Ca2+-ATPase
Thapsigargin selectively inhibits microsomal sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). [1]
Thapsigargin is an inhibitor of the ER calcium-ATPase. [2]
Thapsigargin triggers ER stress by inhibiting the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) activity, leading to depletion of calcium storage from the ER. [4]
Thapsigargin is an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA). [5]

Thapsigargin (0.001-1 μM; for 2 and 4 days) decreases cell proliferation of MH7A human rheumatoid arthritis synoviocytes in a time- and dose-dependent manner [2]. Thapsigargin (0.001-1 μM; for 2 and 4 days). Thapsigargin (0.001-1 μM; for 2 and 4 days) inhibits mTOR activity and increases cyclin D1 expression in MH7A cells [2]. Thapsigargin reduces Ca2+ entrance into human neutrophils [1]. Thapsigargin inhibits carbachol-evoked [Ca2+]i-transients with (IC50=0.353 nM) or without (IC50=0.448 nM) KCl prestimulation, but not in the absence. An extra minor component was found with KCl prestimulation, resulting in low fractionation (IC50=4814 nM). In contrast, KCl-induced transient changes in [Ca2+]i exhibited just one component, with a very low shift to Thapsigargin in the absence of the mean of both components (IC50=3343 nM) and the presence of carbachol prestimulation (IC50 =6858 nM)[3]. Additionally, thapsigargin inhibits TNF-α via phosphorylating p38 MAPK through SOCE influx of Ca2+.

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At concentrations above 500 nM, Thapsigargin inhibits store-regulated Ca2+ influx into Fura-2-loaded human neutrophil granulocytes. A concentration of 2 µM thapsigargin significantly reduced the Ca2+ signal in previously depleted cells, with [Ca2+]i reaching only 200 nM upon CaCl2 addition, and almost completely blocked Mn2+ quenching. [1]
Thapsigargin (0.001, 0.1, and 1 µM) blocks the proliferation of human rheumatoid arthritis synovial cells (MH7A) in a time- and dose-dependent manner. At 1 µM for 4 days, cell death percentage reached 44.6%. It decreased cyclin D1 protein levels (to 16.5% of control at 1 µM for 4 days) and mRNA levels, and reduced the protein levels of mTORC1 downstream indicators pS6K and p4EBP-1. [2]
In HepG2 cells, Thapsigargin (100 nM) induced ER stress, as assessed by up-regulation of BiP and CHOP gene expression and confirmed by ATF-6 shuttling. A dose of 100 nM thapsigargin yielded robust ER stress induction with limited adverse effects on cell viability. Lower concentrations (25 nM, 50 nM) were less consistent in inducing ER stress. [4]
In differentiated 3T3-L1 adipocytes, Thapsigargin doses lower than 100 nM were effective in activating ER stress (e.g., up-regulating BiP, CHOP, DNAjb9, PDAI3), although not to the same degree as tunicamycin. However, 100 nM thapsigargin appeared toxic to adipocytes. [4]
Thapsigargin (1 µM) efficiently inhibits coronavirus (HCoV-229E, MERS-CoV, SARS-CoV-2) replication in different cell types (HuH7, MRC-5, Vero E6). For HCoV-229E in HuH7 cells, 1 µM thapsigargin reduced viral titers by more than 150-fold (from 9.1810^6 to 5.710^4 pfu/ml). The EC50 for MERS-CoV in HuH7 cells was 4.8 nM, and for SARS-CoV-2 in Vero E6 cells was 260 nM. Thapsigargin restored the virus-induced translational shut-down, counteracted the CoV-mediated downregulation of IRE1α and BiP, and induced HERPUD1 and CTH expression. [5]

Thapsigargin (injection; 0.25 μg/g, 0.5 μg/g, and 1 μg/g; 24 hours) caused a 2- to 5-fold increase in the expression of proinflammatory chemokines. The ability to elicit systemic immunological responses is more sensitive in thapsigargin [4].

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To assess ER stress in vivo, we performed interperitoneal injection of either thapsigargin (TG) (0.5ug/g/body weight) or TUN (1ug/g/body weight) in mice. The dose selected for TUN in vivo is in the mid-range of doses used by others and does not elicit cytotoxic effects as assessed by mouse survival from previous studies. Unfortunately, choosing a thapsigargin (TG)  dose in vivo has been difficult as literature of thapsigargin (TG) use in vivo is scarce. In order to proceed with using thapsigargin (TG)  in a mouse model to evaluate the effectiveness of thapsigargin (TG)  in ER stress induction, we needed to demonstrate that our dose of TG (0.5ug/g/body) did not compromise mouse survival. In fact, we found that using thapsigargin (TG)  at a dose of 0.5ug/g/body weight was safe and did not elicit any adverse effects on survival in these mice (Supplementary Figure 2). We then examined the induction of ER stress in the liver and adipose tissue of mice treated with either TG or TUN, by assessing the expression of key ER stress and UPR markers.[4]

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In male Balb/c mice injected intraperitoneally with Thapsigargin (0.5 µg/g body weight), treatment resulted in significant expression of ER stress markers ATF6 and eIF2α in adipose tissue but failed to induce the expression of most ER stress and UPR proteins in the liver, and did not induce hepatic steatosis. [4]

Thapsigargin, an inhibitor of microsomal Ca(2+)-ATPase, is an important tool in inducing store-regulated Ca2+ influx. In the present paper we show that, at concentrations above 500 nM, thapsigargin also has an opposite effect: it inhibits store-regulated Ca2+ influx into Fura-2-loaded human neutrophil granulocytes. As thapsigargin has been frequently applied at concentrations up to 2 microM, its inhibitory action on plasma-membrane Ca2+ fluxes deserves consideration.[1]
Measurement of [Ca2+], Unless otherwise stated, 106 Fura-2-loaded cells were suspended in 3 ml of Ca2+ medium. Changes in fluorescence were recorded in a Deltascan dual-wavelength spectrofluorimeter using 340 nm and 380 nm for excitation and 505 nm for emission wavelength. Measurements were made at 37 'C with continuous stirring. [Ca2+], was calculated from the ratio of fluorescence excited at 340 and 380 nm, by the method detailed in [18]. Measurement of Mn2+ influx This measurement was performed under the same conditions as for [Ca2+1] except that the excitation wavelength was 360 nm, at which fluorescence of Fura-2 is independent of [Ca2+]. Mn2+ influx was initiated by addition of 100 ,uM MnCl2.

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Cell proliferation assay [2]
Cell Types: MH7A human rheumatoid arthritis synoviocytes
Tested Concentrations: 0.001, 0.1 and 1 μM
Incubation Duration: 2 and 4 days
Experimental Results: Prevents cell proliferation in a. time- and dose-dependent manner.

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Apoptosis analysis[2]
Cell Types: MH7A human rheumatoid arthritis synovial cells
Tested Concentrations: 0.001, 0.1 and 1 μM
Incubation Duration: 2 days and 4 days
Experimental Results: Apoptosis was induced in a time and dose dependent manner.

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Western Blot Analysis[2]
Cell Types: MH7A Human Rheumatoid Arthritis Synovial Cells
Tested Concentrations: 0.001, 0.1 and 1 μM
Incubation Duration: 2 and 4 Days
Experimental Results: Impaired mTOR activity and resulted in cyclin D1 expression

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For Ca2+ measurement, human neutrophils were loaded with Fura-2/AM (4 µM) for 30 min at 37°C. Changes in fluorescence were recorded using 340 nm and 380 nm excitation and 505 nm emission. [Ca2+]i was calculated from the ratio of fluorescence. For Mn2+ influx measurement, excitation wavelength was 360 nm, initiated by adding 100 µM MnCl2. [1]
Cell proliferation was measured using the sulforhodamine B (SRB) colorimetric assay. Cells were fixed in trichloroacetic acid, stained with SRB, and the bound dye was solubilized in Tris base for OD reading at 490 nm. [2]
Apoptosis was assessed by Hoechst staining. Cells were directly stained with a Hoechst kit, and cell counting was performed using ImageJ software. [2]
Western blot analysis was performed on cell lysates. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with nonfat dry milk, probed with primary antibodies overnight, and then with HRP-conjugated secondary antibodies. Proteins were detected by enhanced chemiluminescence. [2]
For ER stress induction in vitro, HepG2 and 3T3-L1 cells were treated with various concentrations of Thapsigargin (25-200 nM for HepG2; 25-100 nM for 3T3-L1) for 24 hours. Cells were then harvested for RNA extraction or protein lysis. Immunofluorescence staining for cleaved-ATF6 was performed to confirm ER stress. A cell viability assay (ATP quantitation) was performed to assess cytotoxicity. [4]
To assess protein de novo synthesis, cells were incubated with puromycin (3 µM) for 30 min before harvesting. Total cell extracts were analyzed by immunoblotting using an anti-puromycin antibody, with Coomassie Brilliant Blue (CBB) staining as a loading control. [5]
Immunofluorescence analysis of HCoV-229E-infected cells used antibodies against double-stranded RNA and nonstructural protein 8 (nsp8). [5]
Viral titers in supernatants were determined by plaque assay on confluent cell monolayers. Cells were incubated with serial dilutions of virus, overlaid with medium containing Avicel, fixed with PFA, and stained with crystal violet to count plaques. [5]

Animal/Disease Models: Male balb/c (Bagg ALBino) mouse (20-25 g) [4]
Doses: 0.25 ug/g, 0.5 ug/g, and 1 ug/g
Route of Administration: Injection; 24 hour
Experimental Results: Increased chemokine and pro-inflammatory expression 2 to 5 times.

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Induction of ER stress in vivo[4]
Male Balb/c mice were housed and cared in accordance with the Guide for the Care and Use of Laboratory Animals. Tunicamycin and thapsigargin (TG)  were purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) and diluted in sterile 150mM dextrose to obtain a concentration of 10μg/μl. Male Balb/c mice (20-25g) were injected intraperitoneally with tunicamycin solution (1μg/g body mass) as described previously. For thapsigargin solution a dose response was conducted using (0.25ug/g, 0.5ug/g and 1ug/g body mass). As controls, mice were injected intraperitoneally with control buffer (150mM dextrose containing 1% DMSO). Adipose and liver tissues were harvested 24hrs post treatment, where as liver tissues utilized for Oil Red O staining was harvested 5 days post treatment with the ER stress inducing agents.

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Thapsigargin was dissolved in dimethyl sulfoxide (DMSO) and diluted in sterile 150 mM dextrose to obtain a concentration of 10 µg/µl. Male Balb/c mice (20-25g) were injected intraperitoneally with thapsigargin solution (0.25, 0.5, or 1 µg/g body mass). As controls, mice were injected intraperitoneally with control buffer (150 mM dextrose containing 1% DMSO). Adipose and liver tissues were harvested 24 hours post-treatment for immunoblotting and other analyses. For Oil Red O staining, liver tissues were harvested 5 days post-treatment. [4]

In HuH7 cells, the CC50 of Thapsigargin was 10.7 µM after 24 hours of incubation. The combination of thapsigargin and HCoV-229E infection did not cause additional cytotoxicity (CC50 of 9.7 µM). At 1 µM thapsigargin, a concentration that abolished viral protein translation and replication, cell viability in infected cells was 76.6 ± 7.9%. [5]
In Vero E6 cells, the CC50 of Thapsigargin was 17.22 µM, resulting in selectivity indices (SI, CC50/EC50) of 66.2 for SARS-CoV-2 and 2229.2 for MERS-CoV. [5]

[1]. Thapsigargin inhibits Ca2+ entry into human neutrophil granulocytes. Biochem J. 1995 Jan 15;305 ( Pt 2):525-8.

[2]. Effects of thapsigargin on the proliferation and survival of human rheumatoid arthritis synovialcells. ScientificWorldJournal. 2014 Feb 9;2014:605416.

[3]. Differential thapsigargin-sensitivities and interaction of Ca2+ stores in human SH-SY5Y neuroblastoma cells. Brain Res. 2004 Jun 18;1011(2):177-86.

[4]. Modeling Acute ER Stress in Vivo and in Vitro. Shock. 2017 Apr;47(4):506-513.

[5]. Inhibiting coronavirus replication in cultured cells by chemical ER stress. bioRxiv 2020.08.26.266304;.

[6]. Store-operated calcium entry (SOCE) contributes to phosphorylation of p38 MAPK and suppression of TNF-α signalling in the intestinal epithelial cells. Cell Signal. 2019 Nov;63:109358.

Thapsigargin is an organic heterocyclic tricyclic compound belonging to the hexaoxy-substituted 6,7-guaiacol group, isolated from the roots of Thapsia garganica L., a plant in the Apiaceae family. It is a potent skin irritant and is used in traditional medicine as an anti-irritant. Thapsigargin inhibits the entry of Ca²⁺-transfer ATPase-mediated calcium ion into the sarcoplasmic reticulum, and is therefore often used to study the effects of increased cytoplasmic calcium concentration. It is an EC 3.6.3.8 (Ca²⁺-transfer ATPase) inhibitor and a calcium channel blocker. It is a sesquiterpene lactone, an organic heterocyclic tricyclic compound, and a butyrate ester. Thapsigargin has been reported in Thapsia gymnesica, Thapsia garganica, and Thapsia villosa, with relevant data available. Thapsigargin is a sesquiterpene lactone found in the roots of plants in the Thapsia genus. It inhibits sarcoplasmic reticulum calcium-transfer ATPase.

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Thapsigargin is a tumor-promoting agent that has been shown to selectively inhibit microsomal Ca2+-ATPase. It is an important tool for inducing store-regulated Ca2+ influx (capacitative Ca2+ entry). The effective concentration is extremely low, with maximal effect obtained around 50 nM. However, it has frequently been applied at concentrations up to 2 µM, and its inhibitory action on plasma-membrane Ca2+ fluxes at higher concentrations deserves consideration. [1]
Thapsigargin induces ER stress by depleting ER Ca2+ pools, which leads to the unfolded protein response (UPR). It is a well-characterized ER stress inducer that acts through the inhibition of the SERCA pump. [4]
A protease-cleavable prodrug of thapsigargin, mipsagargin, has been evaluated in phase I and II clinical trials for prostate cancer. [5]

C34H50O12

650.75

650.33

C, 62.75; H, 7.74; O, 29.50

67526-95-8

446378

White to off-white solid powder

1.2±0.1 g/cm3

699.6±55.0 °C at 760 mmHg

209.0±25.0 °C

0.0±5.0 mmHg at 25°C

1.540

6.61

2

12

17

46

1270

8

C(C(=O)O[C@H]1[C@]2([H])[C@@](OC(C)=O)(C[C@@H]([C@@]3([C@](C)(O)C(=O)O[C@]3(C2=C(C)[C@@H]1OC(=O)/C(/C)=C\C)[H])O)OC(CCC)=O)C)CCCCCC

IXFPJGBNCFXKPI-FSIHEZPISA-N

InChI=1S/C34H50O12/c1-9-12-13-14-15-17-24(37)43-28-26-25(20(5)27(28)44-30(38)19(4)11-3)29-34(41,33(8,40)31(39)45-29)22(42-23(36)16-10-2)18-32(26,7)46-21(6)35/h11,22,26-29,40-41H,9-10,12-18H2,1-8H3/b19-11-/t22-,26+,27-,28-,29-,32-,33+,34+/m0/s1

(3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-(acetyloxy)-4-(butyryloxy)-3,3a-dihydroxy-3,6,9-trimethyl-8-{[(2Z)-2-methylbut-2-enoyl]oxy}-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl octanoate

thapsigargin; 67526-95-8; MFCD00083511; Z96BQ26RZD; CHEMBL96926; CHEBI:9516; (-)-Thapsigargin; (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-(acetyloxy)-4-(butanoyloxy)-3,3a-dihydroxy-3,6,9-trimethyl-8-{[(2Z)-2-methylbut-2-enoyl]oxy}-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl octanoate;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~76.83 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: 5 mg/mL (7.68 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (3.20 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.20 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)