Thapsigargin tg

Manufactured by Santa Cruz Biotechnology

Thapsigargin (Tg) is a natural product derived from the plant Thapsia garganica. It acts as a potent and selective inhibitor of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), a crucial enzyme responsible for the uptake of calcium into the endoplasmic reticulum. Tg effectively disrupts calcium homeostasis within cells, leading to the depletion of intracellular calcium stores and the activation of cellular stress responses.

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4 protocols using thapsigargin tg

Thapsigargin and Tunicamycin Effects on Calcium Signaling

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Thapsigargin (Tg, Santa Cruz Biotechnology Inc.) and Tunicamycin (Tun, Sigma Aldrich) were incubated on cells for 24 h at the concentrations indicated. Caffeine (Cf), ATP, 2-Aminoethoxydiphenyl borate (2-APB), ryanodine (Ry) and Ru360 were purchased from Sigma-Aldrich (Milan, Italy). Fluo-4-acetoxymethyl ester and Rhod 2-acetoxymethyl ester were purchased from Thermo Fisher Scientific (Milan, Italy). The cells were loaded with the two probes, then incubated for 10 min with 10 mM Cf or 1 mM ATP with or without 50 µM 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3R), 100 µM Ry (ryanodine, an inhibitor of RYR) or 10 µM Ru360 (an inhibitor of mitochondrial calcium uptake).

Germani S., Marchetti A.C., Guidarelli A., Cantoni O., Sorrentino V, & Zito E. (2022). Loss-of-rescue of Ryr1I4895T-related pathology by the genetic inhibition of the ER stress response mediator CHOP. Scientific Reports, 12, 20632.

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Induction of ER Stress and ISR Pathways

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To induce ER stress, cells were treated with 5 μg/ml tunicamycin (TM; Sigma‐Aldrich) or 1 μM thapsigargin (TG; Santa Cruz Biotechnology) for indicated times. HRI activation was triggered by 5 μM sodium arsenite (Sigma‐Aldrich) treatment for 20 h, PKR activation by 20 μM BEPP (Sigma‐Aldrich) treatment for 6 h and GCN2 activation by 10 nM Halofuginone (Cayman Chemical Company) treatment for 20 h, and ROS was generated by 100 μM menadione (Men; Sigma‐Aldrich) treatment for 4 h. Treatment with 0.5 μM PERK inhibitor GSK2606414 (Sigma‐Aldrich), 40 μM IRE1α inhibitor 4μ8C (Tocris), 75 μM angiogenin inhibitor NSC‐65828 [8‐amino‐5‐(4′‐hydroxybiphenyl‐4ylazo)naphthalene‐2‐sulfonate] (National Cancer Institute (NCI)) and 5 μM mTOR inhibitor Torin1 (Tocris) was started 1 h before induction of ER stress. Treatment with 200 nM ISR inhibitor ISRIB (Selleck Chemicals) was started 2 h before fixation, and treatment with 5 μM HRI inhibitor hemin chloride (Santa Cruz Biotechnology) was started 3 h before fixation. Compounds were dissolved in H₂O (sodium arsenite), 100 mM NaOH (hemin chloride) or DMSO (rest). For all treatments, equal volumes of solvents were added to the controls. To determine ROS levels, cells were incubated with 5 μM CellROX green reagent (Thermo Fisher) 30 min before fixation.

Wolzak K., Nölle A., Farina M., Abbink T.E., van der Knaap M.S., Verhage M, & Scheper W. (2022). Neuron‐specific translational control shift ensures proteostatic resilience during ER stress. The EMBO Journal, 41(16), e110501.

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Myogenic Differentiation Markers

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Fura-2/AM was obtained from Invitrogen. Jet Prime was from Poly Plus. Thapsigargin (TG) was from Santa Cruz. Antibodies of myogenin(F5D, DSHB), MHC(MF-20, DSHB), CyclinD1(A-12, Santa Cruz), CDK4(B-10, Santa Cruz), β-actin(C4, Santa Cruz), MEF2C(MAB6786, R&D Systems) were purchased from the indicated vendor.

Kim K.M., Rana A, & Park C.Y. (2019). Orai1 inhibitor STIM2β regulates myogenesis by controlling SOCE dependent transcriptional factors. Scientific Reports, 9, 10794.

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Calcium Signaling Reagents Protocol

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Ionomycin, Thapsigargin (Tg) and 2-APB were obtained from Santa-Cruz Biotechnologies. ADP, Anisomycin and Dantrolene were obtained from Sigma-Aldrich (UK). Xestospongin C was obtained from Abcam (UK).

Layhadi J.A, & Fountain S.J. (2017). Influence of ER leak on resting cytoplasmic Ca²⁺ and receptor-mediated Ca²⁺ signalling in human macrophage. Biochemical and biophysical research communications, 487(3).

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