Extended Data Fig. 6: Phospho-mimicking mutation of FOXM1 (S376E) attenuates tumor cell growth and metastasis.
From: Targeting FOXM1 condensates reduces breast tumour growth and metastasis
a, Immunoblot (IB) analysis of the parental and FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1 WT, S376A and S376E. b, Growth curves of FOXM1 knock-out MDA-MB-231 cells stable expressing Myc-FOXM1 WT, S376A and S376E; n = 3 independent experiments. c, The relative tumor volume (fold) (left) and tumor weights (right) at the endpoint (day 35) of a. n = 8 mice per group. d, Experimental procedure in vivo: nude mice were subcutaneously injected with FOXM1 WT or S376A MDA-MB-231 cells (2 × 106 cells per mouse) on day 0, followed by intraperitoneal injection of metformin (200 mg/kg) every other day as indicated (top). The visible tumors were measured at the indicated days. Bright view of tumors from each group at week 5 (bottom); n = 6 mice per group. e, Tumor volume from each group was measured at the indicated times in d. f, The relative tumor volume (fold) (left) and tumor weights (right) at the endpoint (day 35) of d. n = 6 mice per group. g, Immunoblot (IB) analysis of the parental and FOXM1 knock-out 4T1 cells stable expressing Myc-FOXM1 WT, S376A and S376E. h-j, Experimental procedure in vivo: nude mice were subcutaneously injected with FOXM1 knock-out 4T1 cells stably expressing FOXM1-WT or FOXM1-S376E (2 × 106 cells per mouse) on day 0 and tumors were grown for 4 weeks (h, top); n = 5 mice per group. The visible tumors were measured at the indicated days. Bright view of tumors from each group at week 4 (h, bottom). Tumor volume from each group was measured at the indicated times (i). The relative tumor volume (fold) (left) and tumor weights (right) at the endpoint (day 28) (j). k, l, Experimental analysis in vivo: 4T1.2 cells stably expressing FOXM1-WT or FOXM1-S376E (2 × 105 cells per mouse) were tail vein-injected into Balb/C mice (n = 5 mice per group). Lung metastasis was measured by bioluminescence imaging (BLI) (k). Photon flux (left) and representative images (right) in each experimental group followed in time (l). Illustration in panel k adapted from ref. 29, Springer Nature. m, Experimental procedure in vivo: nude mice were subcutaneously injected with FOXM1 knock-out 4T1 cells stably expressing FOXM1-WT or FOXM1-S376A (2 × 106 cells per mouse) on day 0, followed by intraperitoneal injection of metformin (200 mg/kg) every other day as indicated (top left). The visible tumors were measured at the indicated days. Bright view of tumors from each group at week 4 (bottom left); n = 6 mice per group. Tumor volume from each group was measured at the indicated times (right). Illustrations in panels d,h,m adapted from ref. 28, EMBO Press. n, The relative tumor volume (fold) (left) and tumor weights (right) at the endpoint (day 28) in m. n = 6 mice per group. o, Expression of p-S376-FOXM1 (left) and p-T172-AMPK (right) of normal tissues (n = 10) and breast cancer patients (n = 100). H score of p-S376-FOXM1 and p-T172-AMPK in every tissue sample was calculated. p, Expression of p-S376-FOXM1 (left) and p-T172-AMPK (right) of normal tissues (n = 10) and breast cancer patients (n = 100) at different malignant stage (I-III). H score of p-S376-FOXM1 and p-T172-AMPK in every tissue sample was calculated. q, Immunoblot (IB) of TCL and anti-FOXM1 IP derived from paired samples of non-tumorous breast (N) and breast tumor (T) from the same patient; n = 6 patients. Data are representative of at least three independent experiments (b). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (c, f, j, n-p) or two-way ANOVA (b, e, i, l, m). Uncropped gel images are provided in Supplementary Fig. 1.
