Fig 1: Proposed mechanism of the inhibitory process. The novel polysaccharide derived from algae extract upregulates the phosphorylation of JNK, activates the downstream cascades of p53, caspase-9 and caspase-3, and then leads to the inhibition of cancer cell proliferation, induces cell apoptosis and cell cycle arrest. By contrast, the polysaccharide does not affect the cancer cell migration, which is mediated through the p38 MAPK signaling pathway or the downstream MMP-9/MMP-2. JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase.
Fig 2: Light micrographs of parts of hepatic lobules at 1 h-control (a, c, e), rGO-15 min (b) and 7 days—rGO treated rats (d, f). Representative photomicrographs of the hepatic parenchyma stained with Hematoxylin-eosin (a, b); treated for immunostaining of anti-caspase 9 (c, d) and stained with Masson’s trichrome staining for detecting collagen type 1 in normal (e) and rGO-treated (f) tissue. Insets depict collagen fibers around the blood vessels. pv portal vein; cv central vein. Bars 100 µm
Fig 3: hDT806 treatment induces apoptosis in the JHU-029 cells. (A) Cells were treated with vehicle (a) or 20 nM hDT806 (b) for 48 h and collected for annexin V and PI staining followed by flow cytometric analysis for apoptotic cells in JHU-029. (B) The populations of apoptotic cells were quantified for JHU-029. Data of four independent experiments are presented as mean ± SD (n = 4). (C) Total protein extracts were prepared from the cells treated with vehicle or hDT806 (20 nM) for 48 h. Western blot analysis was performed for PARP and cleaved PARP (a), p53 (b), caspase-9 (c), active caspase-3 (d), and β-actin (e). (D) Relative band intensities of cleaved PARP/PARP (a), band intensities of p53 (b), caspase-9 (c), and active caspase-3 (d) were quantified relative to the corresponding β-actin bands for comparisons. Data of three independent experiments are presented as mean ± SD (n = 3).
Fig 4: Effect of the novel polysaccharide on cell signaling pathways. MCF-7 cells were pretreated with 5 μM SP600125 (inhibitor of JNK) for 1 h, and then treated with polysaccharide (100 μg/ml) for 48 h. (A) p-JNK, p53, caspase-9, caspase-3 and p38 MAPK were determined by western blot analysis. (B) Effect of polysaccharide on protein expression by densitometry. (C) Effect of polysaccharide and SP600125 on protein expression by densitometry. Magnification, ×400. (D) Effect of polysaccharide and SP600125 on ROS generation in MCF-7 cells using H2-dichlorofluorescin diacetate. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. control; #P<0.05 and ##P<0.01 vs. polysaccharide group. p-, phospho-; JNK, Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.
Fig 5: Renal cortical sections stained with Hematoxylin-eosin (a, b), immunostained for caspase 9 (c, d) and stained with Masson’s trichrome (e, f). The sections were obtained from vehicle-treated (control—1 h) rats (a, c, e) and 15 min (b) and 7 days (d, f) after intravenous injection of rGO. Note that there is no change between the aspect of control and rGO-treated sections in each of the staining assessments. The widening of the tubular lumen and capsular space was observed in control and rGO samples due to perfusion fixation. Insets depict that collagen density (just in large blood veins of renal stroma) was the same for control and rats treated with rGO. 1 Renal glomeruli; 2 proximal tubule; 3 distal tubule; 4 collecting tubule; * Bowman’s space of the renal corpuscle. Arrows point to the immuno-positive apoptotic cells (brown). Bars 100 µm for all panels
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