Extended Data Fig. 3 |. FMD-mediated increase in ET anti-cancer activity is mediated by the reduction in circulating insulin, IGF1 and leptin.

a, MCF7 cells were seeded in 96-well plates and cultured for 96 h with or without STS conditions (1% FBS, 0.5 g/l glucose), low-serum conditions (1% FBS, 1 g/l glucose), TMX or FULV at the indicated concentrations, or combinations of these treatments. Thereafter, cell viability was determined. One representative experiment out of three is presented. Cell viability in each treatment condition was calculated from three (MCF7 cells) or four (T47D, ZR-75–1 cells) biological replicates (wells). b, c, Serum β-hydroxybutyrate, IGFBP1, IGFBP3, adiponectin, TNF and IL-1β concentrations in female 6–8-week-old BALB/c nude mice treated with fasting/FMD (or ad libitum diet) with or without TMX or FULV. In all mouse groups, serum was collected at the end of the fasting/FMD. Data are from biological replicates. d, MCF7 cells were injected into 6–8-week-old female BALB/c nude mice; once tumours became palpable, mice were randomized to be treated with ad libitum diet (n = 6), FULV (n = 6), FULV + weekly FMD (n = 6), FULV + FMD + i.p. insulin (n = 5), FULV + FMD + IGF1 (n = 5), FULV + FMD + leptin (n = 5), or FULV + FMD + combined insulin, IGF1 and leptin (FRFs; n = 7). FRF administration was withdrawn at day 35 (crossover), while it was started in mice that had only been treated with FULV + FMD. Right, tumour volume in each treatment group at day 57; n, number of tumours per treatment group. e, MCF7 cells were seeded in 96-well plates and cultured for 96 h with or without STS, 10 μM FULV, insulin, IGF1, leptin (at the indicated concentrations), combined insulin, IGF1 and leptin, or combinations of these treatments. Cells were then imaged by light microscopy (left) and cell viability was determined (right). One representative experiment out of three is presented. f, At the end of the experiment shown in d, tumour masses were isolated for protein lysate generation. Total and phosphorylated AKT (Ser473) and p70S6K (Thr389) and vinculin (on the same gel) were assessed by immunoblotting. One representative experiment out of three (n = 5 or 6 tumour masses/treatment group were evaluated) is presented. For gel source data, see Supplementary Fig. 1. Data are from biological replicates and represent mean ± s.d. (a–c, e, right) or s.e.m. (d). Data were analysed by two-tailed Student’s t-test.