Immunofluorescence microscope

Place the transwell chamber in 24 well plates to form a two-compartment system. Cells were collected by centrifuge at 3000 rpm, 4 ℃ for 5 min. Cells were then resuspended, washed twice and then resuspended in serum-free medium at a density of 10⁶ cells/ml.
For migration, 100 μl of cell suspension was added to the upper compartment and 0.8 ml medium containing 5% FBS was put in the lower compartment. After 24 h of regular culture, the porous membrane was isolated, fixed with methyl alcohol, and stained with crystal violet. Cells found in at least 20% of the area of the filter was counted under immunofluorescence microscope (Leica, Allendale, NJ, USA).
For invasion, thaw Matrigel on ice and dilute Matrigel with serum free medium by 1:8, and add 40 μl dilutions in the upper compartment (all experiments were conveyed on ice). Then, 100 μl of cell suspension was added to the upper compartment and 0.8 ml medium containing 5% FBS was put in the lower compartment. After 24 h of regular culture, the porous membrane was isolated, fixed with methyl alcohol, and stained with crystal violet. Cells found in at least 20% of the area of the filter was counted under immunofluorescence microscope (Leica, Allendale, NJ, USA).

Ovarian cancer cells were cultured on coverslips in 24-well plates for 48 hours in respective medium containing inhibitor. Cells were fixed with 4% paraformaldehyde, and blocked with a 5% BSA-phosphate buffer solution. The cells were then incubated with rabbit anti-RAD51 polyclonal antibody (Santa Cruz Biotechnology) or rabbit anti-γH2AX^Ser139 polyclonal antibody (Cell Signaling Technology) overnight at 4°C. After washing with PBS, the cells were incubated with secondary antibodies and DAPI at room temperature. Images were acquired and quantified using an immunofluorescence microscope (Leica). The dynamics of γH2AX and RAD51 foci accumulation, as well as percentage of positive cells (more than 5 foci in one cell) were calculated based on analysis of about 200 cells.

Intracellular Ca²⁺ was measured using the Fluo-8 No Wash Ca²⁺ Assay Kit (Abcam, Cambridge, MA), a method previously validated by others [50 (link)-52 (link)]. Briefly, NALM6 cells were incubation with pevonedistat (400 nM) or tunicamycin (400 ng/mL) for 24 h, washed twice with HHBS (1X Hank’s with 20 mM Hepes Buffer, pH 7.0), and seeded (200,000 cells/well in a volume of 100 µL) in 96-well plate with black wall and clear flat bottom. Then, 100 µL of Fluo-8 dye solution was added to each well and plates were incubated for 30 min at 37°C, followed by an additional incubation of 30 min at room temperature. The fluorescence intensity was measured at Ex/Em = 490/525 nm. Thapsigargin (TG, 10 µM) was added to each well and kinetic measured at 27 sec intervals for 20 min (n = 3). For Ca²⁺ measurement using microscopic immunofluorescence, cells were treated with BTP-2 (10 µM) or pevonedistat (400 nM) either alone or in combination (pevonedistat + BTP-2) for 24 h, washed twice with HHBS, and centrifuged at 1000 rpm for 5 min. Fluorescent cell images were captured using a Leica immunofluorescence microscope with a 10× magnification.