Spectramax m2 fluorescence

Cells (in 100 μL of maintenance medium) were seeded into 96-well plates at a concentration of 15 000 (COLO 205, LNCaP) or 10 000 (HCT 116) cells/well. Twenty-four hours later, cells were treated with fresh medium containing either pyrroloiminoquinone alkaloid, positive control TPZ, or DMSO (0.5%, v/v) followed by 18 h (HCT 116, LNCaP) or 48 h (HCT 116, COLO 205) incubation under hypoxic conditions. Each concentration was tested in quadruplicate, and experiments were performed in triplicate. The tested concentration ranges were 0.001−10, 0.05−50, and 0.1 − 10 μM for COLO 205, HCT 116, and LNCaP cells, respectively. The next morning 10 μL of CCK-8 (Dojindo Molecular Technologies, Inc.) was added to each well. After incubation for 2 h at 37 °C, absorbance was read at 450 nm on a SpectraMax M2 fluorescence plate reader (Molecular Devices). The fluorescent signal of each sample was normalized to the average signal of the DMSO-treated controls to calculate percent cell viability.

Phosphatase activity was assessed using the EnzChek Phosphatase Assay Kit (Molecular Probes, Eugene OR) according to manufacturer’s instructions using the fluorescent phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). Upon dephosphorylation, DiFMUP fluoresces at an excitation/emission wavelength of 360/460. TCPTP was first immunoprecipitated from whole cell lysates using anti-TCPTP antibody (see above). Immunoprecipitates were incubated for 15 minutes with DiFMUP after which fluorescence was detected with a SpectraMax M2 Fluorescence Microplate reader using SoftMax Pro v5 Software (Molecular Devices, Sunnyvale, CA). Fluorescence was measured every 15 minutes for the first hour and every 30 minutes thereafter. Measurements were performed in triplicate. A sample from each immunoprecipitation was run on SDS-PAGE and probed for TCPTP to confirm equal protein loading. To account for any differences in overall phosphatase amounts, fluorescence activity units gathered from each assay were compared to TCPTP densitometric values obtained from Western blotting. Thus, values represent the specific activity of TCPTP rather than total quantities of the enzyme.