Fig 1: Mechanism depicting the pathway through which naringin in combination with TMZ causes chemosensitization of TMZ towards glioblastoma cells by inhibiting the DNA repair pathway (PARP-1 and MGMT) and causing apoptosis in tumor cells by increasing the expression of p53, caspase-3, PI3K and by decreasing the expression of Bcl-2. Abbreviation: TMZ - Temozolomide, PARP-1 - Poly [ADP-ribose] polymerase 1, MGMT - O-6-Methylguanine-DNA Methyltransferase, Bcl2 - B-cell lymphoma 2, Phospho PI3K - Phospho Phosphatidylinositol 3-kinase, Bax - Bcl-2 Associated X-protein, Bak - B-cell lymphoma 2 (BCL-2) antagonist/killer, APAF-1 - Apoptotic protease activating factor 1.
Fig 2: Assessment of PARP-1 and MGMT. (A) PARP-1 (B) MGMT (T98-G cells) (C) PARP-1 (U87-MG cells) concentration measurement by Elisa kit. (D) Expression of PARP-1 in T98-G cells by Western blotting. Lanes 1, 2, 3, and 4 are the control (no drug), naringin (243 μM), TMZ (212.5 μM), and naringin + TMZ (243 + 212.5 μM) groups, respectively, in the presentative Western blot picture of PARP-1 protein. The loading control used was GAPDH. For quantification of blots and images ImageJ software was used. (E) Expression of PARP-1 and MGMT by flow cytometry analysis. The percentage of protein expression is represented on the histogram. Results were presented as mean ± SD, n = 3. The D'Agostino-Pearson omnibus post hoc test was used to analyze the results’ normality of distribution. One-way ANOVA and Tukey’s multiple comparison tests were used for every possible comparison between the study groups. (*) p < 0.05, vs control; (#) p < 0.05 vs naringin; ($) p < 0.05 vs TMZ. Abbreviation: PARP-1 - Poly [ADP-ribose] polymerase 1, MGMT - O-6-Methylguanine-DNA Methyltransferase, TMZ - Temozolomide.
Fig 3: Expression of apoptotic and anti-apoptotic proteins in U87-MG and T98-G cells by Western blotting and ICC. (A,B) p53, (C) Caspase - 3 (D) Bcl-2, (E–H) Bar graph showing the fluorescence intensity of p53, caspase-3 and BCL-2. (I) Lanes 1, 2, 3, and 4 are the control (no drug), naringin (243 μM), TMZ (212.5 μM), and naringin + TMZ (243 + 212.5 μM) groups, respectively, in the presentative Western blot picture of several proteins. The loading control used was GAPDH. (J,K) Bar graph showing the expression of p53 and Bcl-2. Scale bars: 50 µm. For quantification of blots and images ImageJ software was used. Results were presented as mean ± SD, n = 3. The D'Agostino-Pearson omnibus post hoc test was used to analyze the results’ normality of distribution. One-way ANOVA and Tukey’s multiple comparison tests were used for every possible comparison between the study groups. (*) p < 0.05, vs control; (#) p < 0.05 vs naringin; ($) p < 0.05 vs TMZ. Abbreviation: ICC - Immunocytochemistry, PARP-1 - Poly [ADP-ribose] polymerase 1, Bcl2 - B-cell lymphoma 2, TMZ - Temozolomide.
Supplier Page from Abcam for Human PARP1 ELISA Kit