Journal: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: Effect of IngC on apoptosis pathway on glioma cell lines. ( A,B ) Panel of 35 proteins related to apoptosis. The data represented by the heat maps show the proteins modulated after 24 and 72 h of IngC treatment (3X IC 50 value (13.09 μM) in glioma cells, GAMG and U373. ( C ) Cells were treated with 3X IC 50 concentrations of IngC (24 and 72 h) for the indicated time periods. GAMG and U373 cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-DR5. The tubulin was used as an internal control to normalize the amount of proteins applied in each lane. This data is representative of three independent experiments. ( D ) After the IngC treatment with 3X IC 50 for 72 h, U373 cells were fixed, stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ( E ) After IngC treatment with 3X IC 50 for 72 h, DNA fragmentation in U373 cell line was measured with the TUNEL assay using flow cytometry. The graphs are representative of at least three independent experiments performed in duplicate. n.s. means non-significant. ** p < 0.005

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Western Blot, Staining, TUNEL Assay, Flow Cytometry

Journal: Molecules

Article Title: Semi-Synthetic Ingenol Derivative from Euphorbia tirucalli Inhibits Protein Kinase C Isotypes and Promotes Autophagy and S-Phase Arrest on Glioma Cell Lines

doi: 10.3390/molecules24234265

Figure Lengend Snippet: IngC promotes autophagy on glioma cells. ( A ) Cells were treated with the IC 50 value of IngC for the indicated time periods. GAMG cell lysates (20 μg per lane) were analyzed using immunoblotting with anti-LC3. ( A , B ) are representative of three independent experiments with GAMG. Tubulin was used as an internal control to normalize the amount of proteins applied in the treatment without bafilomycin A1 (Baf). ( C ) Development of AVO in IngC-treated cells by detecting green and red fluorescence in acridine orange-stained cells using FACS analysis. U373 cells were treated with IngC (IC 50 value), and Baf (20 nM) for 72 h. The graphs are representative of at least two independent experiments. FITC indicates green color intensity, while PerCP shows red color intensity. ( D ) Effect of baf on GAMG and U373 cell viability of IngC-treated cells. At 3 h after exposure to IngC, baf was added and cultured until 72 h and evaluated by MTS assay. The viability of the untreated cells was considered as 100%. Results shown are the means ± S.D. of three independent experiments. ( E ) Effect of Baf on IngC-induced apoptosis. After, IngC and bafilomicyn treatment for 72 h, GAMG cells were stained with annexin V-FITC/PI and analyzed by FACScan. Data shown are representative of three independent experiments. ** p < 0.005 and *** p < 0.0001

Article Snippet: The relative protein expression levels of a panel of 35 proteins related to apoptosis and 26 proteins related to cellular stress were obtained while using the Proteome Profiler Human Apoptosis Array (R&Dsystems- #ARY009) and Proteome Profiler Human Cell Stress Array (R&Dsystems-#ARY018), according to the manufacturer’s instructions and as previously reported [ ].

Techniques: Western Blot, Fluorescence, Staining, Cell Culture, MTS Assay