Plate 24 well black

For all confocal imaging experiments clear bottom black walled plates with optical properties similar to glass coverslips were used (µ-Plate 24 Well Black, IBIDI, Fitchburg, WI). Four hours post-treatment spheroids were fixed using 10% buffered formalin for an hour at 25°C, rinsed with PBS, then permeabilized with 0.25% Triton-X for 45 minutes at 37°C. Non-specific protein interactions were blocked by incubating samples in 5% BSA for 24 hours at 37°C, followed by incubation with anti-STAT3 (phospho S727, 1:500, ab32143, Abcam, Cambridge, MA) for 24 hours at 37°C. Samples were rinsed with PBS and then incubated with secondary antibody (1:350 goat-anti rabbit IgG Alexa Fluor 647) for 24 hours at 37°C protected from light. After rinsing with PBS, samples were incubated with DAPI (1:1000) for 45 minutes at 37°C to visualize cell nuclei. Samples were washed with PBS and imaged on a Nikon A1R confocal laser microscope with a PLAN APO VC 20× 0.75-NA objective. Samples were imaged at the Nyquist optimized step size and phosphorylated STAT3 median fluorescent intensity (MFI) was calculated every five microns for each spheroid using ImageJ software. The edge of the spheroid was defined as the area from the boundary of the spheroid to four nuclei in from the boundary of the spheroid, and the center was defined as the remaining area.