Additional file 1 of A GATA2-CDC6 axis modulates androgen receptor blockade-induced senescence in prostate cancer
Creators: name: Mourkioti, Ioanna
name: Polyzou, Aikaterini
name: Veroutis, Dimitris
name: Theocharous, George
name: Lagopati, Nefeli
name: Gentile, Emanuela
name: Stravokefalou, Vasiliki
name: Thanos, Dimitris-Foivos
name: Havaki, Sophia
name: Kletsas, Dimitris
name: Panaretakis, Theocharis
name: Logothetis, Christopher J.
name: Stellas, Dimitris
name: Petty, Russell
name: Blandino, Giovanni
name: Papaspyropoulos, Angelos
name: Gorgoulis, Vassilis G.
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1: Figure S1. SA-β-gal staining confirms the senescence phenotype of enzalutamide-sensitive and -resistant cells. A SA-β-gal staining of LNCaP cells upon enzalutamide treatment compared to Control. B Quantification of SA-β-gal stainings in A. C Same as A, for C4-2B cells. D Quantification of SA-β-gal stainings in C. E SA-β-gal stainings of C4-2B cells of Fig. 3E. F Quantification of SA-β-gal stainings in E. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). Enzalutamide was used at a 10 μM concentration. ***P &lt; 0.001, of Student’s t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S2. Densitometry accompanying key observations acquired by Western blotting. A Densitomery of indicated markers over control (GAPDH) on immunoblotting presented in Fig. 2B. B Same as A, for Fig. 3A. C Densitometry of indicated markers over control (GAPDH) for immunoblotting in C4-2B cells of Fig. 3D. D Same as C, for PC-3 cells of Fig. 3D. **P &lt; 0.001 and ***P &lt; 0.001, of Student’s t-test (comparing individual lanes to lane 1); n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S3. Loss of CDC6 establishes senescence in enzalutamide- resistant PC-3 cells. A Immunofluorescence for dual GL13/p21WAF1/Cip1 staining and B quantification confirms senescence induction only in CDC6-depleted PC-3 cells. C SA-β-gal staining of PC-3 cells in A and D quantification of SA-B-gal stainings confirm senescence induction upon CDC6 depletion. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). Enzalutamide was used at a 10 μM concentration. *P &lt; 0.05 and ***P &lt; 0.001, of Student’s t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S4. CDC6 loss decreases proliferation of enzalutamide-resistant cells regardless of treatment. A Immunocytochemistry for Ki67 levels in C4-2B cells from Fig. 3D and B quantification of Ki67-positive cells displaying reduced proliferation upon CDC6 loss. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). C Same as A, for PC-3 cells from Fig. 3D. Magnification: 200x (Objective 20x), scale bars: 30 μm. D Same as B, for PC-3 cells from Fig. 3D. E qPCR for EMT markers CDH1, ZEB1 and SNAI1 in the indicated conditions of PC-3 cells from Fig. 3D. Data are normalized to GAPDH expression. Enzalutamide was used at a 10 μM concentration. **P &lt; 0.01 and ***P &lt; 0.001, of Student’s t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S5. Enzalutamide treatment fails to induce senescence in prostate cancer cells acquiring resistance. A GL13 staining assessed by immunofluorescence in enzalutamide-resistant C4-2B cells from Fig. 4A displays no induction of senescence in cells receiving further treatment. B Quantification of positive GL13 cells in A. C Immunocytochemistry for GL13 levels in PC-3 cells from Fig. 4A indicating no senescence induction in cells receiving further treatment. Magnification: 200x (Objective 20x), scale bars: 30 μm. D Quantification of GL13-positive PC-3 cells in C. E SA-β-gal staining in PC-3 cells from Fig. 4A confirming the GL13 staining. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). F Quantification of SA-β-gal-positive cells in E. G Immunohistochemistry for Ki67 levels in C4-2B cells from Fig. 4A. Magnification: 100x (Objective 10x), scale bars: 60 μm. Inset magnification: 400x (Objective 40x). H Quantification of Ki67-positive cells in G. Enzalutamide was used at a 10 μM concentration. N.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S6. Androgen-resistant prostate cancer cells display increased in vitro migration and invasion in response to enzalutamide. A Wound healing assay of control or enzalutamide-treated PC-3 cells, demonstrating migration at the indicated time points post wound formation. The cells were subjected to the assay having already received or not enzalutamide for 72 h. The dashed lines indicate the wound borders in each case. B Quantification of migration in A. C Quantification of invasion of control or enzalutamide-treated PC-3 cells, using a colorimetric invasion assay. The cells were subjected to the assay having already received or not enzalutamide for 72 h. Enzalutamide was used at a 10 μM concentration. Magnification: 50x (Objective 5x), scale bars: 200 μm. ***P &lt; 0.001, of Student’s t-test; n.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S7. Enzalutamide treatment is not accompanied by autophagy or apoptosis in either enzalutamide-sensitive or -resistant prostate cancer cells. A Electron micrographs of control or enzalutamide-treated LNCaP and C4-2B cells showing no autophagy activation following treatment. Autophagic vacuoles were not observed in neither treated nor untreated cells. N: nucleus; m: mitochondrion. Scale bars for LNCaP cells: 1 μm; for C4-2B cells: 500 nm. B Immunocytochemistry for cleaved caspase-3 staining in LNCaP, C4-2B and PC-3 control or enzalutamide-treated cells displaying no induction of apoptosis. PC-3 cells subjected to 5% DMSO treatment for 24 h were used as positive cleaved caspase-3 control. Magnification: 200x (Objective 20x), scale bars: 30 μm. C Quantification of cleaved caspase-3 stainings in B. Enzalutamide was used at a 10 μΜ concentration. Statistics were determined using Student’s t test; Ν.s., non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3). Figure S8. Enzalutamide treatment confers opposite regulation of EMT markers between responsive and resistant cells in vivo. A qPCR for EMT markers CDH1, ZEB1 and SNAI1 in LNCaP mouse xenografts treated or not with enzalutamide (10 mg/kg). Enzalutamide treatment is accompanied by EMT attenuation. B Same as A in C4-2B mouse xenografts. Enzalutamide treatment is accompanied by pronounced EMT compared to control. C Τumor volume curves over days of treatment in mice injected with the indicated cells. D Immunohistochemistry of extracted mouse tumors confirming increased invasiveness of C4-2B cells receiving enzalutamide treatment versus control, into the underlying muscle tissue. Magnification: 200x (Objective 20x), scale bars: 30 μm. **P &lt; 0.01 and ***P &lt; 0.001, of Student’s t-test. Error bars indicate s.e.m. Data shown are representative of at least 3 biological experiments (n ≥ 3).