Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: SOCE measurement in fibroblast lines. ( A ) Protocol used to induce SOCE at the 8-well system using juvenile age-related control fibroblast lines shown as example. Fibroblast lines were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced during 20 min by adding 0.5 µM of TG in 0.1 mM EGTA. Calcium measurements were initiated during the final minute of the TG treatment, followed by re-addition of 2 mM CaCl 2 to trigger SOCE which is represented by the peak F340/F380. Non-normalized SOCE traces in ( A ). ( B ) The delta ratio (indicated by the green dashed line) calculated as the difference between the peak F340/F380 ratio after extracellular Ca 2+ was added and its level immediately before the addition of Ca 2+ as well as the AUC (area filled with gray marks under the line graph) to measure SOCE in juvenile age-related control fibroblast lines. SOCE traces normalized to 1 in ( B ). ( C ) SOCE response in single juvenile age-related control fibroblasts measured in one well. Non-normalized SOCE traces in ( C ). ( D ) Example of average SOCE measurement from different passages and days of calcium imaging of juvenile age-related control fibroblast lines represented by blue and green lines, respectively. Non-normalized SOCE traces in ( D ). Abbreviations: AUC: area under the curve; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid; SOCE: Store-operated calcium entry; TG: thapsigargin

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Control, Imaging

Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: SOCE measurements in fibroblast lines from adult- and juvenile-onset HD compared to age-related controls. Fibroblast lines were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced by 0.5 µM of thapsigargin in 0.1 mM EGTA. Next, 2 mM of Ca 2+ was added to induce SOCE. ( A ) SOCE measured as AUC. PM-HD, EM-HD, M-HD, J-HD vs. age-related controls (A-K, J-K) non-significant (ns). Individual data points represent mean from three individuals per group ( N = 3) except for ( N = 4) for A-K. The results are shown as mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test. ( B - C ) SOCE measured as delta ratio ( B ) or AUC ( C ) was increased in fibroblast lines from PM-HD, EM-HD, and M-HD patients compared to A-K. The results are shown as medians and IQR. Kruskal-Wallis test followed by Dunn’s post hoc test (***, p < 0.001; **, p < 0.01; *, p < 0.05). ( D-E ) SOCE measured as delta ratio ( D ) or AUC ( E ) was decreased in fibroblast lines from J-HD compared to J-K patients. The results are shown as mean ± SEM; Student’s unpaired t-test ( *** , p < 0.001). ( F - G ) SOCE measured as delta ratio ( F ) or AUC ( G ) was increased in fibroblast lines from PM-HD and M-HD patients compared to A-K, while it was decreased in J-HD compared to J-K. SOCE measured as AUC ( G ) was increased in EM-HD patients compared to A-K, while it was non-significant when measured as delta ratio ( F ). Additionally, SOCE, measured as the delta ratio ( F ) and AUC ( G ), was decreased in A-K patients compared to J-K. The results are shown as medians and IQR. Kruskal-Wallis test followed by Dunn’s post hoc test (***, p < 0.001; **, p < 0.01; *, p < 0.05, ns, non-significant). Individual data points in Figures ( B - G ) correspond to the number of analyzed wells (PM-HD: 50; EM-HD: 48; M-HD: 47; A-K: 49; J-K: 46 and J-HD: 47 for delta ratio and PM-HD: 50; EM-HD: 47; M-HD: 47; A-K: 49; J-K: 46 and J-HD: 43 for AUC), which are technical replicates from at least three patient or control fibroblast lines per group. In each well, approximately 50 ROI were measured, where each ROI corresponds to a single fibroblast cell. The average value from each well was then included in the statistical analysis. Abbreviations: A-K: adult age-related controls; AUC: area under the curve; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid; EM-HD: early manifest HD patients; HD: Huntington’s disease; IQR: interquartile ranges; J-HD: juvenile-onset HD patients; J-K: juvenile age-related control; M-HD: manifest-HD patients; PM-HD: premanifest HD patients; SEM: standard error of the mean; SOCE: Store-operated calcium entry; ROI: regions of interest

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Control

Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: Effect of the CAG length on SOCE in fibroblast lines from juvenile-and adult-onset HD. Fibroblast lines were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced by 0.5 µM of thapsigargin in 0.1 mM EGTA. Next, 2 mM of Ca 2+ was added to induce SOCE. ( A - B ) The CAG length of the mutant huntingtin has no effect on SOCE measured as delta ratio ( A ) or AUC ( B ) between fibroblasts from J-HD compared to adult-onset HD groups (PM-HD, EM-HD, M-HD). Individual data points in A-B represents the average SOCE measurement of each HD patient. Fibroblast HD lines indicated as triangles ( A ) or circles ( B ) are summarized in Table with a corresponding reference to their CAG repeat lengths. In Figures ( A - B ) Pearson’s correlation coefficient. ( C - D ) The CAG length of the mutant huntingtin has no effect on SOCE measured as delta ratio ( C ) or AUC ( D ) between fibroblasts from J-HD compared to M-HD. In C , the results are shown as mean ± SEM; Student’s unpaired t-test (ns; non-significant). In D , the results are shown as medians and IQR; Mann-Whitney U test (ns; non-significant). Individual data points in Figures ( C - D ) correspond to the number of analyzed wells (M-HD: 47 and J-HD: 43 for both delta ratio and AUC) representing technical replicates from at least three patient-derived fibroblast lines per group. In each well, approximately 50 ROI were measured, where each ROI corresponds to a single fibroblast cell. The average value of each well was included in the statistical analysis. Abbreviations: A-K: adult age-related controls; AUC: area under the curve; EM-HD: early manifest HD patients; HD: Huntington’s disease; IQR: interquartile ranges; J-HD: juvenile-onset HD patients; J-K: juvenile age-related control; M-HD: manifest-HD patients; PM-HD: premanifest HD patients; SEM: standard error of the mean; SOCE: Store-operated calcium entry; ROI: regions of interest

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Mutagenesis, MANN-WHITNEY, Derivative Assay, Control

Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: Effect of SOCE inhibitors on premanifest HD fibroblast lines. PM-HD fibroblast lines were incubated for 5 min before single-cell Ca 2+ measurements with 10 µM tetrahydrocarbazole in 0.02% DMSO indicated as red points ( A - B ) or 1h with 1 µM EVP4593 in 0.02% DMSO (blue points) ( C - D ) and in 5 min-1h 0.02% DMSO as a control, respectively (black points) ( A - D ). Fibroblast lines in A - D were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced by 0.5 µM of thapsigargin in 0.1 mM EGTA. Next, 2 mM of Ca 2+ was added to induce SOCE. ( A - B ) Tetrahydrocarbazole attenuate SOCE in premanifest HD fibroblast lines measured as the delta ratio ( A ) or AUC ( B ) in PM-HD fibroblast lines treated with tetrahydrocarbazole (T) compared to PM-HD cells treated with DMSO. Individual data points on the graphs correspond to the number of analyzed wells (PM-HD DMSO: 22 and PM-HD COMP T: 21 for delta ratio and PM-HD DMSO: 23 and PM-HD COMP T: 22 for AUC). ( C - D ) EVP4593 attenuate SOCE in premanifest HD fibroblast lines measured as the delta ratio ( C ) or AUC ( D ) in PM-HD fibroblast lines treated with EVP4593 ( E ) compared to PM-HD cells treated with DMSO. Individual data points on the graphs correspond to the number of analyzed wells (PM-HD DMSO: 11 and PM-HD COMP E: 11 for both delta ratio and AUC). In each well in Figures ( A - D ), approximately 50 ROI were measured, where each ROI corresponds to a single fibroblast cell. The average value from each well which was technical replicate was then included in the statistical analysis. In Figures ( A - B ) the results are shown as medians and IQR; Mann-Whitney U test (***, p < 0.0001). In C , the results are shown as mean ± SEM; Student’s unpaired t-test and (***, p < 0.0001). In D , the results are shown as medians and IQR; Mann-Whitney U test (**, p < 0.001). Abbreviations: AUC: area under the curve; DMSO: dimethyl sulfoxide; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid; HD: Huntington’s disease; IQR: interquartile ranges; PM-HD: premanifest HD patients; PM-HD DMSO: premanifest HD cells treated with DMSO; PM-HD COMP E: premanifest HD cells treated with EVP4593; PM-HD COMP T: premanifest HD cells treated with Tetrahydrocarbazole; SEM: standard error of the mean; SOCE: Store-operated calcium entry; ROI: regions of interest

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Incubation, Single Cell, Control, MANN-WHITNEY

Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: SOCE measurement in fibroblast lines from juvenile and adult controls. Fibroblast lines were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced by 0.5 µM of thapsigargin in 0.1 mM EGTA. Next, 2 mM of Ca 2+ was added to induce SOCE. SOCE measured as delta ratio ( A ) and AUC ( B ) was decreased in fibroblast lines from A-K compared to J-K. Individual data points on the graphs correspond to the number of analyzed wells (J-K: 46 and A-K: 49 for both delta ratio and AUC) representing technical replicates from at least three control fibroblast lines per group. In each well, around 50 ROI were measured, where each ROI corresponded to a single fibroblast cell, and then the average value of each well was included in the statistical analysis. The results are shown as medians and IQR; Mann-Whitney U test (*** p < 0.0001). Abbreviations: A-K: adult age-related controls; AUC: area under the curve; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid; IQR: interquartile ranges; J-K: juvenile age-related control; SEM: standard error of the mean; SOCE: Store-operated calcium entry; ROI: regions of interest

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Control, MANN-WHITNEY

Journal: Pharmacological Reports

Article Title: Dysregulation of store-operated calcium entry in fibroblast lines from adult and juvenile-onset Huntington’s disease patients

doi: 10.1007/s43440-025-00820-8

Figure Lengend Snippet: SOCE response in adult- and juvenile-onset HD fibroblast lines and age-related controls. Fibroblast lines were loaded with the Ca 2+ probe Fura-2 AM (2 µM). Cells were treated with 0.1 mM EGTA. Ca 2+ release from the endoplasmic reticulum was induced by 0.5 µM of thapsigargin in 0.1 mM EGTA. Next, 2 mM of Ca 2+ was added to induce SOCE. In Figures ( A - H ) SOCE was calculated as the average of wells with fibroblast lines from each experimental variant: ( A ) A-K, ( B ) PM-HD, ( C ) EM-HD, ( D ) M-HD, ( E ) J-K and ( F ) J-HD. Non-normalized ( A - G ) and normalized to 1 ( H ) SOCE traces. Abbreviations: A-K: adult age-related controls; EGTA: ethylene glycol-bis(β-aminoethyl ether)-N, N,N′,N′-tetraacetic acid; EM-HD: early manifest HD patients; HD: Huntington’s disease; J-HD: juvenile-onset HD patients; J-K: juvenile age-related control; M-HD: manifest-HD patients; PM-HD: premanifest HD patients; SOCE: Store-operated calcium entry

Article Snippet: The low-Ca 2+ medium (Ca 2+ -free solution), containing 0.1 mM EGTA (BioShop cat. no. EGT101.25) in the standard buffer, was then added to the cells for 3 min. To induce SOC channels activity, cells were subjected to the following SOCE protocol: ER Ca 2+ depletion was induced with 0.5 μM thapsigargin (Alomone, cat. no. T-650) in the presence of 0.1 mM EGTA in the standard buffer for 20 min. Calcium measurements began during the final minute of this incubation period, followed by re-addition of 2 mM CaCl 2 to trigger SOCE (Fig. A).

Techniques: Variant Assay, Control

Journal: Cell reports

Article Title: Role of Selenof as a Gatekeeper of Secreted Disulfide-Rich Glycoproteins

doi: 10.1016/j.celrep.2018.04.009

Figure Lengend Snippet: (A) Expression pattern of Selenof and other relevant proteins across mouse tissues. (B-D) Expression of Selenof and other relevant proteins in MEFs from WT, heterozygous, and Selenof KO mice subjected to ER stressors thapsigargin (B), tunicamycin (C), and brefeldin A (D). WT, heterozygous, and KO MEFs were treated with two concentrations of stressors along with control (DMSO treated): thapsigargin (5 nM and 50 nM), tunicamycin (50 ng/mL and 500 ng/mL), and brefeldin A (0.5 μM and 5 mM). Proteins assayed are shown on the left.

Article Snippet: Tunicamycin, brefeldin A, and thapsigargin were from Tocris Bioscience (Bristol, UK); and lipopolysaccharide (LPS; L2637), SIGMAFAST OPD (o-phenylenediamine), and 2-mercaptoethanol were from Sigma (St. Louis, MO, USA).

Techniques: Expressing, Control

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: Frontiers in cell and developmental biology

Article Title: Using Light-Sheet Microscopy to Study Spontaneous Activity in the Developing Lateral-Line System.

doi: 10.3389/fcell.2022.819612

Figure Lengend Snippet: FIGURE 2 | P2yr1 signaling is required for spontaneous activity in supporting cells but not immature hair cells. The spatial patterns of the mean spontaneous calcium activities of the supporting cells (A1–A19) or hair cells (B1–B19) in DMSO and after 15 min of treatment with 1 µM MRS2500. Measurements were performed in immature neuromasts at day 3. The ΔF/F GCaMP6s signals were averaged over each 900 s interval (pre- and post-treatment) and then colorized according to the heat map and superimposed onto a baseline image. The corresponding temporal curves of the mean signal magnitude across the whole neuromast in supporting cells (A2) and in hair cells (B2) in DMSO and after 15 min of treatment with 1 µM MRS2500. The cytosolic baseline calcium in the supporting cells (C1–C19) and hair cells (D1–D19) in DMSO and after 15 min of treatment with 250 nM Thapsigargin. Measurements were performed in immature neuromasts at day 3. The corresponding temporal curves of the mean signal magnitude across the whole neuromast in supporting cells (C2) and in hair cells (D2) in DMSO and after 15 min of treatment with 250 nM Thapsigargin. Scale bar = 5 μm.

Article Snippet: This acquisition speed and spacing was sufficient to measure Drug Mode of action Vendor Working concentration FFA Gap junction blocker antagonist Sigma-Aldrich 25 μM MRS2500 P2yr1 selective antagonist Tocris 1 μM Thapsigargin Potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) Sigma-Aldrich 250 nm α-bungarotoxin (α-Btx) Potent inhibitor of α9 or α10 nicotinic acetylcholine receptor (nAChR) Tocris 10 μM Apamin SK channel blocker Tocris 10 μM Isradipine L-type Ca2+ channel CaV1.3a antagonist Sigma-Aldrich 10 μM BAPTA-tetrasodium salt Disrupts hair-bundle tip links Thermofisher 5 mM Frontiers in Cell and Developmental Biology | www.frontiersin.org April 2022 | Volume 10 | Article 8196123 spontaneous GCaMP6s- and RGECO1-dependent signals near simultaneously within the volumes and optimal to prevent RGECO1 photobleaching.

Techniques: Activity Assay

Journal: Frontiers in cell and developmental biology

Article Title: Using Light-Sheet Microscopy to Study Spontaneous Activity in the Developing Lateral-Line System.

doi: 10.3389/fcell.2022.819612

Figure Lengend Snippet: FIGURE 7 | Locations, mechanisms and developmental timecourse of spontaneous calcium activities in the lateral-line system. Top panel: we reliably detected spontaneous calcium activity in three cell types within the lateral-line system: hair cells, supporting cells and cholinergic (Ach (+)) efferent neurons. Spontaneous calcium activity in the supporting cells and cholinergic efferents is not correlated with calcium signals in hair cells. During development spontaneous calcium signals are robust in all three cell types (left side). Upon maturation, spontaneous calcium signals are decreased in hair cells and efferent neurons, but maintained in supporting cells (right side). Middle panel: Within a neuromast spontaneous calcium signals among populations of hair cells are not correlated (left side); supporting cells are moderately correlated (middle); cholinergic terminals are highly correlated (right side). Bottom panel: In developing hair cells, spontaneous opening of MET channels leads to calcium signals in mechanosensory hair bundles. This apical activity triggers opening of CaV1.3 channels at the presynapse, resulting in spontaneous presynaptic calcium signals (left side). In supporting cells, extracellular ATP acts on P2yr1 receptors. P2yr1 signaling leads to calcium release from the ER, giving rise to a spontaneous calcium signal. Spontaneous calcium signals can be propagated to neighboring supporting cells via gap junction channels (middle). Spontaneous activity in cholinergic efferents may be coupled to activity in spinal motor neurons that is present during development in order to form the central pattern generator required for locomotion (right side).

Article Snippet: This acquisition speed and spacing was sufficient to measure Drug Mode of action Vendor Working concentration FFA Gap junction blocker antagonist Sigma-Aldrich 25 μM MRS2500 P2yr1 selective antagonist Tocris 1 μM Thapsigargin Potent inhibitor of sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) Sigma-Aldrich 250 nm α-bungarotoxin (α-Btx) Potent inhibitor of α9 or α10 nicotinic acetylcholine receptor (nAChR) Tocris 10 μM Apamin SK channel blocker Tocris 10 μM Isradipine L-type Ca2+ channel CaV1.3a antagonist Sigma-Aldrich 10 μM BAPTA-tetrasodium salt Disrupts hair-bundle tip links Thermofisher 5 mM Frontiers in Cell and Developmental Biology | www.frontiersin.org April 2022 | Volume 10 | Article 8196123 spontaneous GCaMP6s- and RGECO1-dependent signals near simultaneously within the volumes and optimal to prevent RGECO1 photobleaching.

Techniques: Activity Assay

Journal: ACS Chemical Neuroscience

Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines

doi: 10.1021/cn500297v

Figure Lengend Snippet: Compound 4hh dose-dependently increases viability in SH-SY5Y and H4 cells. SH-SY5Y or H4 cells were pretreated with 4hh for 2 h and then treated with 7.5 μM thapsigargin for an additional 18 h. Cell viability was assessed using the CellTiter 96 AQ ueous One Solution Cell Proliferation Assay. Wells containing 100 μL of DMEM but no cells were used as background, whereas wells treated with DMSO but no thapsigargin were used as positive controls. The viability (% of control) = 100 × (well value – average of background)/(average of positive control – average of background). Experiments were repeated at least three times, and the data are represented as the average ± SEM.

Article Snippet: After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control.

Techniques: Proliferation Assay, Control, Positive Control

Journal: ACS Chemical Neuroscience

Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines

doi: 10.1021/cn500297v

Figure Lengend Snippet: Benzodiazepinones inhibit thapsigargin-induced p38 MAPK and JNK activation. H4 cells were pretreated with DMSO or benzodiazepinones for 2 h and then treated with 20 μM thapsigargin for one more hour. Cell lysates were collected and analyzed by SDS-PAGE/immunoblotting. Specific antibodies for phospho-p38 MAPK, p38 MAPK, phospho-JNK, and JNK were used. 4hh is an active benzodiazepinone, while 4mm is inactive.

Article Snippet: After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control.

Techniques: Activation Assay, SDS Page, Western Blot

Journal: ACS Chemical Neuroscience

Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines

doi: 10.1021/cn500297v

Figure Lengend Snippet: Compound 4hh exhibits strong cytoprotection only with thapsigargin treatment among different p38 activating cell death inducers. SH-SY5Y cells were pretreated with DMSO or 25 μM compounds for 2 h and then treated with different inducers, including thapsigargin (7.5 μM), tunicamycin (15 μM), staurosporine (2 μM), 6-OHDA (200 μM), DTT (5 mM), and H 2 O 2 (200 μM) for 18 h, and MG132 (5 μM), paraquat (0.5 mM), and DDTD (10 μM) for 48 h. Cell viability was assessed using the CellTiter 96 AQ ueous One Solution Cell Proliferation Assay or ATPlite. Wells with DMSO but no inducer and no compound were used as 100% controls, and wells with DMSO and inducer but no compound were used as negative controls. The experiments were repeated at least three times, and the data are represented as the average ± SEM. Asterisk (*) shows that 4hh exhibits very strong cytoprotective activity against thapsigargin-induced cell death whereas the inactive compound 4mm does not.

Article Snippet: After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control.

Techniques: Proliferation Assay, Activity Assay

Journal: ACS Chemical Neuroscience

Article Title: Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines

doi: 10.1021/cn500297v

Figure Lengend Snippet: Active benzodiazepinone 4hh , but not inactive compound 4ll , inhibits calcium regulator-induced p38 MAPK activation. H4-CHOP-luciferase reporter cells were pretreated with DMSO or compounds for 1 h and then treated with thapsigargin (20 nM), CDDO-IM (600 nM), ionomycin (120 nM), or MG132 (740 nM) for an additional 18 h. The luciferase expression was assessed using the Steady-Glo luciferase assay reagent. The p38 inhibitor was used at 20 μM. The benzodiazepiones 4hh or 4ll (10 μM) were used with thapsigargin or CDDO-IM treatment, while concentrations of 20 and 50 μM were tested with MG132 and ionomycin treatment, respectively. Wells treated with compounds only but no inducers were used as background control correspondingly, and wells treated with inducers but no compounds were used as 100% controls. The RLU (% of control) = 100 × (well value – average of background)/(average of 100% control – average of background). The data were analyzed using MS Excel software. The experiments were repeated at least three times, and the data are represented as the average ± SEM. Statistical significance ( P < 0.05) was determined using one-way analysis of variance. *Significance was compared with inducer treatment groups.

Article Snippet: After 1 h incubation at 37 °C, 2.5 μL of inducers including thapsigargin, 2-cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-IM, Toronto Research Chemicals Inc., C228090), ionomycin (Life Technologies, I-24222), or MG132 (Sigma-Aldrich, C2211) in 2% DMSO was added to reach a corresponding concentration and 2.5 μL of 2% DMSO was used as control.

Techniques: Activation Assay, Luciferase, Expressing, Control, Software

Journal: bioRxiv

Article Title: Gelsolin Counteracts ER Stress-Driven Inflammatory Circuits in Psoriasis-like Dermatitis

doi: 10.1101/2025.11.20.689413

Figure Lengend Snippet: a, b, IMQ cream was applied to the ear lobes of mice. Total RNA was extracted from the ears and the expressions of Xbp1s , Hspa5, and Hsp90b1 mRNA were measured by qPCR ( n = 4) (a). DNA was extracted from the ears and serum, and the amount of mtDNA was measured by qPCR ( n = 4) (b). c, d, e, f, IMQ cream and tunicamycin, an ER stress inducer, were applied daily to the ear lobes of mice. Ear thickness was measured daily ( n = 3) (c). Mouse ears were sampled and subjected to histological analysis (H&E staining) (d). The epidermal thickness in (d) was measured ( n = 10) (e). Total RNA was harvested from the ears, and the expression of Xbp1s , S100a9 , Il17a , Cxcl1, and Defb14 mRNA was measured by qPCR ( n = 3) (f). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P- values were calculated by one-way ANOVA with Tukey’s test (f) or Student’s t -test (a-c and e). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

Article Snippet: Tunicamycin (LKT-Labs) and thapsigargin (Wako) were used to induce ER stress.

Techniques: Cream, Staining, Expressing

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: XBP1s knockdown reversed the effect of TG on gEECs autophagy. The experimental treatment conditions were as follows: pretreatment with TG for 2 h, followed by rapamycin treatment 24 h. (A-D) The relative protein expression of SQSTM1, ATG5, and the LC3II/LC3I were analyzed using western blotting and were quantified by densitometry. (E-F) The relative SQSTM1 and ATG5 levels were quantified through RT-qPCR. Data is represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

Techniques: Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: Effect of PI3K/AKT pathway inhibition by LY294002 on overexpression of XBP1s. The experimental treatment conditions were as follows: pretreatment with LY294002 for 2 h, followed by rapamycin treatment 24 h. (A-D) Quantification of SQSTM1, ATG5, and the LC3II/LC3I band intensities from three independent experiments as determined using densitometric analysis. (E-F) SQSTM1 and ATG5 levels were detected using RT-qPCR. (G) Immunofluorescence images of LC3B expression in gEECs. Representative images of three independent experiments are shown. Scale bar = 10 μm. Data is represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

Techniques: Inhibition, Over Expression, Quantitative RT-PCR, Immunofluorescence, Expressing, Control

Journal: Scientific Reports

Article Title: ERS regulates endometrial epithelial cell autophagy through XBP1s -mediated activation of the PI3K/AKT pathway

doi: 10.1038/s41598-024-84461-6

Figure Lengend Snippet: Effect of PI3K/AKT pathway activation by SC79 on knockdown of XBP1s. The experimental treatment conditions were as follows: pretreatment with SC79 for 2 h, followed by rapamycin treatment 24 h. (A-D) Relative protein expression levels of SQSTM1, ATG5, and the LC3II/LC3I were analyzed and determined using western blotting. (E-F) The relative mRNA expression levels of SQSTM1 and ATG5 analyzed and quantified using RT-qPCR after knockdown of XBP1s. Data are represented as the means ± SEM of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. control group. # , P < 0.05; ## , P < 0.01; ### , P < 0.001 vs. other group.

Article Snippet: The medium was replaced with fresh medium and the following treatments were initiated: (1) rapamycin (50 nM; TargetMol, Boston, MA, USA) for 3 h, 6 h, 12 h, and 24 h; (2) pretreatment with TG (Thapsigargin; 50 nM; an ERS activator; TargetMol) or 4-PBA (4-Phenylbutyric acid; 1 mM, an ERS inhibitor; Abcam, Cambridge, UK) or SC79 (10 µM; a PI3K/AKT pathway activator; TargetMol) or LY294002 (10 µM, a PI3K/AKT pathway inhibitor; TargetMol) for 2 h, followed by rapamycin treatment.

Techniques: Activation Assay, Knockdown, Expressing, Western Blot, Quantitative RT-PCR, Control