For doxorubicin-induced senescence, FRCs were treated with 250 nM of doxorubicin (Cayman Chemical) for 24 h, followed by 83.3 nM of doxorubicin for another 48 h. Cells were assayed 7 days later. EdU staining: Cultured cells were pulsed with 10 mM EdU for 6.5 h in CO2 incubator, followed by fixation in 4% PFA for 10 min and permed in PBS + 0.5% Triton X-100 for 15 min at RT. EdU staining was performed in 0.1 M Tris-HCl (ph 7.5), 1 mM CuSO4, 0.1 M ascorbic acid and 1 µM AlexaFluor488 azide (Life Technologies) for 30 min at RT. Stained cells were washed twice with PBS + 0.5%Triton X-100 and incubated with DAPI for 5 min before immunofluorescence microscopy analysis. Positive staining was quantified in ImageJ. For RT-qPCR, cells were collected at day 14 post-induction. Total RNA and cDNA were prepared using the RNeasy Mini Kit (QIAGEN) and High Capacity cDNA RT kit (Applied Biosystems), according to manufacturers’ protocol and Taqman assays performed. Taqman assays used in these studies are listed in Supplementary Table S3 .
Doxorubicin
Manufactured by Cayman Chemical
Sourced in United States
Doxorubicin is a laboratory chemical used for research purposes. It is a cytotoxic anthracycline antibiotic that is commonly used in cancer research. Doxorubicin is known to intercalate with DNA and inhibit DNA and RNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Nutlin 3a, by Merck Group (5 mentions)
Cisplatin, by Cayman Chemical (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Etoposide, by Cayman Chemical (4 mentions)
Etoposide, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dmem f12 media, by Thermo Fisher Scientific (3 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (3 mentions)
Rpe 1 htert cells, by American Type Culture Collection (3 mentions)
Actinomycin d, by Cayman Chemical (3 mentions)
Dmem with pyruvate, by Thermo Fisher Scientific (3 mentions)
Paclitaxel, by Cayman Chemical (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Cytarabine, by Cayman Chemical (2 mentions)
Cisplatin, by Merck Group (2 mentions)
Celltrace violet, by Thermo Fisher Scientific (2 mentions)
C11 bodipy, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Cellrox green, by Thermo Fisher Scientific (2 mentions)
N acetyl cysteine (nac), by Merck Group (2 mentions)
55 protocols using doxorubicin
1
Doxorubicin-Induced Senescence Assay
2
Determining IC50 of Anti-Cancer Drugs
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The half-maximal inhibitory concentration (IC50) is defined as the concentration of a drug necessary to inhibit the biological activity of the target or pathway by 50%. We determined the IC50 of doxorubicin and sorafenib (both from Cayman Chemical Company, Ann Arbor, MA, USA) in MIHA, Hep3B, and Huh7 cells by treating cells with the indicated concentrations of doxorubicin or sorafenib (Figure 1 ). The IC50 of trametinib and cisplatin (both from Cayman Chemical Company) in Huh7 cells was also determined to examine their combinatorial effects with PAM. Further, 5 × 104 cells seeded in 35 mm dishes were incubated for 18 h before treatment with anti-cancer drugs. Cell viability was measured 72 h after drug treatment using an MTT assay.
3
Inducible Cre-Mediated Leukemia Model
4
Senescent Cell Conditioned Media Assay
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The seeded non-senescent MeT-5A cells were made senescent by incubation with 100 nM of doxorubicin (Cayman chemicals; 15,007) for 48 h. The media along with the remaining doxorubicin was removed and the senescent monolayer was washed with 1 × PBS. Senescent monolayers were incubated with doxorubicin-depleted media for another 24 h and the conditioned media (CM) was collected. Non-senescent MeT-5A monolayers were also incubated with media for 24 h and the CM was collected. A 1:1 mixture of CM from senescent/non-senescent MeT-5A and Medium 199 media (HiMedia; AL014A) along with 10% fetal bovine serum (Gibco; 10,270) were used for conditioned media growth assay with SKOV-3 cancer cells.
5
TP53 Knockout in Mammalian Cells
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CP-A WT cells were grown to 60–70% confluency in 10 cm plates. Next, 5 μg of PX458 plasmid containing TP53-targeting sgRNAs were diluted into 200 µl Opti-MEM I reduced serum medium (Thermo Fisher Scientific, 31985062). Then 15 µl FuGENE HD transfection reagent (for a 3:1 transfection reagent:DNA ratio) (Promega, E2312) was added to the DNA and incubated at room temperature for 15 min. The mixture was then added to a plate drop-by-drop and mixed by shaking. Cells were incubated for 48 h at 37 °C in a humidified incubator. Transfected cells, expressing GFP, were single-cell sorted on a BD FACSAria Fusion instrument (BD Biosciences). Clones were allowed to expand, then individually tested by immunoblotting for TP53 protein levels following 24 h of treatment with 3 µM doxorubicin (Cayman Chemical, 15007).
6
Apoptosis Assessment in MEF Cells
7
Topoisomerase Inhibitors and Oxidative Stress
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B16-F1 and B16-F10 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Topoisomerase inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA; camptothecin, merbarone, teniposide, etoposide, XK469, ICRF-193) and Cayman Chemical (Ann Arbor, MI, USA; doxorubicin, moxifloxacin). NAC, LPA and HYD HCl were from Sigma-Aldrich. Fluorescent probes were from Life Technologies (Waltham, MA, USA; DDAOG, C11-BODIPY, CellROX Green, CellTrace Violet), eBioscience (San Diego, CA, USA; CV450) and Sigma-Aldrich (DAPI). Bafilomycin A1 was from Research Products International (Mount Prospect, IL, USA). Primary antibodies were from Millipore (Billerica, MA, USA; anti-γH2AX, clone JBW301) or Abcam (Cambridge, UK; anti-4-HNE, anti-OHdG). Fluorescent secondary antibodies were obtained from Thermo Pierce (Waltham, MA, USA). Cell culture reagents were from Life Technologies (DMEM, pen-strep solution, trypsin-EDTA) and Gemini Biosciences (West Sacramento, CA, USA; fetal bovine serum, stabilized l -glutamine).
8
Cell Lines and Drug Treatments
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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.
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.
9
Kinase Inhibitor Library for Cell Treatments
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The kinase inhibitor library, composed of 149 selective or broad-spectrum kinase inhibitors dissolved in DMSO (10 mM stock concentration), was purchased from Cayman Chemicals (#10505). The following reagents were used for cell treatments at given concentrations: LPS (Escherichiacoli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml−1), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 µM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 µM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 µM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 µM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 µM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 µM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 µM), Staurosporine (#81590, Cayman, 5 µM), Etoposide (#E1383, Sigma, 5 µM), Doxorubicin (#15007, Cayman, 5 µM), Gemcitabine (#S1714, Selleckchem, 100 nM).
10
Comprehensive Antibody Evaluation Protocol
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Unless otherwise specified, all chemicals in the study, including DMSO (dimethyl sulfoxide) and emetine, were purchased from Sigma (St Louis, MO, USA). Erlotinib, gefitinib, doxorubicin, and cephaeline were purchased from Cayman Chemical (Ann Arbor, MI, USA). KRIBB11 and osimeritnib were purchased from Selleckchem (Houston, TX, USA).
Antibodies against HSF1 (#12972), HSP27 (#2402), HSP90α (#8165), PARP (#8165), phospho-ERK (#9101), ERK (#4695), phospho-MEK (#9121), MEK (#9122), BCL2 (#15071), MCL1 (#4572), caspase-3 (#9662), cleaved caspase-3 (#9664), PARP (#9542), EGFR (#4267), phospho-EGFR (Y1068) (#3777), MET (#8198), HER2 (#4190), and vimentin (#5741) were purchased from Cell Signaling Technology (Danvers, MA, USA), and actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phospho-HSF1 (Ser326) (#ab76076), BAG3 (ab47124) and E-cadherin (ab15148) were purchased from Abcam (Cambridge, MA, USA). N-cadherin antibody (640920) was purchased from BD Bioscience (San Jose, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, mouse anti-rat, and goat anti-mouse IgG antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA).
Antibodies against HSF1 (#12972), HSP27 (#2402), HSP90α (#8165), PARP (#8165), phospho-ERK (#9101), ERK (#4695), phospho-MEK (#9121), MEK (#9122), BCL2 (#15071), MCL1 (#4572), caspase-3 (#9662), cleaved caspase-3 (#9664), PARP (#9542), EGFR (#4267), phospho-EGFR (Y1068) (#3777), MET (#8198), HER2 (#4190), and vimentin (#5741) were purchased from Cell Signaling Technology (Danvers, MA, USA), and actin antibody (sc-47778) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against phospho-HSF1 (Ser326) (#ab76076), BAG3 (ab47124) and E-cadherin (ab15148) were purchased from Abcam (Cambridge, MA, USA). N-cadherin antibody (640920) was purchased from BD Bioscience (San Jose, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit, mouse anti-rat, and goat anti-mouse IgG antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA).
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