Scan r platform

A seven-point dilution series of 116 small molecule inhibitors covering a 1000× concentration range was plated into three 384-well plates using the EP Motion automated dispensing system. Control wells with equal volumes of DMSO were included as negative controls. ACHN cells were grown, trypsinized, counted, and plated directly into warm drug plates using a Multidrop Combi dispenser. Plates were incubated for 72 h and subsequently imaged on an Olympus ScanR Platform at 10× magnification, performing four images per well in 384-well plates. Single-cell nuclear and cytoplasmic fluorescent intensities were calculated using the Olympus ScanR analysis software: The DAPI-positive region of each cell was used as a boundary to quantitate nucleus counts for analysis of cell growth, and integrated nuclear DNA staining intensity was used for cell cycle analysis. A 10-pixel extension of the nuclear region (and not including the nuclear region) was used to quantitate cytoplasmic signal of immunofluorescent staining of p62 protein and phosphorylation of S6. The mean signal intensity of each marker in all cells per well was used as the metric for cytoplasmic marker expression (average intensity of pS6 and p62). Unsupervised hierarchical clustering was used to identify compounds that produced similar pS6 and p62 dose response phenotypes after treatment.

The primary and metastatic tumor tissue samples were formalin-fixed, paraffin-embedded and cut sections were prepared with pathologist assistance. For immunofluorescence staining 5 µm cut sections were de-paraffinized in xylene and passed through a series of graded alcohols. Antigen retrieval was performed in a 0.1 M sodium citrate buffer pH 6 (Sigma) under heat and pressure, followed by blocking with a 1% BSA (ThermoFisher) blocking buffer. Sections were incubated overnight at 4 °C with a primary antibody solution with epithelial marker cytokeratin-19 (KRT19, Dako, Clone RCK108), 1:300), myoepithelial marker cytokeratin-14 (KRT14, Abcam, Clone LL002, 1:300) and proliferating cell marker Ki-67 (DAKO, Clone MIB-1, 1:200) diluted in 1% BSA. Sections were washed in PBS with 0.1% Tween (ThermoFisher) and secondary antibody staining was performed at room temperature for 1 hour with AlexaFluor secondary antibodies against primary host species (1:500, LifeTech) in 1% BSA. 1 μg/ml DAPI (4′,6-Diamidino-2-phenylindole nuclear counterstain, LifeTech) was added to secondary staining buffers. Tissue sections were imaged on an Olympus scan^R platform at 20× magnification.