Cell Line Culture and Manipulation

LNCaP, LNCaP-LN3, C4, C4–2, C4–2B, PC-3M, PC-3M-LN4, PC-3M-Pro4, and MDA-MB453 cells were grown as previously described [30 (link), 67 (link)]. The HEK293T, PC3, and C4–2 cells were cultured in DMEM supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin. The DNA methylase inhibitor 5-Aza-dC (10 μM) (#A3656, Sigma), the 26S proteasome inhibitors MG132 (20 μM) (#S2619, Selleckchem), or N-acetyl-L-leucyl-L-leucyl-L-nor leucinal (LLNL) (25 μM) (#A6185, Sigma), Doxorubicin (Sigma), Talazoparib (SeleckChem), NU7026 (Sigma) were used as described in the text. TGF-β receptor type I (TGF-βRI) kinase inhibitors LY2157299 and LY363947 were bought from Selleckchem.
The expression vectors encoding DACH1 [69 (link)], Ku70 (RFP-Ku70) and Ku80 (RFP-Ku80) [70 (link)], shDACH1[71 (link)] were previously described. The EGFP-DACH1 expression plasmid was made by inserting the human DACH1 cDNA into the HindIII and BamHI sites of the pEGFP-C1 vector.

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Li Z., Jiao X., Robertson A.G., Sante G.D., Ashton A.W., DiRocco A., Wang M., Zhao J., Addya S., Wang C., McCue P.A., South A.P., Cordon-Cardo C., Liu R., Patel K., Hamid R., Parmar J., DuHadaway J.B., Jones S.J., Casimiro M.C., Schultz N., Kossenkov A., Phoon L.Y., Chen H., Lan L., Sun Y., Iczkowski K.A., Rui H, & Pestell R.G. (2023). The DACH1 gene is frequently deleted in prostate cancer, restrains prostatic intraepithelial neoplasia, decreases DNA damage repair, and predicts therapy responses. Research Square.

Publication Preprint 2023

5-Aza-dC S2619 A6185 Doxorubicin NU7026 LY2157299

26s proteasome Cdna Cells Doxorubicin Fetal calf serum Human Inhibitors Kinase Ku70 Leucinal Ly2157299 Methylase Mg132 Nu7026 Penicillin Plasmid Streptomycin Talazoparib Tgf β receptor type i Vector

Corresponding Organization : Baruch S. Blumberg Institute

Other organizations : Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Lankenau Institute for Medical Research, Northern Sydney Local Health District, University of Sydney, Thomas Jefferson University, Institute of Cell Biology and Neurobiology, Mount Sinai Hospital, Memorial Sloan Kettering Cancer Center, Molecular Oncology (United States), Medical College of Wisconsin, Icahn School of Medicine at Mount Sinai, Abraham Baldwin Agricultural College, Massachusetts General Hospital, Harvard University

Top 5 similar protocols

Variable analysis

independent variables

5-Aza-dC (10 μM)
MG132 (20 μM)
N-acetyl-L-leucyl-L-leucyl-L-nor leucinal (LLNL) (25 μM)
Doxorubicin
Talazoparib
NU7026
LY2157299
LY363947
DACH1 expression vector
RFP-Ku70 expression vector
RFP-Ku80 expression vector
ShDACH1
EGFP-DACH1 expression plasmid

dependent variables

Cell growth and proliferation
Gene expression
Protein expression and localization
DNA damage and repair

control variables

HEK293T, PC3, and C4-2 cells cultured in DMEM supplemented with 10% fetal calf serum, 1% penicillin, and 1% streptomycin

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