Fig. 4: Pharmacological inhibition of TFF3 decreases survival of dormancy-like ER+MC models.

a Total cell number: GraphPad Prism was used to generate an IC₅₀ and combination index (CI) table for FC, FD, FR, TC, TD, or TR cells treated with the indicated concentration of AMPC, Fulvestrant (F), or Tamoxifen (T) for 72 h (Fig. S17) (n = 3). Chou-Talalay analysis assessed the combination of AMPC (A) with FUL or TAM. The fraction (%) of live cells was determined by counting the total number of cells. CompuSyn software was used to calculate the logarithmic CI values corresponding to the cell fraction affected (Fa) as described in the methods section. CI value of <1 indicated synergism, =1 indicated additive synergy, and >1 indicated antagonism. b Total cell number: Using GraphPad Prism software, the IC₅₀ values and fold change were calculated for FD or TD cells treated with indicated concentrations of Fulvestrant (F) or Tamoxifen (T) for 72 h (Fig. S20c) (n = 3). c Synergy score: The heatmap represents the combination index of AMPC with all drugs in the Table S1 drug list (Fa=0.9) using Chou-Talalay (left) and Bliss-independent models at 1 µM AMPC (right) in FC, FD, FR, TC, TD, or TR cells treated with indicated concentration of agents for 6 days (Fig. S21, S22) (n = 3). d Oncogenic assay: the heatmap shows foci formation of Fig. S26a, cell viability fraction in 3D growth determined by AlamarBlue assay. FD or TD cells were treated with vehicle (Veh), 1 µM AMPC (A), 1 µM Tamoxifen (T), 1 µM Fulvestrant (F), CDK4/6 inhibitors (Abemaciclib = AB 1 µM, Ribociclib = R 10 µM, Palbociclib = P 1 µM) and combinations thereof for 10 days. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). e Caspase 3/7 and FACS analysis: The heatmap represents CASPASE 3/7 activity, fluorescence-activated cell sorting (FACS) analysis of annexin-V and propidium iodide (PI) staining of apoptotic cell death and ALDH-positive cell population in FD or TD cells. CASPASE 3/7 activity was evaluated using the ApoTox-Glo Triplex Assay Kit in FD or TD cells treated with vehicle (Veh), 1 µM AMPC (A), 1 µM Abemaciclib (AB), 10 µM Ribociclib (B), 1 µM Palbociclib (P) and combinations thereof for 10 days in 3D Matrigel culture. Cells were treated for 72 h to measure apoptosis. The ALDH-positive cell population was determined using the ALDEFLUOR assay after exposure to the treatments for 48 h. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparisons test (n = 3). f Western blot analysis: A heatmap was generated using quantification data of Western blot analysis to evaluate the levels of markers as shown in Fig. S29 (Fig. S34). The data were standardized using the z-score method in the row direction. Statistical analysis was performed using a one-way ANOVA followed by Tukey’s multiple comparison tests (n = 3). The quantitative data are expressed as mean ± SD. The symbols (*) indicate statistical significance compared to the corresponding control group. p < 0.05(*), p < 0.01(**) and p < 0.001(***) were considered statistically significant.