Thapsigargin

Manufactured by Santa Cruz Biotechnology

Sourced in United States

Thapsigargin is a bioactive compound that functions as a potent and selective inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) enzyme. It plays a key role in the regulation of intracellular calcium homeostasis.

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Lab products found in correlation

22 protocols using thapsigargin

Chondrocyte ER Stress Response

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Human primary chondrocytes (PC136121A1-C, Asterand Bioscience, Cambridge, Massachusetts) and
human chondrocyte cell line, C28/I2, were cultured in DMEM/F12 and DMEM mediums, respectively, which
were supplemented with 10% fetal bovine serum and antibiotics (Life Technologies, Grand Island, New
York). Cells were maintained at 37°C and 5% CO2 in a humidified incubator. After ten hours of
serum-free conditions, cells were incubated with 10 ng/ml TNFα (R&D Systems, Minneapolis,
Minnesota) in the presence and absence of 5 µM Salubrinal or 5 µM Guanabenz (TOCRIS
Bioscience, Ellisville, Missouri). One µM thapsigargin (Santa Cruz Biotechnology, Santa Cruz,
California) was used as a positive control (PC) for phosphorylation of eIF2α.

Hamamura K., Nishimura A., Iino T., Takigawa S., Sudo A, & Yokota H. (2015). Chondroprotective effects of Salubrinal in a mouse model of osteoarthritis. Bone & Joint Research, 4(5), 84-92.

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Cell Line Culturing and Inhibitor Treatments

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The prostate cancer cell lines PC-3 and DU-145 were purchased from the ATCC; Dr Vivek Rangnekar, University of Kentucky, Lexington gifted Par-4^+/+ and Par-4^−/− MEFs; Dr Bert Vogelstein, Johns Hopkins University, Baltimore, MD gifted p53^+/+ HCT-116 and p53^−/− HCT-116 cell lines. These cell lines were cultured in RPMI 1640/DMEM medium (Invitrogen Life Technologies, Carlsberg, CA, USA) supplemented with 10% fetal bovine serum, 70 mg/l penicillin, 100 mg/l streptomycin, 6 mm HEPES and 2 mml-glutamine (Sigma-Aldrich, St Louis, MO, USA) at 37 °C and 5% CO_2. The major inhibitors including ER stress inhibitor Salubrinal, JNK inhibitor SP600125 were purchased from Selleck Chemicals, Houston, TX, USA. AKT inhibitor AT7867, caspase inhibitor Fmk-z-VAD and ER stress inducer Thapsigargin were procured from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA.

Rasool R.U., Nayak D., Chakraborty S., Faheem M.M., Rah B., Mahajan P., Gopinath V., Katoch A., Iqra Z., Yousuf S.K., Mukherjee D., Kumar L.D., Nargotra A, & Goswami A. (2017). AKT is indispensable for coordinating Par-4/JNK cross talk in p21 downmodulation during ER stress. Oncogenesis, 6(5), e341-.

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Intracellular Ca2+ Imaging in RPE1 Cells

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For intracellular Ca²⁺ imaging, GJA1 siRNA- or plasmid DNA-transfected RPE1 cells were cultured on fibronectin (Sigma Aldrich, MO, USA)-coated cover glasses. Cells were loaded with 2 µM Fura-2/AM (Invitrogen). Ratiometric Ca²⁺ imaging was performed at 340 and 380 nm in 0 or 2 mM Ca²⁺ Ringer’s solution using an IX81 microscope (Olympus, Tokyo, Japan) at room temperature. Images were processed with Metamorph and analyzed with Igor Pro software. Ca²⁺ peaks were calculated by determining the maximum of each trace upon application of 0.5 μM thapsigargin (Santa Cruz, TX, USA) and Ca²⁺, and baseline values before application were subtracted.

Jang D.G., Kwon K.Y., Kweon Y.C., Kim B.G., Myung K., Lee H.S., Young Park C., Kwon T, & Park T.J. (2022). GJA1 depletion causes ciliary defects by affecting Rab11 trafficking to the ciliary base. eLife, 11, e81016.

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Detailed Biochemical Reagent Protocol

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DMSO, DTT, boric acid, NaCl, Tris, methanol, MgCl₂, sucrose, and methanol were purchased from Sigma-Aldrich (St. Louis, MO). Triton X-100, Tween-20, NP40, and cycloheximide were purchased from Fisher Scientific (Pittsburg, PA). Paraformaldehyde was purchased from Affymetrix/USB Corporation (Cleveland, OH). Thapsigargin and BSA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Phosphatase inhibitors and protease were purchased from Calbiochem (San Diego, CA), and fetal bovine serum (FBS) was purchased from Gibco-Life Sciences (Grand Island, NY).

Kobylewski S.E., Henderson K.A., Yamada K.E, & Eckhert C.D. (2016). Activation of the EIF2α/ATF4 and ATF6 Pathways in DU-145 Cells by Boric Acid at the Concentration Reported in Men at the US Mean Boron Intake. Biological Trace Element Research, 176(2), 278-293.

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Visualization of Proteolytic Activity in Neurons

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For visualization of proteolytically active compartments, neurons at DIV 17 were incubated with 10 µg/mL DQ-BSA Red (Invitrogen) diluted in PBS for 18 h at 37 °C. An equal volume of PBS was added to control cultures. As positive control for LC3 and G3BP stainings, neurons were treated with 30 nM bafilomycin A1 (Sigma-Aldrich) for 1 h or 10 µM thapsigargin (Santa Cruz) for 24 h at 37 °C, respectively. To disrupt microtubules, neurons at DIV 14 were treated with 25 µM vinblastine (Tebu-bio) for 24 h at 37 °C. Equal volumes of dimethyl sulfoxide (DMSO) were included as vehicle control.

Wiersma V.I., van Ziel A.M., Vazquez-Sanchez S., Nölle A., Berenjeno-Correa E., Bonaterra-Pastra A., Clavaguera F., Tolnay M., Musters R.J., van Weering J.R., Verhage M., Hoozemans J.J, & Scheper W. (2019). Granulovacuolar degeneration bodies are neuron-selective lysosomal structures induced by intracellular tau pathology. Acta Neuropathologica, 138(6), 943-970.

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Tunicamycin and Thapsigargin-Induced ER Stress

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Tunicamycin was added at 5 μg/mL, 10ug/mL, and Thapsigargin (Santa Cruz Biotech, Cat# SC 24017) was added at 1 μmol/L and 10 μmol/L to wells in 12‐well plates containing the RT4 cell lines. RT4 cells in plain media not containing the luciferase reporters served as negative controls. Wells were incubated with tunicamycin and Thapsigargin for 6 h, then the media was removed and each well was washed with 1xPBS before adding 150 μL of 1x passive lysis buffer. To assess firefly luciferase activity, we added 50 μL of cell lysate to a luminometer tube and then added 50 μl LARII (Promega, Dual luciferase reporter assay system, Cat# E1910). For the Nano assay, we added 50 μL of cell lysate to the luminometer tube then added 50 μL of Nano substrate solution (Promega, Nano‐glo luciferase assay, Cat# N1110) before taking a measurement value with the luminometer (Monolight 3010).

Bai Y., Wu X., Brennan K.M., Wang D.S., D'Antonio M., Moran J., Svaren J, & Shy M.E. (2018). Myelin protein zero mutations and the unfolded protein response in Charcot Marie Tooth disease type 1B. Annals of Clinical and Translational Neurology, 5(4), 445-455.

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Comprehensive Analysis of P2 Receptor Expression

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All chemicals were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated. Selective antagonists were obtained from Tocris Bioscience (Bristol, UK) (P2Y₁ MRS2500; P2Y₂ AR-C118925XX; P2Y₆ MRS2578; P2Y₁₁ NF340; P2Y₁₂ PSB-0739; P2Y₁₃ MRS2211; P2X4 PSB12062; P2X7 A438079), excluding thapsigargin (sarco-endoplasmic reticulum Ca²⁺-ATPases, SERCA) and U73122 (PLC) (Santa Cruz Biotechnology, Texas, USA). Nucleotides were purchased from Abcam (Cambridge, UK), except ADP (Sigma-Aldrich, Dorset, UK). Primary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) (P2X1 sc-31491; P2X5 sc-15192), Alomone Labs (Jerusalem, Israel) (P2X4, APR-002; P2X7, APR-004; P2Y₁, APR-009; P2Y₄, APR-006; P2Y₆, APR-011; P2Y₁₁, APR-015; P2Y₁₂, APR-020; P2Y₁₃, APR-017) and Abcam (Cambridge, UK) (P2Y₂, ab10270). Phycoerythrin (PE)-conjugated IgG₁ isotype control (400113), CD14 (367103), CD45 (368509), CD73 (344003), CD90 (328109) and CD105 (323205) antibodies were all purchased from Biolegend (San Diego, CA, USA).

Ali S., Turner J, & Fountain S.J. (2018). P2Y2 and P2Y6 receptor activation elicits intracellular calcium responses in human adipose-derived mesenchymal stromal cells. Purinergic Signalling, 14(4), 371-384.

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Assay for ELK1 Transcriptional Activity

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All cell lines described were grown in high-glucose DMEM (Life Technologies) with 10% heat-inactivated fetal bovine serum (FBS) in the presence of 100 U/ml penicillin and 100 g/l streptomycin (Life Technologies). Compounds were obtained from the following companies: BFA (Sigma-Aldrich), GCA (Santa Cruz Biotechnology), MON (Enzo Life Sciences), tunicamycin (Santa Cruz Biotechnology), thapsigargin (Santa Cruz Biotechnology), doxorubicin (Sigma-Aldrich), U0126 (Sigma-Aldrich), and torin2 and MG132 (Selleck Chemicals). The ELK1-luciferase reporter construct (pELK1-Luc, cat. # LR-2061) was purchased from Signosis (USA, distributed by BioCat, Germany).

Baumann J., Ignashkova T.I., Chirasani S.R., Ramírez-Peinado S., Alborzinia H., Gendarme M., Kuhnigk K., Kramer V., Lindemann R.K, & Reiling J.H. (2018). Golgi stress–induced transcriptional changes mediated by MAPK signaling and three ETS transcription factors regulate MCL1 splicing. Molecular Biology of the Cell, 29(1), 42-52.

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Investigating miR-143-3p and miR-155-5p Functions

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Precursors of miR-155-5p and miR-143-3p were purchased from Sigma-Aldrich. Approximately 5 × 10⁴ cells were seeded in P60 culture plates (353002, Falcon^TM, Thermo Fisher Scientific, Waltham, MA, USA) and transfected with 10–20 nM of MISSION^® miRNA mimic hsa-miR143-3p or hsa-miR-155-5p (HMI0221 or HMN0254, Sigma-Aldrich, Saint Louis, MO, USA). As specified in the manufacturer’s protocol (#409-10, Polyplus transfection^®, Strasbourg, France), miRNA expression in transfected cells was assessed 72 h after transfection, whereas protein downregulation was analysed 96 h following transfection. To evaluate insulin signaling in cells transfected with pre-miR-155-5p, they were deprived in 0% FBS medium for 6h and then stimulated with 100 nM insulin (Sigma-Aldrich, Saint Louis, MO, USA) for 10 min. To study the effect of the pre-miR-143-3p on cell apoptosis, HUVECs were deprived of FBS for 6 h or VSMCs were deprived of FBS for 2 h followed by a treatment with thapsigargin for 2 h (100 nM, Santa Cruz Biotechnology, Dallas, TX, USA).

González-López P., Ares-Carral C., López-Pastor A.R., Infante-Menéndez J., González Illaness T., Vega de Ceniga M., Esparza L., Beneit N., Martín-Ventura J.L., Escribano Ó, & Gómez-Hernández A. (2022). Implication of miR-155-5p and miR-143-3p in the Vascular Insulin Resistance and Instability of Human and Experimental Atherosclerotic Plaque. International Journal of Molecular Sciences, 23(18), 10253.

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Modulating Neutrophil Activation Pathways

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Cells were treated with 200nM thapsigargin (Santa Cruz Biotechnologies, Dallas, TX) for 20h. Where indicated, cells were pretreated with 1μM cyclosporine A (Sigma-Aldrich, St. Louis, MO) for 1h. After treatments, cells were harvested, and conditioned media was collected and filtered through 0.45μm filters.
Primary human neutrophils were isolated as described (Zhu et al., 2006 (link)). Isolated cells were resuspended in serum-free RPMI and, where indicated, pretreated with IL8-neutralizing antibody (0.3μg/ml, R&D Systems, Minneapolis, MN) or pS6K1 inhibitor PF-4708671 (5μM; Calbiochem Millipore, Billerica, MA) for 10min prior to treatment with recombinant human IL8 (10ng/ml; R&D Systems) or conditioned serum-free media for 20min before harvesting.

Kaunisto A., Henry W.S., Montaser-Kouhsari L., Jaminet S.C., Oh E.Y., Zhao L., Luo H.R., Beck A.H, & Toker A. (2015). NFAT1 promotes intratumoral neutrophil infiltration by regulating IL8 expression in breast cancer. Molecular Oncology, 9(6), 1140-1154.

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