FIG 1.

Single and double depletions of Hsp90α and Hsp90β on client protein stability. (A) The CRISPR-Cas9 gene editing technique was utilized to create Hsp90α-KO, and a lentiviral infection-mediated shRNA delivery system, FG-12, was used to downregulate Hsp90β or Hsp90α or both in MDA-MB-231 cells. The efficiency of knockout and knockdown of Hsp90β and Hsp90α is shown (panels a and b). (B and C) Parental MDA-MB-231 cells cultured in medium with 10% FBS to 80% confluence were utilized to establish a standard duration and dosage of 17-DMAG treatment. Total lysates of the untreated cells (vehicle alone) and cells treated with 100 nM 17-DMAG for indicated time points up to 72 h (B) and of untreated cells and cells treated with 0 to 1,000 nM 17-DMAG for 48 h (C). All samples were subjected to Western immunoblotting analysis with antibodies against Hsp90α and Hsp90β or five signaling proteins of the mitogenic signaling pathway. Equal loadings of the cell lysates were confirmed with β-actin. Similar results were obtained from three independent experiments, in which the reduced level of the bands in (A, asterisks) was reproducible.