Cellevent caspase 3 7 green detection kit

DMEM, DMEM/F12, MEM and Opti-MEM media, TRIzol and Lipofectamine 2000, MitoSOX, and CellEvent Caspase-3/7 green detection kit were purchased from Invitrogen (Carlsbad, CA); Sorafenib from Enzo Life Sciences, Inc. (Farmingdale, NY), Ultra-low attachment plates and MTT from Millipore Sigma (Burlington, MA), FITC Annexin V Apoptosis Detection Kit from BD Biosciences (San Jose, CA), JC-1 dye, Doxycycline Hydrochloride, and Transwell inserts from Thermo-Fisher Scientific (Waltham MA). The antibodies utilized were obtained from the following sources: Ets-1 (Cell Signaling Technology, #14069), E-cadherin (BD Biosciences, #610181, N-cadherin (BD Biosciences, #610920), Vimentin (Cell Signaling Technology, #5741), Snail (Cell Signaling Technology, #3895), Slug (Cell Signaling Technology, #9585), Zeb2 (Cell Signaling Technology, #97885), GAPDH (Ambion, #AM4300), PARP (Cell Signaling Technology, #9542), Cleaved Caspase 3 (Cell Signaling Technology, #9664), Myc-tag (Cell Signaling Technology, #2276), GPX2 (Abcam, #137431), Cytochrome C (Cell Signaling Technology, #11940), COX IV (Abcam, #14744). RT2 profiler PCR Array Human Transcription Factors (PAHS-075Z) was from Qiagen (Germantown, MD).

Spheroids of PA-1 grown on GCA scaffolds are tested for efficacy of the anticancer drug doxorubicin after day 7 and 14. The spheroids are treated with 0.1, 1, and 5 μM doxorubicin hydrochloride (European Pharmacopoeia Reference standard) for 24 h.^{51 (link)} A control group is maintained under similar experimental conditions as compared to the test group; however, the control group is not treated with drugs. The expression of the early apoptotic marker is checked by staining the spheroids with a CellEvent™ caspase-3/7 green detection kit (Invitrogen USA) and observed under the confocal microscope.

Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236, mTOR, phospho-4EBP1, and GAPDH were from Cell Signaling, Phospho-FAK (Tyr397), HPDL from Thermo Fisher, Paxilin and LAMP2 from BD-bioscience; PAK1 from Proteintech. Secondary antibodies Alexa-fluor 594 anti-Rabbit IgG, Alexa-fluor 488 anti-Rabbit IgG, Alexa-fluor 594 anti-Mouse IgG and Alexa-fluor 488 anti-Mouse IgG were from Cell Signaling, IRDye 800CW and IRDye 680CW were from LI-COR. Alexa fluor TM 555 Phalloidin, Click-iT EdU Imaging Kits, CellEvent Caspase-3/7 Green Detection kit, NHS-Fluorescein, NHS-Alexa Fluor 555, pH-rodo iFL STP ester red and Hoechst 33342 were from Invitrogen. DRAQ5 was from LI-COR. Collagen I and Matrigel were from Corning. Geltrex was from Thermo Fisher. All media and dialyzed FBS were from Gibco, except for DMEM with no amino acid which was from US Biological life science and Plasmax which was kindly provided by Dr Tardito, CRUK Scotland Institute, Glasgow. The details of Plasmax composition were previously described by Vande Voorde and colleagues [18 (link)]. E64d (Aloxistatin) and PF573228 were from AdooQ Bioscience. GM6001 was from APEXBIO. Dynasore, Filipin, and EIPA were from Sigma. FRAX 597 was from bio-Techne. Nitisinone was from MCE. PP2 was from Generon. C13-tyrosine was from Sigma-Aldrich.