Immunofluorescence

For immunofluorescence staining, specimens were fixed in PBS with 4% formaldehyde, washed twice with PBS, and immersed in 0.1% Triton X-100 overnight at 4 °C, followed by washing 3 times with PBS. The following primary antibodies were incubated overnight at 4 °C: anti-phospho-mTOR (Ser2448) (1:200, Bioworld), anti-cleaved caspase-3 (1:200, Epitomics), anti-fibronectin (1:200, Santa Cruz) anti-phospho-ULK1 (ser757) (1:1000, Cell Signaling), anti-phospho-S6 (Ser235/236) (1:200, Cell Signaling) or anti-LC3A (1:50, Cell Signaling). After incubation with primary antibodies, cells were washed with PBS and then incubated with fluorescence-conjugated secondary antibodies (1:5000, FITC, Jackson ImmunoResearch, Westgrove, PA) for 2 hr at room temperature. After nuclear staining with 40,6-diamidino-2-phenylindole (DAPI, D9542, Sigma-Aldrich), spheres were then placed in a Lab-Tek^®II chamber (Nunc, Naperville, IL) and analyzed with a confocal fluorescent microscope (Olympus FV10i). Negative controls without utilizing primary antibodies were also prepared to rule out nonspecific labeling.