected 42 hours later and lysates used for western blot analysis of phosphorylated Akt (Ser473) (P-Akt) expression, as well as of loading controls GAPDH or total Akt expression. (b) The bar graph represents the mean ± SEM of seven independent experiments.

Article Snippet: The Epo-MSCs CM was similarly tested alone or in combination with neutralizing antibodies against IGF-1, VEGF or Epo (MAB959) (R&D Systems), or against both IGF-1and VEGF.

Techniques: Expressing, Western Blot

Journal: Lipids in Health and Disease

Article Title: Deficiency of lysophosphatidic acid receptor 3 decreases erythropoietin production in hypoxic mouse kidneys

doi: 10.1186/s12944-024-02367-8

Figure Lengend Snippet: LPAR3 regulates the HIF-2α‒EPO axis in hypoxic Hep3B cells. A-D Hep3B cells were transfected with nonspecific siRNA (NC siRNA), LPAR3 siRNA-1 or LPAR3 siRNA-2 for 48 h and then exposed to 1% O 2 for 12 h. LPAR3 mRNA (A) and EPO mRNA expression levels (B) were determined via RT‒qPCR, EPO protein levels in culture medium (C) were determined via ELISAs, and HIF-2α protein levels in Hep3B cells (D) were determined via Western blotting. E-G Hep3B cells were starved in serum-free MEM for 24 h, pretreated in the presence or absence of 30 µM Ki16425 for 20 min, and then challenged with 10 µM 2S-OMPT for 1 h, followed by hypoxia exposure (1% O 2 ) for 6 h. EPO mRNA expression levels (E) were determined via RT‒qPCR, EPO protein levels in culture medium (F) were determined via ELISAs, and HIF-2α protein levels in Hep3B cells (G) were determined via Western blotting. n = 3 in each group. The p value was determined by 2-way analysis of variance (ANOVA) followed by a post hoc test. *** p < 0.001, **** p < 0.001 relative to the normoxia control of the same treatment; ## p < 0.01, ### p < 0.001, #### p < 0.0001, between different siRNA-treated or different drug-treated groups; mean ± SD

Article Snippet: EPO levels in mouse plasma were determined by ELISAs (Cat No: KE10031; Proteintech, IL, USA).

Techniques: Transfection, Expressing, Western Blot, Control

Journal: Lipids in Health and Disease

Article Title: Deficiency of lysophosphatidic acid receptor 3 decreases erythropoietin production in hypoxic mouse kidneys

doi: 10.1186/s12944-024-02367-8

Figure Lengend Snippet: LPAR3 promotes the HIF-2α‒EPO axis via the PI3K‒AKT pathway. A Heatmap of the downregulated genes enriched in the PI3K‒Akt signaling pathway in the kidneys of hypoxic mice with LPAR3 deficiency. B The total renal AKT and p-AKT levels in the WT and Lpar3 −/− mice under normoxic or hypoxic conditions (8% O 2 ) were determined by Western blotting, n = 6. The p value was determined by 2-way analysis of variance (ANOVA) followed by a post hoc test. *** p < 0.001 compared with the isogenic normoxia control; # p < 0.05 between different genotype groups; mean ± SEM. C Hep3B cells were transfected with nonspecific siRNA (NC siRNA), LPAR3 siRNA-1 or LPAR3 siRNA-2 for 48 h and then exposed to 1% O 2 for 12 h. Total AKT and phosphorylated AKT (p-AKT) levels were determined via Western blotting. D-F Hep3B cells were starved in serum-free MEM for 24 h, pretreated with or without LY294002 (10 µM) for 20 min, and then challenged with 2S-OMPT for 1 h, followed by hypoxia exposure (1% O 2 ) for 6 h. Total AKT, p-AKT, and HIF-2α protein levels were determined by Western blotting (D) . The EPO mRNA expression levels (E) were determined via RT‒qPCR, and the EPO protein levels in the culture medium (F) were determined via ELISAs. The p value was determined by 2-way analysis of variance (ANOVA) followed by a post hoc test. ** p < 0.01, **** p < 0.0001 relative to the normoxia control of the same treatment; ### p < 0.001, #### p < 0.0001, between different drug-treated groups; mean ± SD

Article Snippet: EPO levels in mouse plasma were determined by ELISAs (Cat No: KE10031; Proteintech, IL, USA).

Techniques: Western Blot, Control, Transfection, Expressing