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. 2016 Oct 19;6:35561. doi: 10.1038/srep35561

Figure 4. Analysis of NAMPT expression in HL-60 cells.

Figure 4

(a) Co-localization experiments of NAMPT (green staining) and PCNA (red staining) in HL-60S and HL-60R using confocal immunofluorescence analysis. Hoechst was used for nuclear staining. This experiment has been performed three times with identical results. (b) Western-blot analysis of cytosolic NAMPT expression in HL-60 cell lines. Cytosols were obtained by nitrogen cavitation and 50 μg of protein loaded per lane. Results were quantified by densitometry of the top band and normalized to β-actin loading control. The data are mean ± SEM of five independent experiments, *p < 0.05 (Student’s t test). (c) Effect of the p21-peptide-YIRS on NAD concentrations in HL-60S and HL-60R. Measurement of NAD concentration after 6 h treatment with the p21-peptide-YIRS or with 3-bromopyruvate (3-BrP) used as a glycolysis inhibitor. The data are mean ± SEM of four independent experiments performed in duplicates. Multicomparison ANOVA test (indicated by*) was used to evaluate the difference between HL-60S and HL-60R and Student’s t test (indicated by #) was used to evaluate the effect of the drug on either HL-60S or HL-60R, *p < 0.05, **p < 0.01, ***p < 0.001. (d) Measurement of the interaction between PCNA and NAMPT using surface plasmon resonnance SPR analysis. A typical sensorgrams showing injection of NAMPT at the indicated concentrations over PCNA covalently immobilized on a CM5 sensor chip is shown (RU, response units). Arrows and stars indicate the injection start and end points for each concentration. A fit representing a typical experiment performed three times is shown, and was obtained by global fitting of the data using the 2-state model. The kinetic parameters of the interaction determined by analyzing response at varying NAMPT concentrations are listed in Supplementary Table 2. (e) P21-peptide binding to PCNA does not interfere with NAMPT–PCNA association. An overlay of the sensorgrams obtained with NAMPT, P21-peptide and a mixture of both on PCNA is showed (RU, response units). Binding assay was performed on a Biacore T200 system as described in Methods. This representative experiment has been performed twice giving identical results.