U2os cell

WI38-hTERT/ER-RAF1 immortalized fibroblastic cell line was kindly provided by C. Mann (48 (link)). They were grown in minimal essential medium supplemented with l-glutamine, nonessential amino acids, sodium pyruvate, penicillin-streptomycin, and 10% fetal bovine serum under 5% CO₂ and 5% O₂. Senescence was induced by treating cells with 20 nM 4-hydroxy-tamoxifen for 3 days. U2OS cells (ATCC) were grown in Dulbecco’s modified Eagle’s medium containing Glutamax supplemented with sodium pyruvate, penicillin-streptomycin, and 10% fetal bovine serum under 5% CO₂. WI38 cells and derivatives were transfected using the siRNA transfection reagent Dharmafect 4 (Dharmacon) following the manufacturer’s instructions, with 100 nM siRNA (unless indicated in a figure legend), and harvested 72 h later. U2OS cells were transfected with the INTERFERin transfection reagent (Polyplus) or with Dharmafect 4 according to the manufacturers’ instructions with 50 nM siRNA and harvested 48 h later. For RNA stability analysis, actinomycin D (Sigma) was added at 10 μg/mL. The siRNAs used are listed in Table 2.

U2OS cells (ATCC, Manassas, Virginia, USA) were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) with pyruvate (Thermo Fisher Scientific, Darmstadt, Germany). RPE-1 hTERT cells (ATCC) were cultured in DMEM:F12 media (Thermo Fisher Scientific). Culture media were supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and penicillin/streptomycin (Thermo Fisher Scientific). Cell lines were tested twice a year for Mycoplasma contamination using the LookOut Detection Kit (Sigma), and all tests were negative.
Cells were treated with DMSO (0.2%; Carl Roth, Karlsruhe, Germany), Nutlin-3a (10 µM; Sigma Aldrich, Darmstadt, Germany), Actinomycin D (5 nM; Cayman Chemicals, Ann Arbor, Michigan, USA), 5-FU (25 μg/ml, Cayman Chemicals), or Doxorubicin (0.2 µg/ml; Cayman Chemicals) for 24 h.

Unless otherwise noted, all chemicals were obtained from Sigma and all enzymes were obtained from New England Biolabs (Ipswich, MA). KAPA Hifi 2x Polymerase (Kapa Biosystems; Wilmington, USA; cat. no. KK2601) was used for all cloning and library production steps. E. coli were cultured at 37°C in Luria broth. All cell culture reagents were purchased from Thermo Fisher Scientific (Waltham, MA) unless otherwise noted. HEK 293T cells (ATCC; Manassas, VA; CRL‐3216) and U‐2 OS cells (ATCC HTB‐96), and derivatives thereof were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1 μg/ml doxycycline (Sigma; St. Louis, MO), unless otherwise noted. hTERT RPE‐1 cells (ATCC CRL‐4000) and derivatives thereof were cultured in F12/DMEM supplemented with 10% FBS, 1 mM PenStrep, and 0.01 mg/ml hygromycin B. For Visual Cell Sorting experiments, DMEM without phenol red was used to reduce background fluorescence. Cells were passaged by detachment with Trypsin–EDTA 0.25%. All cell lines tested negative for mycoplasma in monthly tests. All synthetic oligonucleotides were obtained from IDT, and their sequences can be found in Table EV3. All non‐library‐related plasmid modifications were performed with Gibson assembly. See the Appendix and Table EV3 for construction of the vectors used.

Glioblastoma cell lines (LN229, U118, T98G and A172) were derived from American Type Culture Collection (ATCC; Manassas, VA). U251 was obtained from Sigma Aldrich (St. Louis, MO). Renal cell carcinoma cell lines (ACHN and 786-O) were obtained from ATCC, UM-RC-2 were purchased from Sigma, while RCC4 was a gift from Eric Jonasch in MD Anderson. Meningioma cell lines (IOMM-Lee, JEN and CH-157) were generated in-house (University of Utah) [18 ]. Mouse melanoma cell line (B16) and breast cancer cell line (4T1) were purchased from ATCC. U2OS Cells were obtained from ATCC.

U-2 OS cells (human bone osteosarcoma cells), RRID:CVCL_0042, were obtained from ATCC and propagated in the William Hahn lab; they were not additionally authenticated prior to this experiment. The cell line tested negative for mycoplasma prior to this experiment. HEK293T cells, RRID:CVCL_0063, were obtained from ATCC. The cell line was validated by STR profiling (Genetica DNA Laboratories) and was negative for mycoplasma as measured by MycoAlert Mycoplasma Detection Kit (Lonza, Walkersville, MD).