Journal: Heliyon

Article Title: Botulinum toxin type A activates protective autophagy by modulating endoplasmic reticulum stress in hypoxia/reoxygenation-treated endothelial cells

doi: 10.1016/j.heliyon.2023.e17907

Figure Lengend Snippet: Botulinum toxin type A (BTXA) activated protective autophagy by initiating endoplasmic reticulum (ER) stress and promoted human dermal microvascular endothelial cells (HDMECs) proliferation. (A) Experimental protocol. Thapsigargin (Tg; 1 μM) and bafilomycin A1 (Baf A1; 10 nM) were added in the first and second hours of BTXA pretreatment, respectively. 4-Phenylbutyric acid (4-PBA; 5 mM) was added at the beginning of hypoxia for 8 h. The administration of BTXA, Tg, or 4-PBA is indicated by the down arrow (↓). (B) Optical density (OD) values were tested using the Cell Counting Kit 8 assay and transformed into cell viability rates. (C) To assess DNA replication activity, the ethynyldeoxyuridine (EdU) test was used. The rate of EDU + cells was determined by dividing the number of EdU + cells (orange fluorescence) by the number of nuclei (Hoechst 33,342; blue fluorescence), as shown in D. (E) Western blot was used to assess proteins, and the relative expression levels of associated proteins are shown in panels F, G, and H. All data are presented as mean ± standard derivation. ^P < 0.05 vs Control, ^^P < 0.01 vs Control. *P < 0.05 vs H/R, **P < 0.01 vs H/R. # P < 0.05 vs H/R + BTXA, ## P < 0.01 vs H/R + BTXA. $ P < 0.05 vs H/R + BTXA + Tg, $$ P < 0.01 vs H/R + BTXA + Tg. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: ER stress inducer thapsigargin (Tg; cat. no. HY-13433, MedChemExpress) [ ] and inhibitor 4-phenylbutyrate (4-PBA; cat. no. HY-A0281, MedChemExpress) [ ] and Baf A1 were dissolved in DMSO, respectively, and then stored at −20 °C.

Techniques: Cell Counting, Transformation Assay, Activity Assay, Fluorescence, Western Blot, Expressing, Control

Journal: Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

Article Title: Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

doi: 10.2478/prolas-2018-0065

Figure Lengend Snippet: Fig. 2. Mean FRET efficiency (%) dis- tribution with and without thapsigargin (TG) using ND filters before measure- ment. Condition descriptions shortened from: CFP-STIM1 + YFP-ORAI1 and STIM1-CFP + YFP-ORAI1 with and without thapsigargin (TG). A, Data be- fore testing for Gaussian distribution. B, Data containing only cells that have a FRET pixel frequency conforming to a Gaussian distribution.

Article Snippet: Ten minutes before FRET measurements, cells were washed with PBS and 2 uM thapsigargin (TG) (Alomone Labs, Israel) was applied to induce endoplasmic reticulum calcium depletion, which was necessary for clustering between STIM1 and ORAI1.

Techniques:

Journal: Proceedings of the Latvian Academy of Sciences. Section B. Natural, Exact, and Applied Sciences.

Article Title: Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

doi: 10.2478/prolas-2018-0065

Figure Lengend Snippet: Fig. 4. Distribution of mean FRETeff values (%) for positive (CFP-YFP), negative (CFP + YFP) controls, CFP-STIM1 + YFP-ORAI1 and STIM1-CFP + YFP-ORAI1 with and without thapsigargin (TG). Data from 3 independent experiments were pooled.

Article Snippet: Ten minutes before FRET measurements, cells were washed with PBS and 2 uM thapsigargin (TG) (Alomone Labs, Israel) was applied to induce endoplasmic reticulum calcium depletion, which was necessary for clustering between STIM1 and ORAI1.

Techniques:

Journal: Biological psychiatry

Article Title: Early Stress Prevents the Potentiation of Muscarinic Excitation by Calcium Release in Adult Prefrontal Cortex

doi: 10.1016/j.biopsych.2013.10.017

Figure Lengend Snippet: Muscarinic excitation (acetylcholine [ACh]-elicited inward current [IACh]) is potentiated by ACh-elicited calcium (Ca2+) release from intracellular stores (dF/FACh). (A, B) Muscarinic M1 receptor antagonist pirenzepine (500 nmol/L, 10 min) abolished IACh and dF/FACh in layer V pyramidal cells of adult prefrontal cortex (lower and upper traces, respectively). The middle trace schematically depicts changes in holding potential. Note the brief depolarizing step at the end of the trace, which serves as a control. (C, D) Depletion of intracellular Ca2+ stores with thapsigargin (10 mmol/L, 10 min) attenuates dF/FACh and significantly reduces IACh in calcium responder cells. (E, F) Conversely, in calcium nonresponder cells, a depolarizing pulse (to 0 mV) delivered before ACh application could trigger Ca2+ entry and subsequent dF/FACh, presumably due to the replenishment of intracellular stores. TG, thapsigargin.

Article Snippet: Pirenzepine, thapsigargin, and KB-R7943 were obtained from Tocris (R&D Systems Inc., Minneapolis, Minnesota).

Techniques: Control

Journal:

Article Title: Multiple forms of LTP in hippocampal CA3 neurons use a common postsynaptic mechanism

doi: 10.1038/10180

Figure Lengend Snippet: Ca2+ release from internal stores when fast synaptic transmission is blocked. (a) In the presence of kynurenate (8–10 mM), L-HFS-3 elicited a rise in [Ca2+]i. An individual experiment in which the mGluR1 antagonist AIDA (500 μM) blocked the rise in [Ca2+]i during L-HFS. After washout (30 min), a large rise in [Ca2+]i was evoked in the proximal dendrite by L-HFS. In this case, a sustained depolarization was observed both with and without AIDA. (Waveforms are from single stimulation trains.) (b) Ryanodine (ryn; n = 3), thapsigargin (thaps; n = 2), MCPG (n = 3), AIDA (n = 6) or CPCCOEt (n = 3) blocked the rise in postsynaptic [Ca2+], consistent with the block of Ca2+ release from internal stores. (c) AIDA (n = 3) or CPCCOEt (n = 3) blocked mossy fiber LTP when fast synaptic transmission was blocked (83.1 ± 4.3%, n = 6; data were combined for the two drugs). These results were significantly different from LTP induced without mGluR antagonists present (p < 0.05). Horizontal bar indicates drug application.

Article Snippet: Where specified, one or more of the following drugs was included in the media: DCG-IV (Tocris), prepared from a stock solution dissolved in 10 mM DMSO; kynurenate (Sigma), dissolved directly into the media (pH 7.2 with NaOH); ryanodine or thapsigargin (RBI), prepared from stock solutions dissolved in 20 mM or 5 mM DMSO, respectively; MCPG (RBI), AIDA (Tocris), or CPC-COEt (Tocris), dissolved directly into the media.

Techniques: Transmission Assay, Blocking Assay

Journal: Osteoarthritis and Cartilage Open

Article Title: Iron triggers the early stages of cartilage degeneration in vitro: The role of articular chondrocytes

doi: 10.1016/j.ocarto.2021.100145

Figure Lengend Snippet: Fig. 3. Iron loading induces morphological alter- ations in chondrocytes. Transmission electron micro- scopy images illustrate the ultrastructure of chondrocytes in freshly collected cartilage (A) or in cartilage cultured for 9 days with sodium citrate (control) (B) or ferric citrate (iron loading condition) (C–F). Whilst the morphology of control chondrocytes was indistinct from that of cells from freshly collected cartilage, iron-treated chondrocytes presented side- rosomes (white arrows) and a number of morpho- logical changes consistent with cell death by chondroptosis. (D) is a magnification of the area within the square in (C), showing a siderosome and abundant dilated cisternae of rough ER. A chon- droptotic cell with convoluted nucleus with condensed chromatin, abundant Golgi and vacuoles (arrow heads), and extrusion of cellular material into the extracellular space (black arrows) is represented in (E). An empty lacuna is depicted in (F), where the remnants of a dead chondrocyte are identified by a dotted line. Samples were contrasted with uranile acetate and lead citrate. Original magnification: 15000 . Bar¼1 μm.

Article Snippet: Cartilage was also incubated with ER stress/ chondroptosis inducers (tunicamycin or thapsigargin, Santa Cruz Biotechnology) or with ferroptosis inducers (erastin; RSL-3; buthioninesulfoximine, BSO) for 9 days.

Techniques: Transmission Assay, Cell Culture, Control

Journal: Cell Reports Methods

Article Title: Deuterium labeling enables proteome-wide turnover kinetics analysis in cell culture

doi: 10.1016/j.crmeth.2025.101104

Figure Lengend Snippet: Experimental validation of deuterium-incorporation sites and single-time-point kinetics analysis (A) Experimental validation of predicted labeling sites using acid hydrolysis and high-resolution MS. (B) Mass spectra from acid hydrolysis of unlabeled samples, showing no detectable d -alanine. N.D., not detected. (C) As in (B), but for alanine in fully labeled samples, showing detectable d -Ala at m/z 91.0612 with 9.3% relative intensity to monoisotopic Ala ( m/z 90.0550). Inset: IsoSpec2-predicted d -Ala relative intensity (0.1%, 6.5%, 12.8%, 19.2%) relative to the monoisotopic peak at S Ala = 0–3, respectively. (D) As in (B), but for Arg. (E) As in (C), but for Arg, showing no detectable d -Arg ( m/z 176.1252) in cell culture. (F) Relative intensity of d -Ala, Asp, Glu, Gly, Leu/Ile, Pro, and Arg in unlabeled and fully labeled samples, using amino acid analysis with high-resolution MS. Asn and Gln are deamidated during acid hydrolysis ; hence, measured values are a mixture of Asp/Asn and Glu/Gln. (G) Application of D 2 O labeling to proteome-wide turnover kinetics in normal and stressed AC16 cells. (H) Point ranges of protein half-life measured by D 2 O and SILAC in AC16, showing comparable ranges between methods. D 2 O recapitulates a slowdown in cell proliferation and protein synthesis upon thapsigargin-induced ER stress. Error bars: SD. (I) Scatterplot of D 2 O and SILAC measurements after 1 μM thapsigargin, revealing strong correlation ( r: 0.94) and stress-response genes with high turnover flux upon ER stress.

Article Snippet: Thapsigargin , SelleckChem , S7895.

Techniques: Biomarker Discovery, Labeling, Cell Culture, Multiplex sample analysis

Journal: bioRxiv

Article Title: Monoclonal anti-AMP-antibodies reveal broad and diverse AMPylation patterns in cancer cells

doi: 10.1101/2020.06.23.164731

Figure Lengend Snippet: A Reproduction of previously published data confirms loss of Bip-AMPylation upon ER stress by thapsigargin in WB. 20 µg treated (as indicated) ChoK1 cell lysate per lane or 50 ng recombinant Bip-AMP were analyzed in WB by antibody 17G6 and anti-Bip antibody. B Successful IP with antibody 17G6 on recombinant BiP-AMP confirms that antibody 17G6 is AMP-specific. C Successful IP of endogenous Bip-AMP with antibody 17G6 from treated (as indicated) ChoK1 cell lysates confirms applicability in immunoprecipitation. 50 ng recombinant Bip-AMP were blotted as control. D Using antibody 17G6 on various immortalized and cancer cell lines reveals diverse cellular AMPylation. 20 µg cell lysate per lane as indicated were blotted and probed with antibody 17G6 using 1 mM MnCl 2 as additive. Afterwards cells were treated with 1 M hydroxylamine to cleave ADP-ribosylation at aspartate and glutamate residues and reprobed with antibody 17G6 using 1 mM MnCl 2 . Antibodies against Bip, GAPDH and Histone H3 serve as loading control.

Article Snippet: 90% confluent cells were stimulated by either 0.5 μM thapsigargin (Biosynth Carbosynth) for 2 h or 100 μg/ml cycloheximide (Sigma-Aldrich) for 4 h. Cells were washed twice with Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich) and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with cOmplete EDTA free protease inhibitor (Roche, Basel, Switzerland).

Techniques: Recombinant, Immunoprecipitation, Control