Extended Data Fig. 2: Verification of PROTAC design of AU-15330 and confirmation of on-target growth effects.
From: Targeting SWI/SNF ATPases in enhancer-addicted prostate cancer
(a) Immunoblots for indicated proteins in normal (RWPE) or PCa cells (LNCaP, VCaP, 22RV1, and LAPC4) treated with AU-15330 at varied concentrations. Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. (b) Western blot analysis showing the time-dependent effect of AU-15330 on SMARCA2, SMARCA4, and PBRM1 in RWPE, LNCaP, and VCaP cells. Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. (c) Immunoblots in LNCaP and VCaP cells examining time-dependent cleavage of PARP upon AU-15330 treatment. Vinculin is the loading control probed on a representative immunoblot. This experiment was repeated independently twice. (d) Dose-response curves of VCaP, LNCaP, PNT2, PNT2, BPH1, Bin67, and HEK293 cells treated with AU-15330, AU-15139, or AU-16235. Data are presented as mean +/− SD (n = 6) from one-of-three independent experiments. (e) Growth curves of non-neoplastic or PCa cells upon treatment with increasing concentrations of AU-15330. Bottom, rightmost panel shows real-time assessment of apoptotic signals in LNCaP cells after treatment with DMSO or increasing AU-15330 concentrations. Data are presented as mean +/− SD (n = 5) from one-of-three independent experiments. (f) (top) Chemical structure of AU-15330, AU-16235 (an epimer control of AU-15330), and AU-15139 (parent bromodomain-binding ligand of AU-15330). (bottom) Immunoblots for SMARCA4 and PBRM1 in LNCaP and VCaP cells treated with AU-15330, AU-15139, or AU-16235 at indicated concentrations. Vinculin is the loading control probed on all immunoblots. This experiment was repeated independently twice. (g) Immunoblots of SMARCA4 and PBRM1 in VCaP and LNCaP cells pre-treated with VL285, MLN4924, bortezomib, or thalidomide for 1h, then treated with AU-15330 at noted concentrations for 4h. Vinculin is the loading control probed on all immunoblots. This experiment was repeated independently twice. (h) Real-time measure showing the rescue effect of VHL ligand on AU-15330-mediated growth inhibition in VCaP and LNCaP cells. Data are presented as mean +/− SD (n = 4) from one-of-three independent experiments.
