Journal: NPJ Breast Cancer

Article Title: The interplay between FOXO3 and FOXM1 influences sensitivity to AKT inhibition in PIK3CA and PIK3CA/PTEN altered estrogen receptor positive breast cancer

doi: 10.1038/s41523-025-00752-9

Figure Lengend Snippet: A Cell viability assay of T47D CTRL, MCF7 CTRL, T47D PTEN-KO and MCF7 PTEN-KO treated for 120 h with DMSO, 0.5 μM capivasertib and 0.5 μM alpelisib and MCF7 CTRL and MCF7 PTEN-KO treated for 120 h with DMSO, 1 μM capivasertib and 1 μM alpelisib. Data were normalised to DMSO; plotted as mean ± SEM ( n = 3). Statistical analysis 2-way ANOVA test vs vehicle-treated, alpelisib treated or capivasertib treated * p < = 0.05, ** p < = 0.01, *** p < = 0.001, **** p ≤ 0.0001. B Western blot using MCF7 CTRL, MCF7 PTEN - KO, T47D CTRL and T47D PTEN - KO protein lysates after 96 h treatment, characterising modulation of biomarkers of the PI3K-AKT pathway. All MCF7 cells were treated with 1 μM capivasertib and 1 μM alpelisib. All T47D cells were treated with 0.5 μM capivasertib and 0.5 μM alpelisib. βactin was used as loading control. C Gene expression analysis relative to vehicle treatment in T47D CTRL, T47D PTEN-KO, MCF7 CTRL and MCF7 PTEN-KO of FOXM1 target genes following 5 days treatment with capivasertib and alpelisib. All MCF7 cells were treated with 1 μM capivasertib and 1 μM alpelisib. All T47D cells were treated with 0.5 μM capivasertib and 0.5 μM alpelisib. Statistical analysis 2-sided students t -test vs vehicle-treated * p ≤ 0.05, ** p < = 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 ( n = 6). D Comparison of the DNA synthesis (S-phase) in MCF7 PTEN-KO and T47D PTEN-KO after 5 days treatment with capivasertib and alpelisib. MCF7 cells treated with 1 μM capivasertib and 1 μM alpelisib. T47D cells treated with 0.5 μM capivasertib and 0.5 μM alpelisib. Data are mean of 2 independent experiments ± SD. E Cell viability assay of PDXO models (CTC174 and CTG3283) treated with 1 μM capivasertib and 1 μM alpelisib for 5 days. Data was normalised to DMSO and plotted as mean ± SEM ( n = 3). Statistical analysis one-way ANOVA test vs DMSO-treated * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. F PDXO CTG3283 characterisation and comparison with PDX CTG3283 by western blot after 5 days treatment to validate biomarker modulation upon treatment with 1 μM capivasertib and 1 μM alpelisib. βactin was used as loading control.

Article Snippet: FOXM1 CST #20459 , 1:500.

Techniques: Viability Assay, Western Blot, Control, Gene Expression, Comparison, DNA Synthesis, Biomarker Discovery

Journal: NPJ Breast Cancer

Article Title: The interplay between FOXO3 and FOXM1 influences sensitivity to AKT inhibition in PIK3CA and PIK3CA/PTEN altered estrogen receptor positive breast cancer

doi: 10.1038/s41523-025-00752-9

Figure Lengend Snippet: A FOXO3 immunofluorescence staining and quantification of its localisation in T47D PTEN-KO cells treated for 3 days with DMSO, 0.5 μM capivasertib and 0.5 μM alpelisib. Images were captured using the confocal microscope Yokogawa CV8000, 63× magnification. Immunofluorescences were performed to visualise FOXO3 localisation using a specific secondary antibody (AF488) (green) and F-actin with DyLight 594 Phalloidin (orange). Nuclei were stained with Hoechst (blue). Scale bar 20 µm. Data was normalised to vehicle and plotted as mean ± SD. Statistical analysis one way ANOVA vs vehicle-treated, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. B Characterisation by western blot of T47D PTEN-KO and MCF7 PTEN-KO where FOXM1 and FOXO3 were depleted after 3 days from guides transfection. β actin was used as loading control. C FOXM1 mRNA analysis in PTEN-KO MCF7 and T47D cells following FOXO3 or FOXM1 depletion (after 3 days from transfection). Data normalised to CTRL. Data are presented as mean of n = 2 ± SD. Statistical analysis t test, * p < 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. D Characterisation by western blot of T47D PTEN-KO FOXO3-KO and MCF7 PTEN-KO FOXO3-KO cells treated 96 h with DMSO, capivasertib and alpelisib (MCF7 PTEN-KO/FOXO3-KO treated with 1 μM capivasertib and 1 μM alpelisib; T47D PTEN-KO/FOXO3-KO treated with 0.5 μM capivasertib and 0.5 μM alpelisib). Lysates from PTEN-KO CTRL and FOXM1-KO cells were used as control. βactin was used as loading control. E Cell viability assay of T47D PTEN - KO/ FOXO3-KO and T47D PTEN-KO/ FOXM1-KO and MCF7 PTEN-KO/ FOXO3-KO and MCF7 PTEN-KO/ FOXM1-KO after 5-day treatment with DMSO, 1 μM capivasertib and 1 μM alpelisib (MCF7); and 0.5 μM capivasertib and 0.5 μM alpelisib (T47D). Data was normalised to DMSO; plotted as mean ± SD ( n = 3). Statistical analysis 2-way ANOVA test vs vehicle-treated, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. F Cell viability assay of T47D PTEN-KO/ FOXO3-KO / Ctrl KO after 5-day treatment with DMSO, 0.5 μM capivasertib and 100 μM fulvestrant. Data was normalised to DMSO treatment; statistical analysis one-ways ANOVA test vs vehicle-treated plotted as mean ± SD * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 ( n = 3). G Characterisation by western blot of FOXM1 protein levels, lysates from PTEN- KO cells modulated to transiently overexpress FOXM1 (FOXM1_OE) or GFP (GFP_OE). βactin was used as loading control. H Cell proliferation assay (Day 5) of T47D PTEN-KO FOXM1_OE or GFP_OE after treatment with DMSO, 0.5 μM capivasertib monotherapy and 0.5 μM capivasertib + 100 nM fulvestrant (combination). Data was normalised to DMSO; plotted as mean ± SD * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001 ( n = 6).

Article Snippet: FOXM1 CST #20459 , 1:500.

Techniques: Immunofluorescence, Staining, Microscopy, Western Blot, Transfection, Control, Viability Assay, Proliferation Assay

Journal: NPJ Breast Cancer

Article Title: The interplay between FOXO3 and FOXM1 influences sensitivity to AKT inhibition in PIK3CA and PIK3CA/PTEN altered estrogen receptor positive breast cancer

doi: 10.1038/s41523-025-00752-9

Figure Lengend Snippet: A Characterisation by western blot of T47D and MCF7 capivasertib resistant lines (capiR) compared to T47D and MCF7 parental cell lines. 2 or 3 pools were generated per each cell line (capiR R1, R2 or R3). Vinculin was used as loading control. Parental cells +10 μM capivasertib (24 h), capiR with continuous 10 μM capivasertib treatment (cc), capiR without capivasertib treatment for 120 h (cw), capiR without capivasertib treatment for 96 h and re-dosing (10 μM capivasertib) for 24 hours (24 h). B Dose response graphs of T47D capiR R1 line treated for 7 days with 10 μM capivasertib. Dose responses were calculated using Graphpad Prism. C Characterisation by western blot of FOXM1_KO vs control_KO in T47D capiR R1 line (expressing Cas9). βactin was used as loading control. D Depletion of FOXM1 (FOXM1_KO) in T47D capiR R1 cells. 3day-KO followed by a treatment with 0.5 μM capiva growth curve plotted as mean ± SD ( n = 3). Statistical analysis 2-way ANOVA test vs CTRL * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Representative images plotted for each time point and condition. Scale bar 400 µm. E Schematic graph showing AKT, FOXM1 and FOXO3 roles in PI3K/AKT pathway inhibition.

Article Snippet: FOXM1 CST #20459 , 1:500.

Techniques: Western Blot, Generated, Control, Expressing, Inhibition

Journal: NPJ Breast Cancer

Article Title: The interplay between FOXO3 and FOXM1 influences sensitivity to AKT inhibition in PIK3CA and PIK3CA/PTEN altered estrogen receptor positive breast cancer

doi: 10.1038/s41523-025-00752-9

Figure Lengend Snippet: Oligonucleotides

Article Snippet: FOXM1 CST #20459 , 1:500.

Techniques: Sequencing

Journal: NPJ Breast Cancer

Article Title: The interplay between FOXO3 and FOXM1 influences sensitivity to AKT inhibition in PIK3CA and PIK3CA/PTEN altered estrogen receptor positive breast cancer

doi: 10.1038/s41523-025-00752-9

Figure Lengend Snippet: Antibodies

Article Snippet: FOXM1 CST #20459 , 1:500.

Techniques: