Fig 2: BIM is a downstream effector for Uev1A to promote cell survival under serum starvation conditions in breast cancer cells. a, c pCMV6-FOXO1 was transfected into UEV1A-overexpressed MDA-MB-231-TR (a) or NCF7 (b) cells. The protein levels of ectopic FOXO1 and BIM were detected by western blot. The protein levels of ectopic Uev1A were monitored by an anti-HA antibody. b, d pcDNA4.0/TO/HA(+)-BIM was transfected into UEV1A-overexpressed MDA-MB-231-TR (b) or MCF7 (d) cells. The protein levels of ectopic BIM were monitored by western blot. Ecotopic Uev1A levels were monitored by an anti-HA antibody. e, f Growth curve of four groups of MDA-MB-231-TR (e) or MCF7 (f) cells under serum-deprived conditions. Vector only (CK), UEV1A-overexpressed (UEV1A), FOXO1- and UEV1A-overexpressed (UEV1A + FOXO1) and BIM- and UEV1A-overexpressed (UEV1A + BIM). All MDA-MB-231-TR cells were cultured with doxycycline. Each sample was measured in triplicate and repeated at least 2 times. **P < 0.01

Fig 3: FoxOs attenuate osteoclast progenitor proliferation and decrease mature osteoclast lifespan.(a) Representative pictures (left) and number of TRAP-positive multinucleated cells (MNCs) generated from BMMs (right, triplicates). Scale bar, 500 μm. (b) Cathepsin K (CatK), TRAP and calcitonin receptor (calcr) mRNA levels in osteoclast cultures (triplicates). (c) Representative pictures of BrdU (green) and propidium iodide (red) staining (left) and percentage of BrdU-positive cells in BMMs cultures (right; triplicates). Scale bar, 20 μm. (d–f) Same as a–d using cells from FoxO3C;LysM-Cre and LysM-Cre littermates. (g) Caspase-3 activity in mature osteoclast cultures (triplicates). AFU, arbitrary fluorescent units. (h) Cyclin D1 mRNA and (i) protein levels in BMM cell cultures (triplicate). Bars represent mean and s.d.; *P<0.05 versus respective FoxO1,3,4f/f or LysM-Cre cells by Student’s t-test.

Fig 4: Effects of silencing GCN2 on clonogenicity and protein expression in MCF-7, MCF-7-EpiRand MCF-7TaxRtreated with GSK2606414. MCF-7, MCF-7EpiR and MCF-7TaxR cells were transfected with either control or GCN2 siRNA and then treated with GSK2606414. A. MCF-7, MCF-7EpiR and MCF-7TaxR cells were treated with different concentrations of GSK2606414 every 48 h for 10 days after GCN2 depletion using siRNA. Fresh media were added and cells maintained for 10 days then stained with crystal violet in clonogenic assays. Representative images are shown. The clonogenic results of the cells were normalised to that of MCF-7, MCF-7-EpiR or MCF-7-TaxR cells without GCN2 silencing and GSK2606414 treatment (right panel). Data are representative of 3 independent experiments. Data represent means ± SEM. Significant *P < 0.05, ***P < 0.001. (n = 3; 2-way ANOVA). Significant *P < 0.05, ***P < 0.001. B. Expression levels of protein were then analysed after transfection with either control or GCN2 siRNA with and without 1 μM GSK2606414 treatment. Western blotting was performed to determine the protein expression levels for GCN2 (220 kDa), FOXO3 (85 kDa), P-PERK (140 kDa), PERK (140 kDa), P-eIF2a (38 kDa), eIF2a (38 kDa), P-p38 (38 kDa), p38 (38 kDa), P-JNK (54 kDa), JNK (54 kDa), p27Kip1 (27 kDa) and β-Tubulin (55 kDa).

Fig 5: a) Changes in signaling molecules in HK-2 cells previously treated with diatrizoate and incubated for an extended period of 22 h in the presence of darbepoetin.HK-2 cells were incubated with 75 mgI/mL sodium diatrizoate only for 2 h (designated as NaD) or with pre-treatment 0.25ug/mL or 1.0ug/mL darbepoetin for 2.5 h (designated as 0.25D and 1.0D respectively), prior to addition of sodium diatrizoate. Then the cells were cultured in fresh serum-free medium for a further 22 h (in the presence of darbepoetin), after which the cells were harvested and cell lysates were prepared and subjected to SDS-PAGE and Western blotting, as explained in the Materials and Methods section.b) Histograms of the densitometric measurements of the Western blots of phosphorylated p38 and Akt kinases, STAT-3, FOXO3a as represented in Fig. 3a (shown as the mean ± SE). Densitometric measurements of each protein band were normalized to the corresponding actin bands of the same blot. A pvalue < 0.05 indicating a significant difference was found for the following groups:PARP-1: NaD vs 0.25D; NaD vs 1.0D; 0.25D vs 1.0D.PARP-cp: NaD vs 1.0D.p-FOXO3a: NaD vs 1.0D.p-p38: NaD vs 0.25D; NaD vs 1.0D; 0.25D vs 1.0D.p-STAT-3: NaD vs 0.25D; NaD vs 1.0D.pAkt: NaD vs 1.0D.