Bx50 microscope

The assay was performed by plating in triplicates 1×10⁵ OVCAR3 or OVCAR3-sfRon cells per well of 96-well plate in serum deprived medium (0.5% FBS in RPMI 1640). Cells were incubated for 2h at 37°C followed by washing away unattached cells. Adherent cells were fixed with ice-cold methanol for 10 min and then stained with 0.5% crystal violet solution (made in 25% methanol and stored at room temperature) for 10 min in room temperature. Next, culture wells were rinsed with ddH₂O until purple color was no longer coming off while rinsing and culture plates were dried overnight. Dried culture plates were imaged on an Olympus Bx50 microscope with a Canon EOS Rebel XSI camera using EOS imaging software and quantified at OD 595 nm after extraction using FLUOstar Omega Microplate Reader (BMG LABTECH, Cary, NC)

Two mice bearing PDX-0113 from each group were sacrified 3h after drug(s) administration. Harvested tumors were fixed in 10% neutral buffered formalin, paraffin embedded, and hematoxylin–eosin (H&E) stained according to our standard protocols [7 (link), 9 (link)]. Tumors were analyzed by IHC for expression of the following markers: anti-human cytokeratin (1:400, DAKO #Z0622), PAX8 (1:1000, Abcam, #ab189249), WT1 (1:1250, Cell Signaling, #83535), phospho-S6 (1:200, Cell Signaling, #4856), Ki67 (1:200, Thermo Scientific #RM-9106-S1) or cleaved caspase-3 (1:250, Cell Signaling #9661). Staining was visualized by 3,3-diaminobenzidine (DAB), with hematoxylin as a counter-stain. Slides were imaged on an Olympus Bx50 microscope with a Canon EOS Rebel XSI camera using EOS imaging software. For Ki67 or cleaved caspase-3 quantification, 3 images per tumor from two different animals from each treatment group were analyzed with ImageJ software [51 ]. The relative proliferation (Ki67) or apoptosis (cleaved caspase-3) were expressed as a number of positively stained nuclei in each image, as previously described [52 (link)].