Hcs cell mask

N2a cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (PAA). HEK 293T/17 cells were grown in Iscove's modified Dulbecco's medium (Sigma) supplemented with 1% glutamine, 10% FCS, and 3.024 g/ml NaHCO₃. Chemicals were purchased from Sigma or Carl Roth, unless stated otherwise. Monoclonal antibody (MAb) rat anti-HA clone 3F10 (1:1,000) (Roche Diagnostics), MAb 4A5 rat anti-NM (M domain) (1:10) (81 (link)), MAb mouse anti-Myc epitope (Miltenyi Biotec), MAb antiactin clone C4 (1:5,000) (MP Biomedicals), MAb mouse anti-HA (1:200) (Santa Cruz), MAb mouse anti-HA–Alexa Fluor 647 (1:500) (Biozol), or polyclonal antibody (pAb) rabbit anti-Myc tag (Abcam) was used. Horseradish peroxidase-conjugated antibodies were from Dianova, and fluorescently labeled antibodies were from Life Technologies. Cytoplasm was stained with HCS CellMask (Life Technologies). Effectene (Qiagen) or Lipofectamine 2000 (Thermo Scientific) was used for transfection.

HCT116 and RPE1 cells and their aneuploid derivatives were seeded to a 96-well plate (HCT116: 10⁴ cells per well, RPE1: 10³ cells per well) and incubated at 37 °C and 5% CO₂ on the day before experiments. For cell fixation, a 3% formaldehyde solution was used for 15 min at room temperature. Next, cells were permeabilized with 0.1% Triton for 20 min at room temperature. Then, cells were preblocked with 3% bovine serum for 30 min at room temperature. Primary antibodies were diluted in blocking solution (Supplementary table 2) and incubated overnight at 4 °C. The next day, cells were washed and secondary antibodies were added in a concentration of 1 mg per ml followed by incubation for 1 h in the dark at room temperature. SYTOX® green (Invitrogen) or DAPI (Invitrogen) were used to localize the nucleus. For cytoplasmic staining, the HCS Cell Mask (H327 12 Component, 701618, Invitrogen) was applied. The cells were covered with SlowFade Gold Antifade (Invitrogen). To localize mitochondria, MitoTracker^TM Red CMXRos (M7512, Invitrogen) was used (100 nM for 45-h incubation at 37 °C). For lysosome localization, we also used LysoTracker^TM Red DND-99 (L7528, Thermofisher Scientific) according to the manufacturer’s protocol. Information regarding the used antibodies can be found in Supplementary table 2.

HT1080 AID-mCherry transfectants were grown in 96-well μclear microplate (Greiner Bio One) for 24 hr prior to treatment with LMB, MG132, or both at indicated times. Following treatment, cells were fixed in 3.7% formaldehyde and stained with whole cell stain (HCS CellMask, Invitrogen) and DAPI. Fixed cells were then washed with PBS and were imaged by Thermo Scientific ArrayScan VTI HCS reader. Cells with very low or very high mCherry signals were eliminated, gating based on the mock transduction control (low) and eliminating cells with signals more than 5 SD from the mean (high). The HCS Colocalization BioApplication protocol was used to determine nuclear and whole cell boundaries in individual cells as defined by DAPI and HCS CellMask, respectively, thereby defining the cytoplasmic region as the region between nuclear and whole cell boundaries. The average signal in the nuclear and cytoplasmic compartments was determined in individual cells by measuring the total intensity of mCherry signal divided by area within each compartment. The ratio of nuclear to cytoplasmic signal (N/C) was calculated as the ratio of the average signals of nuclear and cytoplasmic mCherry.