Extended Data Fig. 5: EGR1, PTEN and reduced AKT activation mediate the cooperation between ET and fasting/FMD in BC cells.

a–d, MCF7 cells were transduced with either one of two independent EGR1-targeting shRNAs (#1 or #2). In a, d, cells were seeded in 96-well plates and cultured with or without STS conditions, TMX or FULV at the indicated concentrations (5 μM TMX or 10 μM FULV in a) or their combinations for 96 h and then cell viability was detected (a, d). In a (upper panel), b, c, cells were plated in 6-well plates and cultured with or without STS, 5 μM TMX, 10 μM FULV or their combinations for 48 h. Afterwards, cells were subjected to protein lysate generation, and EGR1, PTEN and phosphorylated (Ser473) and total AKT, as well as GAPDH, were detected by immunoblotting. e, MCF7 cells transduced with a control vector (n = 12), an EGR1- (n = 10) or a PTEN-targeting (n = 10) shRNA, or myr-AKT (n = 11) were injected into 6–8-week-old female BALB/c nude mice. Once tumours became palpable, mice were treated with FULV and weekly cycles of FMD, and tumour volume was monitored. n, number of tumours per treatment group. f, MCF7 cells engineered to express either a control scrambled shRNA or an EGR1-targeting shRNA (shRNA#1) were seeded in 96-well plates. Twenty-four hours later, cells were cultured with or without TMX (5 μM), STS, GDC0068 (1 μM), AZD5363 (500 nM), LY294002 (2 μM) or their combinations. Cell viability was detected after 96 h. g, h, MCF7 cells were transduced with either a PTEN-targeting shRNA or myr-AKT. Cells were plated in 6-well plates and subjected to protein lysate generation. PTEN (g), AKT (h) and vinculin (g, h) were detected by immunoblotting. Thereafter, cells were seeded in 96-well plates and cultured with or without STS, TMX (5 μM), FULV (10 μM) or their combinations for 96 h and then cell viability was detected. i, MCF7 cells were seeded in 6-well plates and 24 h later were cultured with or without STS + TMX (5 μM), STS + TMX + combined insulin (400 pM), IGF1 (5 ng/ml) and leptin (50 mg/ml) or STS + TMX + 17β-oestradiol (100 nM). Forty-eight hours later, cells were subjected to protein lysate generation and EGR1, PTEN, 4E-BP1 and vinculin were detected by immunoblotting. j, k, MCF7 cells that were engineered with either a control vector or myr-AKT were cultured for 48 h with or without TMX (5 μM), FULV (10 μM), STS or their combinations. Thereafter, cells were subjected to protein lysate generation and monitoring of EGR1 and GAPDH levels by immunoblotting (j) or for RNA extraction and EGR1 and PTEN mRNA quantification (k). In a (lower panel), d, f, g (lower panel), h (lower panel), k, data are from biological replicates (in d, data are from four biological replicates (wells) per treatment condition). In a (upper inset), b, c, g (upper inset), h (upper inset), i, j, one representative experiment out of three is presented. Loading controls (GAPDH, vinculin) were always run on the same gels that were used to detect other proteins. For gel source data, see Supplementary Fig. 1. Data are presented as mean ± s.d. (a, d, f–h, k) or s.e.m. (e). In a, d, f–h, k, P values were determined by two-tailed Student’s t-test. In e, data were analysed by two-way ANOVA with Bonferroni post-hoc test and by two-tailed Student’s t-test (tumour volumes at day 40).

Source data