Journal: Cells

Article Title: Caffeine Mitigates Adenosine-Mediated Angiogenic Properties of Choroidal Endothelial Cells Through Antagonism of A 1 Adenosine Receptor and PI3K-AKT Axis

doi: 10.3390/cells15010087

Figure Lengend Snippet: NECA promotes AKT phosphorylation in ChEC, which is dependent on PI3K but not VEGFR2 activity. ( A ). The expression levels of phosphorylated AKT, total AKT, and β-actin were detected by Western blot following incubation with DMSO (con), NECA (N), or LY294002 + NECA (Ly + N) treatment. ( B ) Relative expression of p AKT and t AKT (total AKT) was quantified using ImageJ and Prism software (* p < 0.05; ** p < 0.01; NS: not significant). NECA-mediated enhanced AKT phosphorylation was independent of VEFGR2 activity. ( C ) The expression of phosphorylated AKT, total AKT, and β-actin was detected by Western blot after DMSO (Con), NECA (N), or SU5402 + NECA (SU + N) treatment. ( D ) Relative expression of p AKT and t AKT was quantified using ImageJ and Prism software (* p < 0.05; NS: not significant, n = 5). NECA promotes the migration of ChEC, which is mitigated by incubation with the PI3K inhibitor. ( E ) Phalloidin (green) and DAPI (blue) staining were used to detect migrated ChEC following incubation with DMSO (con), NECA (N), or LY294002 + NECA (Ly + N) within Transwells. ( F ) The cells that were transferred through the Transwell were counted using ImageJ, and data were analyzed using the Prism software (* p < 0.05, n = 8).

Article Snippet: Caffeine citrate (51754-0500-1; Exela Pharma Sciences, Lenoir, NC, USA), Bz-ATP (2′(3′)-O-(4-benzoylbenzoyl) adenosine -5ʹ -triphosphate, tri(triethylammonium) salt (HY-136254), NECA (5′-N-ethylcarboxamidoadenosine, HY-103173), DPCPX (A 1 AR antagonist, HY-100937), Istradefylline (A 2A AR antagonist, HY-10888), MRS1754 (A 2B AR antagonist, HY-14121), MRS1523 (A 3 AR antagonist, HY-121119), SU5402 (VEGFR2 inhibitor, HY-10407) (Med Chem Express, Monmouth Junction, NJ, USA), LY294002 (PI3K inhibitor, L9908; Sigma), and AZ 11645373 (P2X7 receptor antagonist, 3317; R&D Systems, Minneapolis, MN, USA) were prepared with the desired concentration in EC growth medium.

Techniques: Phospho-proteomics, Activity Assay, Expressing, Western Blot, Incubation, Software, Migration, Staining

Journal: International Journal of Molecular Sciences

Article Title: Direct and Indirect Downstream Pathways That Regulate Repulsive Guidance Effects of FGF3 on Developing Thalamocortical Axons

doi: 10.3390/ijms26157361

Figure Lengend Snippet: FGF3 enhances the expression of repellent molecule Slit1 . ( A , A’ ) Western blot analyses showed that, compared with blank controls, FGF3 (300 ng/mL) in the culture media significantly increased the expression of Slit1 (n = 6). ( B , B’ ) In contrast, the FGFR inhibitor SU5402 (20 µM) reduced the expression of the repellent factor Slit1 (n = 6). ( C – H ) Enhanced immunofluorescence staining of DAPI (blue) and Slit1 (green) shows FGF3 upregulates Slit1 expression: explants of E4 diencephalon were cultured either for two days alone ( C , D ), with FGF3 ( E , F ), or with FGF3+SU5402 in the media ( G , H ). ( I ) Quantification of fluorescent signal from immuno-labelled Slit1 per volume of the thalamic explant. ( J ) Top GO items of RNA-seq were shown according to the gene counts. ( K ) Heatmap visualizes mRNA expression patterns of relative genes across different groups (controls and FGF3-treated). ( L ) Bar graphs for specific genes at mRNA transcription level ( Vglut2 , Mki67 , Pcna ), comparing their expression between controls and FGF3-treated explants. ns: Not-significant; FPKM, fragments per kilobase of transcript per million mapped reads; GO, gene ontology; **: p < 0.01; ***: p < 0.001; Scale bars = 40 µm.

Article Snippet: FGF3 protein was used, based on standards, at concentrations of 300 ng/mL or 500 ng/mL; its antagonist SU5402 (MedChemExpress, Monmouth Junction, NJ, USA) was used at 20 μM.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Cell Culture, RNA Sequencing