Semin Ophthalmol 2001; 16:201C206

Semin Ophthalmol 2001; 16:201C206. verteporfin and CA3, attenuate the MCS cell phenotype. Verteporfin or CA3 Dabrafenib Mesylate treatment S1PR2 reduces YAP1/TEAD level/activity to suppress MCS cell spheroid formation, matrigel invasion, migration and tumor formation. These brokers also increase MCS cell apoptosis. Moreover, constitutively-active YAP1 expression antagonizes inhibitor action, suggesting that loss of YAP1/TAZ/TEAD signaling is required for response to verteporfin and CA3. These brokers are active against mesothelioma cells derived from peritoneal (epithelioid) and patient-derived pleural (sarcomatoid) mesothelioma, suggesting that targeting YAP1/TEAD signaling may be a useful treatment strategy. Implications: These studies suggest that inhibition of YAP1 signaling may be a viable approach to treating mesothelioma. (9157), LATS1 (9153), YAP1-(13008), YAP1 (4912), TAZ (4883), TEAD (13295) and Snail (3895) were purchased from Cell Signaling Technologies (Danvers, MA). Anti-TAZ-(sc-17610) was obtained from Santa Cruz Technologies (Dallas, TX). Peroxidase-conjugated anti mouse IgG (NA931V) and anti-rabbit IgG (NA934V) were obtained from GE Healthcare (Buckinghamshire, UK), and anti-goat IgG (PA1C28664) was obtained from Invitrogen (Carlsbad, CA). These secondary antibodies were used as a 1:5000 dilution. Dabrafenib Mesylate Matrigel (354234) and BD BioCoat Millicell inserts (353097) were purchased from BD Bioscience (Franklin Lakes, NJ). The YAP1 signaling inhibitors, CA3 (CIL56) [17] and verteporfin [16] were purchased, respectively, from SelleckChem (Houston, TX), and Tocris (Bristol, UK). For use in cell culture, the compounds were dissolved in DMSO at a 1000-fold stock. YAP(S127A) (Addgene plasmid # 27370) is a plasmid encoding a constitutively active form of YAP1 donated by Kunliang Guan. Cell culture and proliferation assay Meso-1 and NCI-Meso-17 are immortal mesothelioma cell lines generated, respectively, from malignant epithelioid peritoneal and malignant sarcomatoid pleural mesothelioma, were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FCS [9,22]. Meso-1 cells have been in culture for several years. NCI-Meso-17 cells are short-term passage cells derived from a pleural mesothelioma tumor that has been used for patient-derived xenograft studies [22]. The NCI-Meso-17 cell line was kindly provided by Dr. Raffit Hassan (National Institutes of Health). Monolayer cells were plated at 200,000 cells per 9.6 cm2 plate (#353001, Corning, Tewksbury, MA) in RPMI 1640 medium supplemented with 10% heat-inactivated FCS. After attachment, plates were treated with 0 C 5 M of verteporfin and cell number was counted in day two and expressed as mean SEM. The cells lines are periodically confirmed as authentic using short tandem repeat profiling. Cell lines were also tested for mycoplasma contamination at the time of initiation of the studies, and are generally retested yearly. Spheroid formation, invasion and migration For spheroid Dabrafenib Mesylate formation assay, Meso-1 or NCI-Meso-17 cell cultures (near-confluent) were dissociated Dabrafenib Mesylate with trypsin, collected by centrifugation, resuspended in RPMI1640 medium containing 10% FBS. Single cell suspension was plated for spheroid growth at 40,000 cells per 9.5 cm2 ultra-low attachment dishes (#4371, Corning, Tewksbury, MA) and allowed to form spheroids for 0 C 5 d. We count the total number of spheroids and/or average spheroid Dabrafenib Mesylate diameter. We define a spheroid as a clonal collection of cells achieving a diameter greater than or equal to 25 microns. Verteporfin and CA3 impact on spheroid growth/survival was monitored using growing and/or pre-formed spheroids. To monitor impact on nascent spheroid growth, cells were seeded on ultra-low attachment plates, the compound was added the next day and spheroid expansion was monitored thereafter. Alternately, spheroids were permitted to pre-form for 3 C 5 d prior to initiation of drug treatment. To measure invasion, BioCoat Millicell inserts (d = 1 cm, 8 M pore size, #353097) were coated with 120 l of 250 g/ml Matrigel. Cells (20,000) were seeded atop the matrigel in 500 l of RPMI1640 containing 1% fetal calf serum. The lower chamber.