Pladienolide b

LNCaP, 22RV1, C4-2, and RWPE-1 cell lines were obtained from the American Tissue Culture Collection (ATCC) and PNT1 cell line was acquired from Sigma. LN95 cell line was kindly provided by Professor Stephen Plymate (University of Washington). LNCaP, 22RV1, C4-2 and PNT1 cells were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS). RWPE-1 cells were cultured in keratinocyte serum-free media, while LN95 cells were maintained in phenol red-free RPMI supplemented with 10% charcoal-stripped FBS. All cell lines were allowed to adhere for at least one day prior to the treatment with inhibitors. THZ531, OSMI-4, TG003 and GSK3326595 were obtained from MedChemExpress. KH-CB19, Pladienolide B and SRPIN340 were purchased from Tocris.

Cell culture, splicing inhibition, and FKBP5 induction HeLa (kind gift of the lab of Dr. Phillip Sharp, MIT) and A549 (human lung carcinoma, A549, ATCC CCL-185) cells were cultured in DMEM (Gibco) supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin, and 10% fetal bovine serum (FBS, Fisher). Splicing inhibition was accomplished by treating HeLa cells with 1uM Pladienolide B (Tocris Biosciences, 6070500U) for 4 hours, as described by (16) . HeLa cells were then fixed and used for RNA FISH as described below. FKBP5 was induced by treating A549 cells with 25nM dexamethasone (Sigma, D2915) for the specified lengths of time. A549 cells were then fixed and used for RNA FISH as described below.