Imagexpress micro 4

The 5-ethynyl-2’-deoxyuridine assay (EdU) assay was performed according to the manufacturer’s instructions (YF^®488/555/594/647A Click-iT EdU Imaging Kits, US Everbright^® Inc., Suzhou, China). Briefly, after a 2-hour incubation with EdU, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% Triton-X. After Click-iT reaction buffer treatment, cell nuclei were stained with Hoechst 33342. Images were obtained using a high-content imaging system (ImageXpress Micro 4, Molecular Devices, Shanghai, China), and cell proliferation was assessed as the percentage of EdU-positive cells.

Toxic effects of oleanic acid, betulinic acid and tolcapone on mitochondrial membrane potential of LO-2 cells were examined. The cells were cultured as described in the Section 2.3. Then the cells were incubated with 100 μL tested compounds of various concentrations (from 0.25 to 25 μM prepared in FBS-free culture medium) for 24 h. To assess the mitochondria membrane potential, JC-10 (5 μg/mL) was incubated with the cells for 30 min, and Hochest (10 μg/mL) was used to stain nuclei. After removal of culture medium and rinsing three times with PBS, cell imaging and quantitative analysis were performed by high-content imaging analysis (Molecular Devices^® ImageXpress Micro 4, the USA).

Cells were plated at a density of 2 × 10⁵ per well and allowed to adhere overnight. Cells in about 70% confluence were transfected with mRFP-GFP-LC3 double-labeled adenovirus (Ad-mRFP-GFP-LC3) to label autophagosome (Hanbio Biotechnology Co., Shanghai, China) according to the manufacturer's instruction. After 2 h of transfection, the cells were cultured in a fresh medium for 48 h then washed with pre-cooled PBS twice and stained with DAPI. The intracellular autophagy was observed by a high-content imaging system (ImageXpress Micro 4, Molecular Devices, San Francisco, CA, United States). Double labeling of LC3 (green) and mRFP (red) immunofluorescence corresponds to changes in autophagic flux. When autophagy and lysosome fusion occurs, LC3-GFP fluorescence is quenched, and only red fluorescence can be detected. After merging the red and green fluorescence images, yellow spots in the cell image symbolize autophagosomes.

24 hr after co-culture, a random 96-well-plate was fixed and stained. 2 channels (DAPI, MAP2), 4 stacks.field^–1, each stack separated by 0.5 µm, 25 fields.well^–1, 60 wells.96-well plate^–1 were acquired at ×20 (Plan Apo λ, NA 0.75, air objective, Nikon) with a high-content screening confocal microscope (ImageXpress Micro 4, Molecular Devices). Acquired images were transferred and stored on a Columbus (PerkinElmer) server. A Columbus Acapella software (PerkinElmer) algorithm was designed to discriminate hN nuclei from hpA nuclei. DAPI and MAP2 channels of each field were merged in a single plane to create maximum projection images. DAPI projected images were filtered using a Gaussian method and the nuclei population was detected based on the C method (diameter >20 µm). Intensity and morphological properties of each identified nuclei were calculated. Among all nuclei, the hN nuclei were identified according to their intensity, contrast, as well as area, roundness, and distinct MAP2-positive soma (Figure 1—figure supplement 2). This script allowed quantification of the mean number (and the standard deviation) of hNs identified in each well, and the coefficient of variance (%) per plate. Thresholding for plating consistency was set above 4000 hNs per well and a covariance below 8% per plate.

The EdU cell proliferation assay was performed according to the manufacturer’s instructions. Briefly, LX2 cells were transfected with KCNMA1-expressing plasmids or KCNMA1 siRNA for 48 h, and then plated in 96-well plates and incubated for 24 h. After incubation, the EdU solution (10 mM) was added directly to the culture medium and incubated for a further 30 min to ensure capture of most of the proliferating cells. The cells were subsequently fixed in 4% paraformaldehyde. Incorporation of EdU was performed by incubating the fixed cells with 2% bovine serum albumin (BSA) in PBS for 30 min and Alexa Fluor 488 for a further 30 min under Cu(I)-catalyzed click reaction conditions. The cells were washed with PBS and counterstained with Hoechst 33342 (1:1000) in PBS before detection by fluorescence microscopy using ImageXpress^® Micro 4 (Molecular Devices, San Jose, CA, United States).

To evaluate the ability of tumor cells to grow in suspension and form spheres, we cultured UM-HACC-2A cells (5 × 10³) in ultra-low attachment round-bottom plates (Corning^® Elplasia^® 96-well Black/Clear Round Bottom Ultra-Low Attachment—Thermo Fisher Scientific, Waltham, MA, USA) [44 (link),45 (link),46 (link)]. Cells were incubated for 3 days to allow spheres to form. Following this, we added the histone modifier drugs identified in the HTS to the plates at a final concentration of 10µM and closely monitored the spheres for 48 h. Images of the spheres were obtained and quantified using Molecular Devices ImageXpress Micro 4.

Cells in 70–80% confluence were irradiated with 6 Gy X-rays. For BrdU assay, cells were incubated with 20 mM BrdU for one and half-cell cycles (36 h) prior to IR. Two hours after irradiation, cells were washed with cold PBS, fixed with 4% formaldehyde for 10 min, permeabilized with 0.5% Triton X-100 for 10 min and then incubated in 0.1% PBS-Tween solution containing 1% BSA, 10% normal goat serum, and 0.3M glycine for 1 h to block non-specific protein-protein interactions. Cells were then incubated with primary antibodies detecting γ-H2AX, Rad51, and BrdU (1:200, Cell Signaling Technology) overnight at 4 °C, and followed by Alexa Fluor 488 or 555-conjugated secondary antibody (1:1000, Cell Signaling Technology) for 1 h. DAPI was used to stain the cell nuclei at a concentration of 1.43 μM. Cell fluorescence image was photographed with a high content screening system (ImageXpress Micro 4, Molecular Devices, San Jose, CA, USA).