Journal: Cell death & disease

Article Title: Caspase-dependent cell death-associated release of nucleosome and damage-associated molecular patterns.

doi: 10.1038/cddis.2014.450

Figure Lengend Snippet: Figure 7 Calpain activity is a prerequisite for the release of nucleosomes and DAMPs in dying cells. HeLa cells were incubated in amino acid-depleted medium (HBSS) or staurosporine-containing medium (Stau) (1 μg/ml) for 24 or 8 h, respectively, in the presence of solvent control, BAPTA-AM (50 μM) or A23187 (1 μg/ml). Released DNA was quantified with PicoGreen (a). Amount of intracellular free Ca2+ was measured in cells treated with solvent control or staurosporine at the indicated time periods by eFluor 514 (b). Cells were treated with staurosporine for 8 h in the presence of control solvent, ALLN (20 μM), or PD150606 (20 μM), and released DNA or protein was detected by PicoGreen staining (c) or western blot for histones and DAMPs (d), respectively. Genomic DNA was separated in agarose gel electrophoresis from staurosporine-treated cells with or without PD150606 (e). The cells treated with staurosporine and PD150606 for 8 h, were stained for lamin A/C and histone H1, and examined by confocal microscopy; the arrows indicate nuclear fragmentations (f). Cells treated with control solvent, zVAD-fmk (20 μM), or PD150606 with or without staurosporine for 8 h were measured for viabilities with Calcein assay (g). Cells treated with staurosporine were measured for calpain activity, caspases 3 and 7 activity, or released DNAs at the indicated time periods (h). HeLa cells transfected with non- targeting shRNA (NT), calpastatin shRNA, calpain S1 shRNA, or overexpressing calpain 1, calpain 2, or calpastatin were incubated with staurosporine for 8 h and the released DNAs were measured by PicoGreen staining (i). Cells expressing calpain 1 or calpain 2 tagged with Flag at the N-terminus and HA at the C-terminus were treated with staurosporine and western-blotted with anti-Flag antibody or anti-HA antibody (j). HeLa cells expressing halide-sensitive YFP were treated with staurosporine and/or PD150606 for 4 h, and their chloride ion currents were measured by fluorometry by detecting quenching of YFP fluorescence (k). Data performed in triplicate are presented as mean± S.D. (a–c and g–i)

Article Snippet: Cl-amidine was purchased from Cayman Chemical (Ann Arbor, MI, USA). zVAD-fmk, zDEVD-fmk, Necrostatin-1, BAPTA-AM, diphenyliodonium, PD150606, ALLN and A23187 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Incubation, Solvent, Control, Staining, Western Blot, Agarose Gel Electrophoresis, Confocal Microscopy, Transfection, shRNA, Expressing, Fluorescence

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: In vitro characterization of CG LLPS. (A) Scheme used for purifying GFP and 6x-His-tagged CGA or CGB, respectively. Stable lines expressing CGB-GFP and CGA-GFP under a doxycycline-inducible promoter are treated with doxycycline and the calcium ionophore A23187 to induce secretion of the respective proteins in serum-free medium, which is then used for purification using Ni-NTA affinity columns. (B) Plot of CGA generated using PONDR depicting disordered regions in the proteins. Almost 90% of CGA is disordered when analyzed using the VL-XT algorithm. (C) Coomassie-stained gel depicting purified CGA-GFP. Images showing different droplets of CGA-GFP at different protein concentrations at pH 6.1 in presence of 5% PEG 8000. Note that the size of the condensates decreases with decreasing protein concentration. (D) Representative images of solutions containing CGA-GFP buffered at either pH 6.1(left) or pH 7.3 (right). Droplet formation occurs at pH 6.1 and not at pH 7.3. Droplets of CGA-GFP were induced at 4 µM protein concentration in presence of 5% dextran. (E) Images obtained from plating a solution of CGB-GFP (2.5 µM final concentration) to monitor the presence or absence on liquid-like condensates either without or with 250 µM or 5 mM calcium. Note that droplet formation is induced only in the presence of 5 mM calcium. (F) Phase diagram obtained by varying calcium and CGB concentrations after plating solutions on imaging dishes and observing after 15–20 min. Red circles indicate conditions where no droplets were seen, and green circles indicate conditions which favored presence of CGB droplets. (G) Images obtained from plating a solution of CGA-GFP (2.5 µM final concentration) in presence of either 250 µM, 20 or 40 mM calcium to test for the presence or absence of droplet formation. No droplets are seen even at 40 mM which is the highest calcium concentration. (H) Representative images of CGB-GFP (2.5 µM) in presence of different concentrations of zinc. At high concentrations zinc induces formation of insoluble aggregates but at low concentration, it induces CGB-GFP droplets. Source data are available for this figure: .

Article Snippet: Fully confluent cells were induced for protein expression by the addition of doxycycline monohydrate (1 μg/ml; LKT Laboratories) in serum-free DMEM high glucose also containing proteinase inhibitor aprotinin (1 μg/ml; Sigma-Aldrich) and antibiotic A23187 (1 μg/ml; Thermo Fisher Scientific Alfa Aesar) for 15–20 h. Medium was collected and pre-cleared of cells by centrifugation and then by filtration using a 0.45 μm filter.

Techniques: In Vitro, Expressing, Purification, Generated, Staining, Protein Concentration, Concentration Assay, Imaging

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Calcium binding property of TgpCaBP. ( A ) Western blot analysis of native, HIS- and GST-tagged TgpCaBP using a rat anti-TgpCaBP antibody. ( B ) Calcium-dependent solubility was detected using western blotting. Parasites were lysed in 1% Triton X-100 in 5 mM EGTA or 5 mM CaCl 2 and fractionated by centrifugation. TgpCaBP was detected with a rat anti-TgpCaBP antibody, a mouse anti-IMC1 antibody was used as a control for the protein in the pellet (P) and a rabbit β-Tubulin monoclonal antibody was used as a control for the protein in the supernatant (S). ( C ) TgpCaBP migrated faster in the presence of Ca 2+ . Ca 2+ was replaced by Mg 2+ and K + in the control group, and EGTA was used to chelate-free calcium in the extracellular environment (Fig. S3A and B). Parasites were lysed in 1% Triton X-100 and incubated with EGTA, a combination of EGTA and CaCl 2 , or CaCl 2 . The native protein was extracted from T. gondii RH tachyzoites. ( D ) The expression of TgpCaBP in the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites (with two batches of proteins from each parasite line) was analyzed using western blotting with a rat anti-TgpCaBP antibody to verify the presence and absence of TgpCaBP protein in different parasite lines.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Binding Assay, Western Blot, Solubility, Centrifugation, Control, Incubation, Expressing

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: TgpCaBP was associated with intracellular growth and gliding motility of T. gondii . ( A ) Plaque assays were conducted for parental (P), ΔTgpCaBP ( Δ ), and ΔTgpCaBP-C (C) parasites. Quantification of plaque sizes from three independent biological experiments using one-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. Scale bar, 2 mm. ( B ) Red/green assays of parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites calculating the invasion efficiency. The invasion capacity of the ΔTgpCaBP parasites was significantly lower than that of the parental and the gene-complemented parasites. Quantification of invasion from three independent biological experiments using one-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( C ) Egress assays of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites stimulated by 3 µM A23187. The egress of the ΔTgpCaBP parasites was significantly lower than the other two parasite lines. Quantification of egress from three independent biological experiments using two-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( D ) Effect of A23187 on gliding of T. gondii . Indirect immunofluorescence microscopy demonstrated that the length of trails deposited during gliding with A23187 treatment in the parental and ΔTgpCaBP-C parasite was longer and more complete than the ΔTgpCaBP parasite. Trails were visualized with a mouse anti-SAG1 antibody and conjugated to Alexa 488. Scale bar, 5 µm. ( E ) Quantification and statistical analysis of the trail length of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites treated with A23187 and dimethyl sulfoxide control. The motility was impaired in the ΔTgpCaBP parasites and rescued in the ΔTgpCaBP-C parasites. Values are means ± SEM; ( n = 3). P > 0.05: not significant. ( F ) Replication assays of the parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites. There was no significant difference between the replication of the ΔTgpCaBP parasites and the other two parasite lines. Quantification of replication from three independent biological experiments using two-way ANOVA with Tukey’s multiple comparisons. Values are means ± SEM; ( n = 3). Ns: not significant. ( G ) Survival of mice challenged with 50, 500, 5 × 10 3 , or 5 × 10 5 parental and ΔTgpCaBP parasites. ( H ) Survival of mice challenged with 50 parental, ΔTgpCaBP, and ΔTgpCaBP-C parasites. The virulence of the ΔTgpCaBP parasites decreased significantly compared to the parental parasites. The virulence was rescued in the ΔTgpCaBP-C parasites. All mice were monitored for 28 days. All data were analyzed using simple survival analysis (Kaplan-Meier), P < 0.0001.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Immunofluorescence, Microscopy, Control

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Effect of the Ca 2+ ionophore A23187 on microneme secretion of T. gondii . ( A ) Western blot of supernatants secreted by parasites after stimulation with A23187 at 37°C for 5 min, showing a significant difference in MIC2 secreted by the parental and the ΔTgpCaBP parasites. The pellets were monitored by a mouse anti-IMC-1 monoclonal antibody. Stimulation with 2% ethanol was used as a positive control, and DMSO was used as a solvent control. ( B ) Quantification and statistical analysis of MIC2 secreted by the parental and the ΔTgpCaBP parasites after treatment with 2% ethanol, DMSO, and different concentrations of A23187. Secretion of MIC2 was inhibited in the ΔTgpCaBP parasites. Values are means ± SEM; ( n = 3). P > 0.05: not significant.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Western Blot, Positive Control, Solvent, Control

Journal: Microbiology Spectrum

Article Title: Calcium-binding protein TgpCaBP regulates calcium storage of the zoonotic parasite Toxoplasma gondii

doi: 10.1128/spectrum.00661-24

Figure Lengend Snippet: Role of TgpCaBP in the efflux of cytosolic Ca 2+ of T. gondii . ( A ) Intracellular calcium concentration ([Ca 2+ ] i ) (nM) of the parental parasites, the ΔTgpCaBP and the ΔTgpCaBP-C parasites in the presence of 100 µM EGTA or 2 mM CaCl 2 . Values are means ± SEM; ( n = 6–10). P > 0.05: not significant. ( B ) Measurements of dynamic cytosolic Ca 2+ in the presence of Fura-2AM in tachyzoites of the parental (RH), the ΔTgpCaBP and the ΔTgpCaBP-C parasites. The buffer contains 100 µM EGTA to chelate contaminating Ca 2+ , and 2 mM Ca 2+ was added to the suspension at 120 s. The pink box indicates the area used for the quantification presented in ( C ). ( C ) Quantification and statistical analysis of the change in cytosolic Ca 2+ during the first 20 s after the addition of extracellular Ca 2+ . Values are means ± SEM; ( n = 7–12). P > 0.05: not significant. ( D ) Cytosolic Ca 2+ increases after adding Thap (1 µM), following Ca 2+ influx resulting from adding 2 mM extracellular Ca 2+ at 260 s. Thap: thapsigargin. The pink boxes indicate the area used for the quantification presented in ( E ) and ( F ). ( E ) Quantification and statistical analysis of the change in cytosolic Ca 2+ (Δ[Ca 2+ ] cyt ) at 50 s after adding Thap. Values are means ± SEM; ( n = 9–12). P > 0.05: not significant. ( F ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt at 20 s after adding 2 mM of Ca 2+ . Values are mean ± SEM; ( n = 9–16). P > 0.05: not significant. ( G ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt at 20 s after adding Ca 2+ in the presence or absence of Thap in the parental (P) and the ΔTgpCaBP parasites. Values are means ± SEM; ( n = 7–12). P > 0.05: not significant. ( H ) Quantification and statistical analysis of cytosolic Ca 2+ increase stimulated by Zap (100 µM) in the presence of 2 mM extracellular Ca 2+ . Values are means ± SEM; ( n = 5–6). P > 0.05: not significant. Zap: zaprinast. ( I ) Quantification and statistical analysis of the Δ[Ca 2+ ] cyt during the 20 s after adding Ca 2+ without additions (ND) or after adding Thap or Zap. Values are means ± SEM; ( n = 5–15). P > 0.05: not significant. Two-way ANOVA with Tukey’s multiple comparison test for A and I and one-way ANOVA with Tukey’s multiple comparison tests for C, E, F, G, and H.

Article Snippet: Vero monolayers were infected with 1 × 10 6 tachyzoites in 12-well plates for 2 h and cultured at 37 °C with 5% CO 2 for 24 h. Accurately, 3 µM Ca 2+ ionophore A23187 (MCE) was added at 37 °C for 3 min and DMSO was used as a control.

Techniques: Concentration Assay, Suspension, Comparison

Journal: Aging Cell

Article Title: The mitochondrial ATP synthase is a shared drug target for aging and dementia

doi: 10.1111/acel.12715

Figure Lengend Snippet: J147 modulates resting Ca 2+ homeostasis to activate the AMPK / mTOR axis. (a) J147 increases the levels of cytosolic Ca 2+ in HT 22 cells. A23187 was used as a positive control (* p = .0162, *** p = .0002, *** p < .0001, ANOVA ). (b) The Cam KK 2 inhibitor STO ‐609 attenuated J147‐induced activation (100 n m ) of AMPK targets in rat primary cortical neurons. Phosphorylation targets include AMPK (α‐Thr172), Raptor (Ser792), ACC 1 (Ser79), and S6 (Ser235/236); corresponding quantifications for each are shown below. (c) J147 protection against glutamate (2.5 m m ) is reduced in AMPK KO fibroblasts demonstrating a direct role for AMPK in the protective effects of J147 (**** p < . 0001 (10 n m J147),**** p < . 0001 (25 n m J147),*** p < .0001 (100 n m J147))

Article Snippet: For calcium ionophore experiments, A23187 (Tocris 1234) and ionomycin (Cayman 10004974) were added at the indicated concentrations along with J147 for 1 hr.

Techniques: Positive Control, Activation Assay, Phospho-proteomics

Journal: Investigative Ophthalmology & Visual Science

Article Title: Canonical and Truncated Transglutaminase-2 Drive TGF-β1 Autocrine Loop to Induce Fibrosis in Graves’ Orbitopathy

doi: 10.1167/iovs.66.5.22

Figure Lengend Snippet: Positive feedback loop between TGM2 and TGF-β1 drives fibrosis in OFs, and truncated TGM2 amplifies this effect. ( A ) Western blot analysis ( n control = 3, n test = 3) demonstrates the impact of various GTP concentrations on COL1A1 and α-SMA expression following TGF-β1 treatment (10 ng/mL). ( B ) Western blot results ( n control = 3, n test = 3) indicate that ZM39923 significantly affects the expression of COL1A1 and α-SMA induced by TGF-β1. ( C ) The expression changes of COL1A1 and α-SMA after TGF-β1 (10 ng/mL) treatment in the presence of A23187 are shown ( n control = 3, n test = 3). ( D ) FITC-tyramine staining reveals TGM2 activity ( n control = 3, n test = 3), while the right panel presents the quantitative results of TGM2 activity. S cale bar : 20 µm. ( E ) A colorimetric assay quantifies TGM2 activity following TGF-β1 treatment combined with various concentrations of ZM39923 and GTP ( n control = 3, n test = 3). ( F ) Colorimetric analysis evaluates the effects of ZM39923 and GTP on TGF-β1–induced TGM2 activity in cells where different TGM2 variants (TGM2_V1 and TGM2_V2) were reintroduced after TGM2 knockdown ( n control = 3, n test = 3). ( G ) Western blot analysis ( n control = 3, n test = 3) examines the effects of GTP or ZM39923 on COL1A1 and α-SMA expression after restoring full-length TGM2 (TGM2_V1) and truncated TGM2 variants (TGM2_V2) in fibroblasts with TGM2 knockdown. * P < 0.05, ** P < 0.01, *** P < 0.001 compared to the stimulation group; P < 0.05, P < 0.01, P < 0.001 compared to the control group (ANOVA), KD, knockdown; ns, not significant.

Article Snippet: Cells were treated with TGF-β1 (5 ng/mL; R&D Systems, Minneapolis, MN, USA); RepSox, ERKi, ZM39923, GTP, LDN-27219, cystamine, and A23187 (all from TargetMol, Boston, MA< USA); and rh-TGM2 (MedChemExpress, Monmouth Junction, NJ, USA) in DMEM with 1% FBS.

Techniques: Western Blot, Control, Expressing, Staining, Activity Assay, Colorimetric Assay, Knockdown