Additional file 9 of Distinct mechanisms of resistance to fulvestrant treatment dictate level of ER independence and selective response to CDK inhibitors in metastatic breast cancer
Creators: name: Kaminska, Kamila
name: Akrap, Nina
name: Staaf, Johan
name: Alves, Carla L.
name: Ehinger, Anna
name: Ebbesson, Anna
name: Hedenfalk, Ingrid
name: Beumers, Lukas
name: Veerla, Srinivas
name: Harbst, Katja
name: Ehmsen, Sidse
name: Borgquist, Signe
name: Borg, Åke
name: Pérez-Fidalgo, Alejandro
name: Ditzel, Henrik J.
name: Bosch, Ana
name: Honeth, Gabriella
Publisher: name: figshare
Year: 2021
Subjects: subject: Medicine
subject: Cell Biology
subject: Pharmacology
subject: Biotechnology
subject: Ecology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 9. Figure showing cell cycle regulation and response to CDK inhibitors. A) Cell cycle profiles for parental (-P) and fulvestrant-resistant (-FR) CAMA-1 and MCF7 cells showing the proportion of cells in G0/G1, S, and G2/M phase, respectively, after treatment with 100 nM fulvestrant for 72 h (+F) or untreated (ctrl). Combined data from two biological replicates. Statistical differences between the proportion of cells in G0/G1-phase were calculated using student’s t-test. * represents p-value ≤0.05, ns represents no statistical differences. B) Western blotting for expression of cell cycle regulating proteins in parental and fulvestrant-resistant T47D and EFM-19 cells after treatment for 24 h with 100 nM fulvestrant (+F) or DMSO control (-F). Representative data from three biological replicates. β-actin was used as loading control. Quantification of band intensities is presented in Additional file 4 L-M. C) Palbociclib IC50 values in parental and fulvestrant-resistant cells. Calculated from graphs presented in (D) and in Fig. 5b. D) Dose-response curves for parental and fulvestrant-resistant T47D and EFM-19 cells in response to 6-days treatment with increasing concentrations of the CDK4/6 inhibitor palbociclib (1 nM to 10 μM). Graphs represent combined data (average ± SEM) from three biological experiments with three technical replicates each. IC50 values are shown in (C). E) CDKi3 IC50 values in parental and fulvestrant-resistant cells. Calculated from graphs presented in (F) and in Fig. 5c. F) Dose-response curves for parental and fulvestrant-resistant T47D and EFM-19 cells in response to 6-days treatment with increasing concentrations of the CDK1/2 inhibitor CDKi3 (100 pM to 50 μM). Graphs represent combined data (average ± SEM) from three biological experiments with three technical replicates each. IC50 values are shown in (E). G) Time in weeks needed for each parental and fulvestrant-resistant cell line to develop resistance to palbociclib, counted from initial exposure to 100 pM dose until the cells were able to proliferate in presence of 1 μM palbociclib. H) Dose-response curves for parental (-P, solid black lines), fulvestrant-resistant (-FR, solid red lines), palbociclib-resistant (-PalbRes, black dotted lines) and double fulvestrant- and palbociclib-resistant (-FR-PalbRes, red dotted lines) CAMA-1 and MCF7 cells in response to 6-days treatment with palbociclib (1 pM to 10 μM). Graphs represent combined data (average ± SEM) from two biological experiments with three technical replicates each. IC50 values are shown (I). I) Palbociclib IC50 values for graphs in (H). J) Fulvestrant IC50 values for graphs presented in Fig. 5d. P-values in (C, E, I and J) were calculated using Extra sum-of-squares F test.