Fig 1: FK866 selectively inhibits the proliferation of A549. (A) The protein–protein interaction (PPI) of differentially expressed genes (DEGs) between H1975 and A549. The protein in the pink circle was less expressed in A549 than in H1975, and protein in the blue circle was more expressed in A549. (B) The top is the metabolic pathway of NAD+. The text in the box represents metabolite, and the text around the arrow represents metabolic enzyme. The middle part is expression level of NAMPT in H1975 and A549, the corresponding quantitative result is in the bar plot at the bottom (p-value=0.001, ***p < 0.005). (C) The efficacy of FK866 in vitro. H1975 and A549 were cultured in different concentrations of FK866 for 72h. The cell viability comparison of H1975 and A549 in the same dose group was calculated by t-test. The significant difference started from 10 nM (p < 0.0001: ****ns: not significant). (D) The efficacy of FK866 in vivo. Mice inoculated with A549 were given FK866 (20 mg/kg/day) or vehicle for eight days. The tumor volume at the same time point was compared by t-test in these two groups, and the significant difference started from the fourth day (p < 0.005: ***p < 0.0001: ****ns: not significant).
Fig 2: Effects of PNGL on Nampt expresssition and NAD + levels in the ischemic brains. (A) Representative images of Nampt (red) with DAPI (blue) staining in rat ischemic brains after MCAO/R injury, measured by the immunofluorescence assay; scale bar, 200μm. (B) The enlarged images of Nampt (Red) and the beta-III tublin(Green) with DAPI (Blue) staining in rat ischemic cortex regions, measured by the immunofluorescence assay, scale bar, 10μm. (C) The statistical data of Nampt fluorescence value, analyzed by using the Image J 2.44 softwae. (D) (E) The NAD and NADH concentrations in hippocampus and cortex in MCAO/R rat brains, determined by ELISA and specific assay kits. (n = 5-8 in each group). Mean values ± SEM; ∗p < 0.05, ∗∗p < 0.01 versus MCAO/R group; #p < 0.05, ##p < 0.01, versus sham group.
Fig 3: Effects of PNGL on the downstream SIRT1/2/3-MnSOD/PGC-1α signaling pathway mediated by the target Nampt in the ischemic brains. (A) The protein bands and relative expression levels of the PGC-1α and MnSOD in hippocampus, respectively, examined by western blot analysis and analyzed by using the Gel-Pro analyzer software. (B) The protein bands and relative expression levels of the PGC-1α and MnSOD in cortex. (C) the sirtuins, SOD2, and NADPH concentrations and levels in the ischemic hippocampus regions, detected by the ELISA assay kits. (D) the sirtuins, SOD2, and NADPH concentrations and levels in the ischemic cortex regions, detected by the ELISA assay kits Mean values ± SEM (n = 3); p < 0.05, ∗∗p < 0.01, versus MCAO/R group; #p < 0.05, ##p < 0.01 versus sham-operated group.
Fig 4: Effects of PNGL on the Nampt-NAD-SIRT1/2/3 signaling pathways in the ischemic brain. (A-B) The protein bands of Nampt and SIRT1/2/3 in the ischaemic brain sections examined by the western blot analysis. (C–F) the relative expression levels of Nampt, Sirt1, Sirt2 and Sirt3 proteins, respectively, quantified and analyzed by using Gel-Pro analyzer software. Mean values ± SEM (n = 3); p < 0.05, ∗∗p < 0.01, versus MCAO/R group; #p < 0.05, ##p < 0.01 versus sham-operated group.
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