Cytochrome c

Manufactured by Santa Cruz Biotechnology

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Cytochrome c is a heme-containing protein that plays a crucial role in the electron transport chain within the mitochondria of eukaryotic cells. It is responsible for the transfer of electrons between the electron transport complexes, facilitating the production of adenosine triphosphate (ATP), the primary energy currency of the cell. Cytochrome c is a highly conserved protein across various species and is essential for cellular respiration and energy metabolism.

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Anti-cytochrome c (53 citations) Cytochrome c antibody (11 citations) Anti-cytochrome c antibody (10 citations) Cytochrome c (CYT C), (8 citations) Mouse anti-cytochrome c (5 citations) Cytochrome c (Sc-13156) (3 citations) Cytochrome C (sc‐13561 (2 citations) Anti-cytochrome c (A-8, (2 citations) Rabbit polyclonal anti-cytochrome C (2 citations) Andanti-cytochrome c oxidase (COXIV-1) antibody (2 citations)

170 protocols using cytochrome c

Antioxidant and Apoptosis Pathway Study

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EGCG was purchased from Sigma Aldrich (St. Louis, MO, USA). The antibody against Nrf2 was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against heme oxygenase-1 (HO-1) was purchased from Abcam (Cambridge, UK). Goat anti-Bcl-2, Bax, cytochrome c, cleaved caspases 9 and 3, and β-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology. Anti Kim-1 rat polyclonal antibody was purchased from Thermo Scientific (Pittsburgh, PA, USA). Sodium Fluoride (NaF, molecular weight 41.98) and all other chemicals and solvents were of certified analytical grade and purchased from S.D. Fine Chemicals (Mumbai, India) or Hi Media Laboratories Pvt. Ltd. (Mumbai, India). Reagent kits were obtained from Span Diagnostics (Mumbai, India). The structure of EGCG is shown in Fig. 1.Fig. 1

Chemical structure of EGCG (C₂₂H₁₈O₁₁).

Thangapandiyan S, & Miltonprabu S. (2014). Epigallocatechin gallate supplementation protects against renal injury induced by fluoride intoxication in rats: Role of Nrf2/HO-1 signaling. Toxicology Reports, 1, 12-30.

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Western Blot Analysis of Cellular Proteins

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Proteins from different groups were separated in 10% or 12% SDS-polyacrylamide gel and transferred to the nitrocellulose membrane (Pall). Then the membranes were washed with TBS-T and blocked in 5% powdered-milk solution in TBS-T for 1 h. After washing with TBS-T, the membranes were separately probed with antibodies of anti-FLAG (sigma, F3165), β-actin (Cell Sigmaling, #4970), cytochrome c (Santa Cruz Biotechnology, sc-13156) for 1 h. After washing with TBS-T, the membranes were incubated with the secondary HRP-labeled anti-mouse antibody (SouthernBiotech, 6170-05) or anti-rabbit antibody (SouthernBiotech, 4030-05) for 1 h at room temperature. Proteins were visualized by the ECL Plus Western Blotting Substrate (Thermo) according to the manufacturer's directions.

An X., Ji B, & Sun D. (2020). TRIM34 localizes to the mitochondria and mediates apoptosis through the mitochondrial pathway in HEK293T cells. Heliyon, 6(1), e03115.

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Molecular Profiling of Cancer Cell Lines

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DMEM‐F12 (Dulbecco's modified Eagle's medium‐F12) was purchased from Sigma (Sigma‐Aldrich). FBS (foetal bovine serum), ITS (insulin‐transferrin‐selenium), L‐glutamine, DMSO (dimethyl sulfoxide), MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazoliumbromide), penicillin, streptomycin and sirtinol were purchased from Sigma (Sigma‐Aldrich). Antibodies against sirt1, ERα, IGF1R, CCND1 (cyclin D1), vimentin, N‐cadherin, bax (bcl2 associated x), bcl‐2 (B‐cell lymphoma 2), parp‐1 (poly(ADP‐ribose) polymerase 1), cytochrome c, pCREB‐1^Ser133 (phospho cyclic AMP response element binding) and GAPDH (glyceraldehyde 3‐phosphate dehydrogenase) were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc, Heidelberg, Germany). Antibody against VDAC1 (voltage‐dependent anion‐selective channel protein 1)/porin) was purchased from Abcam (Abcam, Cambridge, UK). HRP (Horseradish peroxidase)‐conjugated secondary antibodies were purchased from Bethyl (Bethyl Laboratories, Montgomery, TX, USA) and ECL (Electrochemiluminescence) Western blotting detection system from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz CA, USA). The TRIzol reagent, High Capacity cDNA Reverse Transcription Kit and PowerUp™ SYBR™ Green Master Mix were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific).

Chimento A., De Luca A., Nocito M.C., Sculco S., Avena P., La Padula D., Zavaglia L., Sirianni R., Casaburi I, & Pezzi V. (2021). SIRT1 is involved in adrenocortical cancer growth and motility. Journal of Cellular and Molecular Medicine, 25(8), 3856-3869.

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Western Blot Analysis of IEC Proteins

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About 50 μg total proteins of small IECs was denatured in sample buffer containing SDS and β-mercaptoethanol, separated on a 4–20% gradient SDS-PAGE gel, and electroblotted onto nitrocellulose membranes. Nonspecific binding sites of the membrane were blocked using defatted milk protein. The relative amount of primary antibody was detected with peroxidase-conjugated secondary antibody. Densitometry was used to quantify protein abundance. Similar procedures were carried out with antibodies against PTEN (Sigma Chemical Co), cytochrome c, Histone(H3), p65, β-actin (all from Santa Cruz Biotechnology), cleaved caspase-3, cleaved caspase-8, cleaved caspase-9, COX IV (all from Cell signaling Technology, Danvers, MA, USA) and Bax.

Liu Z., Jiang J., Yang Q., Xiong Y., Zou D., Yang C., Xu J, & Zhan H. (2016). MicroRNA-682-mediated downregulation of PTEN in intestinal epithelial cells ameliorates intestinal ischemia–reperfusion injury. Cell Death & Disease, 7(4), e2210-.

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Antibody Immunoblotting for Cellular Proteins

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Antibodies to Lamin A, VDAC, Bax, Bad and Cytochrome C were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Mouse monoclonal Grb2 was obtained from BD Biosciences (San Jose, CA), anti-α-tubulin clone B512 was obtained from Sigma-Aldrich (Sigma-Aldrich Co, St. Louis, MO), and anti-Mannosidase II-Golgi marker was from ABCAM (Abcam Inc. MA).

Darbinian N., Darbinyan A., Merabova N., Selzer M.E, & Amini S. (2020). HIV-1 and HIV-1-Tat Induce Mitochondrial DNA Damage in Human Neurons. Journal of HIV and AIDS, 6(1), 176.

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Apoptosis and ROS Induction Assay

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RPMI 1640, Fetal Bovine Serum (FBS), Trypsin-EDTA, Penicillin-Streptomycin, and MTT were obtained from Thermo Fisher Scientific (Waltham, MA, USA). AnnexinV-FITC apoptosis detection kit and Caspase-6 and -9 colorimetric assay kits were bought from Biovision (Mountain View, CA, USA). Fluorescent Reactive Oxygen Species detection kit was obtained from Marker Gene Technologies (Eugene, OR, USA). The antibodies against Bax, Bcl-2, and cytochrome c were bought from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). JC-1 was purchased from Enzo Life Sciences (Farmingdale, NY, USA).

Hamzeloo-Moghadam M., Aghaei M., Abdolmohammadi M.H., Khalaj A, & Fallahian F. (2018). Cytotoxic effect of Drimia maritima bulb extract and induction of mitochondrial apoptotic signaling in human breast cancer cells, MCF-7 and MDA-MB-468. OncoTargets and therapy, 11, 7669-7677.

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MTT Assay for Cytotoxicity Evaluation

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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is the product of SRL, N-acetyl cysteine (NAC) was purchased from Sigma Aldrich, and HCT116 and HEK293 cell lines were obtained from the National Centre For Cell Science (NCCS), Pune, Govt. of India. HCT 116 cell lines were cultured in DMEM Dulbecco's Modified Eagle's Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin antibiotics, where trypsin and ethylenediaminetetraacetic acid (EDTA) were all purchased from Gibco BRL. Tissue culture plastic wares were obtained from NUNC (Roskidle, Denmark). DAPI (4′,6-diamidino-2-phenylindole dihydrochloride), acridine orange (AO), and ethidium bromide (EtBr) were obtained from Invitrogen (California) and BCA protein assay reagent from Fermentus (EU). The primary antibodies for Bcl2, Bax, caspase 3, cleaved caspase-3, caspase-9, cleaved caspase-9, cytochrome c, P53, cleaved PARP, and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). DCF-DA was obtained from Sigma-Aldrich (MO, USA). An Annexin-V FITC apoptosis detection kit was obtained from Calbiochem (CA, USA). Doxorubicin was purchased from Sigma Aldrich.

Das T., Mishra S., Nag S, & Saha K.D. (2022). Green-synthesized gold nanoparticles from black tea extract enhance the chemosensitivity of doxorubicin in HCT116 cells via a ROS-dependent pathway. RSC Advances, 12(15), 8996-9007.

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Apoptosis Pathway Protein Analysis

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The following antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA): BCL-2 (2876, 1:1000), Caspase-3 (9662, 1:1000) and PARP (9532, 1:1000). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Cytochrome C (7H8: sc-13560, 1:1000), Bcl-xL (H-5: sc-8392, 1:500), Mcl-1 (S-19: sc-819, 1:1000), Bax (N-20: sc-493, 1:500), Bak (G-23: sc-832, 1:500), Bid (FL-195: sc-11423, 1:1000), p53 (DO-1: sc-126, 1:500), and the actin antibody (ACTN05, 1:2000) from Thermo Scientific, (Rockford, IL, USA). Protein concentrations were determined using the Micro bicinchoninic acid (BCA) protein assay (Pierce Chemical Company, Rockford, IL, USA). PNT2258 and associated reagents were provided by ProNAi Therapeutics (Ann Arbor, MI).

Ebrahim A.S., Kandouz M., Liddane A., Sabbagh H., Hou Y., Li C, & Al-Katib A. (2016). PNT2258, a novel deoxyribonucleic acid inhibitor, induces cell cycle arrest and apoptosis via a distinct mechanism of action: a new class of drug for non-Hodgkin's lymphoma. Oncotarget, 7(27), 42374-42384.

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Andrographolide-Induced Apoptosis Pathway

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Andrographolide was procured from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in DMSO and kept at 4 °C at a concentration of 50 mM. AnnexinV-FITC Apoptosis Detection Kit was purchased from BD Pharmingen (Pharmingen, USA). Caspases fluoremetric assay kit was purchased from Chemicon International Corporation (USA). Ac-DEVD-CHO (caspase-3 inhibitor), and Ac-LEHD-CHO (caspase-9 inhibitor) were from Calbiochem (La Jolla, USA). Primary antibodies (Bcl-2, Bcl-xL, Bax, Apaf-1, cytochrome c, β-actin and COX IV) and polyclonal secondary antibody were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, USA). The fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and antibiotics were purchased from Gibco BRL (Grand Island, USA). Plastic wares for cell culture were procured from NUNC (Roskilde, Denmark). Other chemicals including 4, 6-diamidino-2-phenylindole (DAPI), 3[4-dimethylthiazol-2-71]-2-5-diphenyl tetrazolium bromide (MTT) were from Sigma-Aldrich (St. Louis, USA).

Banerjee M., Chattopadhyay S., Choudhuri T., Bera R., Kumar S., Chakraborty B, & Mukherjee S.K. (2016). Cytotoxicity and cell cycle arrest induced by andrographolide lead to programmed cell death of MDA-MB-231 breast cancer cell line. Journal of Biomedical Science, 23, 40.

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Colorectal Cancer Cell Line Maintenance

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Human colorectal cancer HCT116 and HT-29 cell lines were purchased from the American Type Culture Collection. Cells were maintained in 10% fetal bovine serum (FBS)-supplemented RPMI 1640 medium (GIBCO, Grand Island, NY, USA) and 1% penicillin-streptomycin (GIBCO) at 37°C in a humidified incubator containing 5% CO₂. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI), zVAD and all of the other chemical reagents were purchased from Sigma Chemical (St. Louis, MO, USA). Antibodies against various proteins were obtained from the following sources: PARP (Poly-ADP-ribose polymerase), Mcl-1, Bcl-2, Bcl-XL, survivin, cytochrome c, Bax, Bak, Bim, anti-mouse and anti-rabbit IgGs were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Phospho-Akt (Ser473), phospho-GSK-3β, Akt, phospho-p44/42 MAPK (1/2 Erk) (Thr202/Tyr204), p44/42 MAPK (1/2 Erk), caspase8, caspase 9, γH2AX, p21 and acetyl-α-tubulin were obtained from Cell signaling (Danvers, MA, USA)., Actin was obtained from Chemicon (Billerica, MA, USA). Acetyl-histone H3 and GPADH were from Millipore (Billerica, MA, USA). Caspase 3 and was obtained from IMGENEX (San Diego, CA, USA).

Chen C.H., Lee C.H., Liou J.P., Teng C.M, & Pan S.L. (2015). Molecular mechanisms underlying the antitumor activity of (E)-N-hydroxy-3-(1-(4-methoxyphenylsulfonyl)-1,2,3,4-tetrahydroquinolin-6-yl)acrylamide in human colorectal cancer cells in vitro and in vivo. Oncotarget, 6(34), 35991-36002.

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