Following antibodies were used: WIP1 antibody (clone F-10, sc-376257), p21 (sc-397), p53 (clone D01, sc-126), BRCA1 (sc-6954), rabbit-53BP1 (sc-22760), RAD51 (sc-6862) and TFIIH (sc-293, used as loading control) from Santa Cruz Biotechnology (Dallas, TX, USA); phoshpo-Thr543-53BP1 (#3428), phospho-S15-p53 (#9284) from Cell Signaling Technology (Danvers, MA, USA); RPA2 (clone 9H8, ab2175), and phospho-Ser1524-BRCA1 (ab2401) from Abcam (Cambridge, UK), γH2AX (05-636), and mouse monoclonal 53BP1 (MAB3802) from Merck Millipore (Burlington, MA, USA); phospho-S824-KAP1 (GTX63711), KAP1 (GTX62973) and PP4C (GTX114659) from Genetex (Irvine, CA, USA); secondary Alexa Fluor conjugated antibodies from Thermo Fisher Scientific (Waltham, MA, USA). WIP1 inhibitor GSK2830371 (here referred to as WIP1i and used at 0.5 μM unless stated otherwise), olaparib and camptothecin (all Medchemexpress, New York, NJ, USA) and mitomycin C (Santa Cruz Biotechnology, Dallas, TX, USA) were dissolved in DMSO and used at indicated concentrations.
Camptothecin
Manufactured by MedChemExpress
Sourced in United States
Camptothecin is a chemical compound derived from the Camptotheca acuminata tree. It is a naturally occurring alkaloid with potent anti-cancer properties. Camptothecin acts as a topoisomerase I inhibitor, which is a key enzyme involved in DNA replication and transcription. This compound has been extensively studied and used as a lead molecule for the development of various anti-cancer drugs.
Automatically generated - may contain errors
Lab products found in correlation
γh2ax 05 636, by Merck Group (1 mentions)
Alexa fluor conjugated antibody, by Thermo Fisher Scientific (1 mentions)
Gsk2830371, by MedChemExpress (1 mentions)
Mitomycin c, by Santa Cruz Biotechnology (1 mentions)
Olaparib, by MedChemExpress (1 mentions)
Guava easycyte 8ht flow cytometry system, by Merck Group (1 mentions)
Live dead fixable green dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Solarbio (1 mentions)
T 5224, by Apexbio (1 mentions)
Hyclone 5 fetal bovine serum, by GE Healthcare (1 mentions)
Anti gapdh g9545, by Merck Group (1 mentions)
Anti lamp2 sc 18822, by Santa Cruz Biotechnology (1 mentions)
Hy 16560, by MedChemExpress (1 mentions)
Anti mouse igg 7076, by Cell Signaling Technology (1 mentions)
Anti flag f3165, by Merck Group (1 mentions)
Cisplatin, by MedChemExpress (1 mentions)
Atm inhibitor ku55933, by MedChemExpress (1 mentions)
Topotecan, by MedChemExpress (1 mentions)
Oxaliplatin, by MedChemExpress (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using camptothecin
1
DNA Damage Response Pathway Profiling
2
Apoptosis induction in cell lines
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Cells were seeded in 6-well plates at a density of 1.0 × 105 cells/well, serum-starved for 24 h, and treated with 10 % of FBS, L-PRP, or P-PRP for 7 days. The cells were then incubated with 10 μM camptothecin (MedChem Express, Monmouth Junction, NJ, USA) for 6 h to induce apoptosis. Cell viability and apoptosis was analyzed by staining with Annexin V and PI [37 (link)]. Briefly, cultured cells were detached, centrifuged, resuspended in phosphate buffered saline (PBS; Gibco, Carlsbad, CA, USA) and stained with an Annexin V-FITC/PI staining kit (Cell Signaling Technology, Denvers, MA, USA). Data was acquired and analyzed by flow cytometry and guavaSoft (Guava easyCyte 8HT flow cytometry system, Millipore, MA, USA).
3
Apoptosis Induction and Viability Assay
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Media of different pHs were added to the cells. After 24 h of incubation, the cells were incubated with 10 μM camptothecin (MedChem Express) for 6 h to induce apoptosis. Cell viability was then analysed using a LIVE/DEAD Fixable Green Dead Cell Stain Kit (Molecular Probe) or an Annexin V/PI apoptotic analysis kit (Cell Signalling Technology) according to the manufacturers' instructions.
4
Drug Treatment and Cell Line Analysis
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The human leukemia (HEL 92.1.7, K562) and epithelial-like HEK293T (CRL-3216) cell lines were obtained from ATCC (US) and tested negative for mycoplasma. These cell lines were cultured and maintained in Dulbecco’s Modified Eagle Medium supplemented with HyClone 5% fetal bovine serum (GE Healthcare, US).
For drug treatment, cells were treated with Camptothecin (MedChemExpress, CN), T5224 (APExBIO, CN) and R406 (Beyotime Biotechnology, CN) for indicated times and used for cell proliferation analysis. Generation of K562-fli1 cells was previously described [21 (link)]. For FLI1 induction, cells were treated with 5μM of doxycycline (Solarbio, CN).
For drug treatment, cells were treated with Camptothecin (MedChemExpress, CN), T5224 (APExBIO, CN) and R406 (Beyotime Biotechnology, CN) for indicated times and used for cell proliferation analysis. Generation of K562-fli1 cells was previously described [21 (link)]. For FLI1 induction, cells were treated with 5μM of doxycycline (Solarbio, CN).
5
Molecular Mechanisms of Autophagy Regulation
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Nocodazole (HY-13520), paclitaxel (HY-B0015), cisplatin (HY-17394), chloroquine (HY-17589A), doxorubicin (HY-15142) and camptothecin (HY-16560) were all purchased from MedChem Express. The following antibodies were used: anti-CGAS (15102), anti-LC3B (3868), anti-H2AX (7631), anti-LAMP1 (9091), anti-CAT/catalase (12980), anti-VDAC (4661), anti-CALR/calreticulin (12239), anti-phospho-H2AX (9718), anti-GFP (2956), anti–ATG7 (8558), horseradish peroxidase (HRP)-conjugated goat anti-rabbit (7074) or anti-mouse (7076) IgG (all from Cell Signaling Technology); anti-hemagglutinin (HA; H6908), anti-FLAG (F3165) and anti-GAPDH (G9545) (all from Sigma-Aldrich); anti-LC3B (PM036) and anti-LC3B (M152-3) were obtained from Medical & Biological Laboratories; anti-LAMP2 (sc-18822) was purchased from Santa Cruz Biotechnology. GFP-Trap Agarose (gtd-20) was purchased from ChromoTek GmbH. The plasmid containing the DNA encoding human CGAS (F122086), CGAS mutation (site-directed mutation from F122086), LC3B (F102982) and TEME-192 (F100950) were obtained from Changsha Youbio Tech and cDNAs were then subcloned into the pCDH vector (Changsha Youbio Tech, VT1480). YFP-Atg8-family proteins encoding plasmids were a kind gift from Dr. Felix Randow (MRC Laboratory of Molecular Biology, UK). cgas knockout mice (026554) were obtained from Jackson Laboratory.
6
Investigating Targeted Combination Therapy
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Camptothecin (CPT), Cisplatin (CDDP), Topotecan, ATM inhibitor (KU55933), and WDR5 inhibitor (OICR-9429) PRMT5 inhibitor (GSK591) were purchased from MedChemexpress (Monmouth Junction, NJ, USA). Human recombinant His-JDP2 protein was purchased from Abcam (Cambridge, MA, USA).
7
Cytotoxicity Evaluation of Anticancer Agents
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BCI hydrochloride, irinotecan hydrochloride, oxaliplatin, KU60019, and camptothecin were purchased from MedChemExpress LLC (TriMen Chemicals, Lodz, Poland). Except for oxaliplatin, each of these compounds was dissolved in DMSO; in the case of oxaliplatin, the solvent was H2O. Culture media, phosphate-buffered saline (PBS), penicillin–streptomycin mixture, and trypsin/EDTA were provided by Biowest (CytoGen, Zgierz, Poland). Fetal bovine serum (FBS), DMSO, MTT, and propidium iodide were purchased from Merck/Sigma Aldrich Chemical Co (Burlington, MA, USA). Caspase-Glo® 3/7 Assay System was provided by Promega (Madison, WI, USA). Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit was purchased from Invitrogen (Eugene, OR, USA).
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