Fig. 4: The NDI1-NAD⁺-SIRT3 axis impairs tumorigenicity of neurofibromin-deficient cells.

A Spectrophotometric analysis of the NAD⁺/NADH ratio following NDI1 expression in Nf1^+/+ and Nf1^−/− MEFs. B Effect of NDI expression on soft agar growth of Nf1^−/− cells. Measurement of colony area (left) and representative pictures of colonies (right) are reported. NDI: cells expressing the pWPI-NDI1 construct; EV: cells expressing the pWPI empty vector. C Effect of nicotinic acid (NIC 5 mM, 3 weeks) on colony growth in soft agar of Nf1^−/− cells. D WB analysis of SIRT3 protein levels after expression of the pFUGW-GFP/SIRT3 construct (upper part) or SIRT3 knocking-out (sgSIRT3, lower part). Negative controls were cells expressing pFUGW-GFP (GFP, upper part) or scrambled single guide targeting EGFP (sgEGFP, lower part). E Effect of SIRT3 upregulation on soft agar colony formation of Nf1^−/− cells. F WB analysis of NDI1 expression in SIRT3 wild-type and knock-out (sgEGFP and sgSIRT3, respectively) neurofibromin-deficient cells. G Effect of NDI1 expression on soft agar growth of SIRT3 wild-type (sgEGFP) or knock-out (sgSIRT3) Nf1^−/− MEFs. In D, F, actin and calnexin were used as loading controls. Data are reported as mean ± SD values (n ≥ 3); ***p < 0.001; **p < 0.01 and *p < 0.05 with a Student’s t test analysis or One-way ANOVA followed by Bonferroni post-test (G).