Additional file 1 of Suppression of TNBC metastasis by doxazosin, a novel dual inhibitor of c-MET/EGFR
Creators: name: Kim, Seongjae
name: Park, Jung Min
name: Park, Soeun
name: Jung, Eunsun
name: Ko, Dongmi
name: Park, Minsu
name: Seo, Juyeon
name: Nam, Kee Dal
name: Kang, Yong Koo
name: Lee, Kyoungmin
name: Farrand, Lee
name: Kim, Yoon-Jae
name: Kim, Ji Young
name: Seo, Jae Hong
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: Additional file 1: Supplementary Fig. S1. Effect of DOXA on apoptosis in mouse TNBC 4T1 cells. (A-B) Cells were treated with DOXA (0–20 µM) for 48 h. (A) The sub-G1 population was quantified using flow cytometry (**p&lt;0.01). (B) The proportion of early and late apoptotic cells in the presence or absence of DOXA was determined by annexin V/PI staining (***p&lt;0.001). Results are presented as mean values ± SEM of at least three independent experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Supplementary Fig. S2. Effect of DOXA on the expression of apoptosis-related proteins in 4T1 cells. Immunoblot analyses of PARP, cleaved caspase-3 and cleaved caspase-7 expression in 4T1 cells following exposure to DOXA (0-20 µM, 48 h). Quantitative graphs of protein content relative to GAPDH expression are shown in the right panel (**p&lt;0.01). Results are presented as mean values ± SEM of at least three independent experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Supplementary Fig. S3. Effect of amivantamab on cell viability in TNBC cells. (A) MDA-MB-231 and (B) BT549 cells were treated with various concentrations of amivantamab (1-1000 μg/ml) or control vehicle (DMSO) for 72 h. (C) Cell viability, 50% inhibitory concentration (IC50) and 95% confidence interval (CI95) values were determined by MTS assay (*p&lt;0.0001). Supplementary Fig. S4. Immunoblotting analysis for c-MET expression in TNBC cells after treatment with DOXA, corresponding to Fig. 2D in the main text. Quantitative graphs represent the ratio of p-c-MET/total-MET expression levels in the presence or absence of DOXA (30-40 μM, 48 h) in MDA-MB-231 and BT549 cells (***p&lt;0.001). Results are presented as mean values ± SEM of at least three independent experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Supplementary Fig. S5. Effect of DOXA on p-glycoprotein mRNA expression in MDA-MB-231 cells. The relative mRNA expression of P-glycoprotein (P-gp) was analyzed by quantitative RT-PCR in MDA-MB-231 cells after treatment with DOXA (40 μM, 0-48 h). The quantitative graph represents the ratio of P-gp/GAPDH mRNA expression. Results are presented as mean values ± SEM of at least three independent experiments and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Supplementary Fig. S6. Immunoblot analysis of immunoprecipitated with EGFR for CD44 expression in TNBC cells after treatment with DOXA. MDA-MB-231 and BT549 cells were treated with DOXA (40 μM) for 24 h. Cell lysates were immunoprecipitated (IP) with anti-EGFR antibody and analyzed by immunoblotting (IB) with CD44 antibody. Whole lysate used for immunoprecipitation indicates input group and IgG means normal mouse immunoglobulin G. Supplementary Fig. S7. Impact of DOXA on BCSC-like property in 4T1 cells. (A-B) Cells were treated with DOXA (0-20 μM) for 48 h. (A) CD44high/CD24low populations were determined by flow cytometry. The quantitative graph represents the percentage of CD44high/CD24low populations (****p&lt;0.0001). (B) Aldefluor-positivity was assessed and quantified (*p&lt;0.05). DEAB was defined as the baseline of Aldefluor fluorescence with flow cytometry. Results are presented as mean ± SEM of at least three independent experiments and analyzed by one-way ANOVA followed by Bonferroni's multiple comparison test. Supplementary Fig. S8. Effect of DOXA on cell migration in 4T1 cells. (A-C) Cells treated with DOXA (0-40 μM) for 24 h. (A) The kinetic analysis of cell migration was determined using the IncuCyte™ Live-Cell Imaging System and quantified for the indicated time duration (*p&lt;0.05). (B) The quantitative graph represents the relative wound density (%) in 4T1 cells at 24 h (*p&lt;0.05). (C) Representative images of wound closure by cell migration at 0 and 24 h after treatment with DOXA (40 μM). The yellow dotted line indicates the edge of the scratched wound. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Supplementary Fig. S9. Influence of DOXA on CD49f expression in allograft tumors derived from 4T1 mammospheres. Tumor tissues were immunostained for CD49f (green) with DAPI (nuclei, blue). Quantitative graphs of signal intensities are shown in the right panel (****p&lt;0.0001). The results are presented as mean ± SEM and data were analyzed by unpaired Student’s t-test.