Xtt assay

Cell viability was tested as previously described [31 (link)]. In brief, mouse macrophages RAW-246 were cultured overnight in Dulbecco's Minimum Essential Medium (DMEM, Sigma-Aldrich) supplemented with 10% inactivated fetal calf serum (FCS, Biological Industries, Beit-Ha’emek, Israel), 1% L-glutamine (Biological Industries) and 1% streptomycin (Biological Industries), at 37°C in 5% CO₂. Each formulation of biodegradable polymer and lipopeptide within plastic inserts (Rosenshein, Israel) that were previously sandblasted, was added to eight wells of a 96-well microtiter plate. Then 200 μL of cell suspension were added to the wells and after 24 hrs the XTT assay (Biological Industries) was initiated by the addition of 50 μL activated XTT solution to each well. The microtiter plate was incubated for 2–4 hrs and then monitored by measuring the absorbance of the supernatant at 450 nm in a VERSAmax microplate reader.

Cells were transfected with si-NT or si-m/hVDAC1, and at 24 h post-transfection, were counted and seeded in 96-well plates. Then, cells were subjected to the XTT assay according to the manufacturer’s instructions (Biological Industries; Beit Haemek, Israel). Absorbance at 450–500 nm, and 630–690 as a reference, were measured using an Infinite M1000 plate reader (Tecan, Männedorf, Switzerland).

Cells (1–2 × 10³) were seeded in a well of 96-well plate in the presence of puromycin (2 μg/ml) with or without doxycycline (1 μg/ml). Cell proliferation was analyzed using the XTT assay (Biological Industries) and spectrophotometrically quantified. Relative cell growth was determined by taking the XTT result of the seeded cells as 100%. The results obtained by the XTT assay were confirmed by direct counting of cells over the period of PSMD1 KD induction. When indicated, a pan-caspase inhibitor QVD (BioVision) or JNK inhibitor SP600125 (Enzo) was added to the growth medium the next day following doxycycline induction.
In the recovery experiment, MDA-MB-231 cells (2 × 10³) were seeded in a well of 96-well plate in the presence of puromycin (2 μg/ml) and doxycycline (1 μg/ml). After 72 h, the cells were washed twice with the culture medium and supplemented with fresh medium with puromycin but without doxycycline for an additional 7 days. The ability of cells to recover from PSMD1 KD was compared to growth characteristics of cells that continued to be supplemented with 1 μg/ml doxycycline after the washing step or were not induced at all.

NLFs were cultured in DMEM supplemented with 10% FCS. At 70% confluency, cells were transfected with Accell Delivery Media (GE Dharmacon; B-005000) supplemented with 1 µM Accell SMARTpool mouse Myc siRNA (Dharmacon; E-040813) or Accell Control Pool nontargeting siRNA (Dharmacon; D-001910) for 96 hr. Accell SMARTpool contains a mixture of four siRNAs targeting one gene and provides extended duration of gene knockdown with only minimal effects on cell viability and the innate immune response. The efficiency of Myc siRNA knockdown was analyzed by qRT-PCR.
For individual siRNA experiments, NLFs were cultured and transfected as described, utilizing individual Myc targeting siRNA constructs (Dharmacon, A-040813-20, A-040813-18, A-040813-17) or control siRNA.
For overexpression of Myc, cells were transiently transfected with a plasmid of Myc (MGC Mouse Myc cDNA pCMV-SPORT6: mammalian expression insert sequence: BC006728, #MMM1013-202763479) or with a control plasmid. Cells were transfected with jetPRIME (polyplus transfection, 114-01) according to the manufacturer’s protocol. All experiments were performed 24 hr following transfection.
XTT assay (Biological Industries, 20-300-1000) was performed 24 hr following transfection according to the manufacturer’s protocol.

Cell viability was determined by XTT assay (Biological Industries Ltd., Kibbutz Beit Haemek, Israel) used as instructed by the manufacturer. We seeded 2 × 10³ cells/cm² in 96-well plates with different concentrations of BP (0, 15, 30, 60, 120, 240 μg/ml for ordinary KURAMOCHI cells, and 0, 12.5, 25, 50, 100, 200, and 400 μg/ml BP for ALDH+ KURAMOCHI cells, 0, 12.5, 25, 50, 100, 200 μg/ml for both OVASAHO ALDH+ and ALDH- cells) for 48h. Then the half-maximal inhibitory concentration (IC50) of both types of cells was obtained. The four-parameter logistic regression (4PL) method described in the previous literature was used 21 (link). The equation is expressed as follows: Y=d+(a-d)/(1+(X/c)^b), where Y is the response, and X is the concentration. The variable a is the bottom of the curve, and d is the top of the curve. The variable b is the slope factor, and c is the concentration corresponding to the response midway between a and d 22 (link). The XTT solutions and N-methyl dibenzopyrazine methyl sulfate (PMS) were defrosted immediately in a water bath at 37°C. To each 100-μL culture in wells of 96-well plates was added 50 μL XTT/PMS. After 2-5 h of incubation at 37°C, plates were analyzed by spectrophotometry to determine the optical density of the solutions at a wavelength of 450 nm (reference wavelength, 650 nm).