*2.2. Cell Culture*

Human neuroblastoma cell line SK-N-SH was obtained from ATCC (Manassas, VA, USA). SK-N-SH cells were cultured in Eagle minimum essential medium (ATCC), supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin—streptomycin (Gibco, Paisley, UK). Fibroblasts were obtained from dermal human fibroblasts, per protocol #7044 (approved by the Institutional Review Board at Sheba Medical Center, Tel Hashomer, Ramat Gan, Israel). Fibroblasts cells were cultured in high glucose DMEM supplemented with 20% FBS, penicillin-streptomycin, 1% non-essential amino acids and 0.2% β-mercaptoethanol (Invitrogen, Life Technologies, Grand Island, NY, USA). Normal human astrocytes (HA) obtained from Science Cell Research Laboratories (Carlsbad, CA, USA). HA cells were cultured in HA medium (Catalog #1801) with supplement from Science Cell Research Laboratories (Carlsbad, CA, USA). All cells were routinely tested for the presence of mycoplasma. The cell lines were cultured at 37 ◦C in a humidified atmosphere containing 5% CO2. As they approached confluence, the cells were sub-cultured following treatment with 6% trypsin-EDTA.

## *2.3. MTT Test*

The effect of HU-600 and HU-585 on the SK-N-SH NBL cell line was studied using the 3-(4, 5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich Co., St. Louis, MO, USA). SK-N-SH cells (4 × 10<sup>3</sup> cells/well) were plated (200 μL) in triplicates in flat bottom 96-well plates in appropriate medium as mentioned above. The cells were allowed to adhere to the plate surface overnight and then cultured with increasing doses of HU-600 or HU-585 (0–200 μM) for 72 h. Cell viability was then determined using MTT assay, which measures reduction of MTT to formazan by mitochondria of viable cells. Formazan was measured spectrophotometrically by absorption at 560 nm in a PowerWaveXTM (BioTek, Santa Clara, CA, USA) plate reader. All experiments were repeated at least 3 times. Cell morphologies were assessed daily by light microscopy.

#### *2.4. Colony-Forming Assay (CFA)*

CFA was used to determine the effect of HU-600 and HU-585 treatment on the ability of a single cell to grow into a colony. SK-N-SH cells (500 cells/well) were plated in triplicates in 6-well plates (3 mL) and were treated according to treatment regimen mentioned below with 12.5, 37.5, and 75 μM of HU-600 or HU-585 for 14 days. Subsequently, colonies were fixed with ethanol 70%, stained with crystal violet solution (0.5% *w*/*v*), rinsed extensively by tap water, dried, and counted using EPSON scan software. Colonies were counted using ImageJ software.

#### *2.5. Cell Migration Assay*

Wound healing assay was performed to compare the effect of HU-600 and HU-585 on cell migration ability. SK-N-SH cells (1 × 10<sup>6</sup> cells/well) were plated in duplicates in 6-well plates (3 mL) and were allowed to adhere overnight, then treated with 75 μM of HU-600 or HU-585, and were allowed to grow into a confluent cell monolayer. Cells then were starved using starvation medium (FBS free) overnight. A single scratch along each well was made by micropipette, fresh medium was supplemented, and cells were incubated for 24 h. Cell migration was quantified by taking pictures with a regular inverted microscope at two time points: right after scratching and 24 h later. Gap intervals were measured using ImageJ software, and the percentage of migration and gap closure was calculated using the formula:

% *migration* = *average wound width* 0*h* − *average wound width* 24*h average wound width* 0*h* × 100

#### *2.6. Western Blot Assay*

To assess apoptotic activity following treatment with HU-585, caspase-3 and Bcl-2 protein levels were evaluated by western blot assay. One day before HU-585 treatment (100 μM for 24 h and 48 h), cells were plated (1 × 10<sup>6</sup> cells per 9-cm plate). Cells were harvested and proteins were extracted with RIPA buffer supplemented with phosphatase and protease inhibitors (Sigma–Aldrich). Protein concentrations were calibrated using the BCA Protein Assay Reagent Kit (Pierce, Rockford, IL, USA). Equal amounts of protein (30 μg) were loaded onto 12.5% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad, Rishon Le Zion, Israel). The blots were reacted using caspase-3 (9662S) or Bcl-2 (D55G8, 4223S) rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) as the primary antibody. The secondary antibody, horseradish peroxidase conjugated goa<sup>t</sup> anti-rabbit antibody (Jackson ImmunoResearch Laboratories, Farmington, CT, USA), was detected by chemiluminescence. Signals were detected using an ECL Kit (CYANAGEN, Bologna, Italy) and visualized using the ChemiDocTM MP Imaging System. Quantification of caspase-3 and Bcl-2 was done by Image Lab software.

#### *2.7. Senescence-Associated β-Galactosidase Activity Assay*

Senescence-associated β-galactosidase (SA-β-gal) activity was measured with a βgalactosidase staining kit (Senescence B-Galactosidase Staining KIT, Cell Signaling Technology, #9860) according to the manufacturer's instructions. Briefly, SK-N-SH cells (5 × 10<sup>4</sup> cells\well) were plated in 6-well plates (3 mL) and treated according to the previously described treatment regimen [19] with 50, 75 and 100 μM of HU-600 or HU-585 for 48 h and then fixed and incubated overnight at 37 ◦C in CO2 free environment. Accumulation of a distinctive blue color in senescent cells was then observed by microscope (Olympus Scientific Solutions, Waltham, MA, USA). Pictures of three representative fields of each well were taken, blue colored cells were counted by ImageJ software, and the percentage of β-galactosidase positive cells was determined and normalizing to the control.

#### *2.8. In Vitro Senolytic Studies*

In vitro senolytic studies were performed using Navitoclax (ABT-263) dissolved in DMSO (Sigma-Aldrich, Rehovot, Israel). SK-N-SH cells (2 × 10<sup>4</sup> cells/well) were plated in triplicates in 96-well plates (200 μL) and were cultured according to the previously described regimen, with increasing concentrations of HU-600 or HU-585 (0–200 μM) for 48 h. Subsequently, cells were cultured with ABT-263 (2.5 μM) for 24 h, then an MTT test was performed according to the MTT assay mentioned above. ABT-263 concentration was chosen following several dose response MTT assays. The maximal dose with no effect on survival was 2.5 μM (data not shown).

#### *2.9. Murine Xenograft Therapeutic Studies*

In vivo experiments were carried out according to protocols approved by the Ethical Committee of the Chaim Sheba Medical Center. Female athymic nude mice (Foxn1nu) 6–8 weeks of age (ENVIGO RMS, Jerusalem, Israel) were used for the tumor xenograft (Xn) model. In total, 5 × 10<sup>6</sup> SK-N-SH mCherry expressing cells were subcutaneously inoculated in the right flank of each mouse. Following cell injection, tumor burden was determined once a week by a Spectrum Animals in vivo imaging system (IVIS ®), and the mice were allocated into four homogeneous groups according to the intensity of the average total radiant efficiency signal measured by IVIS: Control vehicle; HU-585 (120 mg/kg); ABT-737 (75 mg/kg); combined treatment with HU-585 (120 mg/kg) and ABT-737 (75 mg/kg), (*n* = 8 per group). ABT-737 (S1002, Selleckchem, Houston, TX, USA) was formulated in a mixture of 30% propylene glycol, 5% Tween 80, and 65% DsW (5% dextrose in DDW). Treatment started 4 weeks following cell injection and was administered intraperitoneally (IP) once daily for a total of 21 days. Tumor burden was determined once a week by the IVIS system and by external electronic caliper measurements throughout treatments. Tumor volumes were calculated by the following formula: A × B2/2, where A is the greatest diameter, and B is the diameter perpendicular to A. At the end of treatment, the animals were then euthanized, and tumor Xns were immediately removed, weighed, stored, and fixed.
