Journal: The American Journal of Pathology

Article Title: MYC-Mediated Ribosomal Gene Expression Sensitizes Enzalutamide-resistant Prostate Cancer Cells to EP300/CREBBP Inhibitors

doi: 10.1016/j.ajpath.2021.02.017

Figure Lengend Snippet: Validation of MYC, RPL29, and RPL36 expression. A: Representative Western blots and quantification of parental and enzalutamide-resistant DuCaP cells probed for MYC, RPL29, and RPL36 ( n = 6). B: Representative Western blots and quantification for DuCaP EnzaR cells treated with 10 μM C646, 10 μM I-CBP112, or dimethyl sulfoxide (DMSO) for 48 hours, probed for MYC, RPL29, and RPL36 ( n = 6). C: Pooled siRNAs either targeting MYC (siMYC) or nontargeting control (siCTRL) were used to establish and validate MYC knockdown together with mock treatment (Mock). Knockdown efficiency was validated by Western blot with an antibody for MYC. D: The blots were probed with antibodies specific for RPL36 and RPL29. For quantification, MYC, RPL36, and RPL29 were normalized to mock-treated cells ( n = 3). E: PC3 cells were treated for 48 hours with 10 μM C646 or 10 μM I-CBP112 ( n = 3). Blots were probed with MYC, RPL36, and RPL29 specific antibodies. Numerical data were analyzed via one-way analysis of variance. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: The following antibodies were used at the indicated dilutions: CREBBP (7389S, Cell Signaling Technology, Danvers, MA; 1:2000), EP300 (ab10485, Abcam, Cambridge, UK; 1:1000), FKBP5/FKBP51(A301-430A, Bethyl, Montgomery, TX; 1:2000), alpha tubulin (sc-5286, Santa Cruz Laboratories, Dallas, TX; 1:2000), MYC (D84C12, Cell Signaling; 1:1000), RPL29 (AP20452c-ev, ABcepta, San Diego, CA; 1:500), RPL36 (ELA-E-AB-32803 to 60, Elabscience Biotechnology, Houston, TX; 1:500), and glyceraldehyde-3-phosphate dehydrogenase (ABS16, Millipore, Burlington, MA; 1:5000).

Techniques: Biomarker Discovery, Expressing, Western Blot, Control, Knockdown

Journal: The American Journal of Pathology

Article Title: MYC-Mediated Ribosomal Gene Expression Sensitizes Enzalutamide-resistant Prostate Cancer Cells to EP300/CREBBP Inhibitors

doi: 10.1016/j.ajpath.2021.02.017

Figure Lengend Snippet: Next-generation bromodomain inhibitor and ribosomal neogenesis inhibitor in prostate cancer cell lines. A: Dose–response curve for parental and resistant DuCaP and LNCaP cells after 72 hours of CPI637 treatment. B: Representative Western blots for DuCaP EnzaR cells treated with 2 μM CPI637 for 48 hours, probed for MYC, RPL29, and RPL36 ( n = 3). C: Dose–response curve for parental and resistant DuCaP and LNCaP cells after 72 hours of CX-5461 treatment. D: Fifty percent inhibitory concentration (IC 50 ) of CPI637 and CX-5461 for DuCaP and LNCaP parental and enzalutamide-resistant cell lines 72 hours after treatment based on a viability assay ( n = 3). P value was calculated using extra-sum-of-square-F-test for IC 50 . E: Boyden chamber assay with parental and LNCaP EnzaR cells, with or without 2 μM CX-5461 for 72 hours. Cells were stained with Calcein AM. Upper row shows migrated cells, and lower row shows total cells. ( F ) Graph shows relative fluorescence intensity to untreated cells, normalized to wells with same treatment but without insert. Numerical data were analyzed via one-way analysis of variance. Bars indicate SEM ( n = 3) ( A and C ). ∗∗∗ P < 0.001. Scale bars: 100 μm ( E ). DMSO, dimethyl sulfoxide.

Article Snippet: The following antibodies were used at the indicated dilutions: CREBBP (7389S, Cell Signaling Technology, Danvers, MA; 1:2000), EP300 (ab10485, Abcam, Cambridge, UK; 1:1000), FKBP5/FKBP51(A301-430A, Bethyl, Montgomery, TX; 1:2000), alpha tubulin (sc-5286, Santa Cruz Laboratories, Dallas, TX; 1:2000), MYC (D84C12, Cell Signaling; 1:1000), RPL29 (AP20452c-ev, ABcepta, San Diego, CA; 1:500), RPL36 (ELA-E-AB-32803 to 60, Elabscience Biotechnology, Houston, TX; 1:500), and glyceraldehyde-3-phosphate dehydrogenase (ABS16, Millipore, Burlington, MA; 1:5000).

Techniques: Western Blot, Concentration Assay, Viability Assay, Boyden Chamber Assay, Staining, Fluorescence