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Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM <t>ionomycin</t> (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
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Images

1) Product Images from "Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii"

Article Title: Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii

Journal: Scientific Reports

doi: 10.1038/srep42746

Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
Figure Legend Snippet: Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

Techniques Used: Fluorescence, Standard Deviation, Microscopy

2) Product Images from "Adenosine-Induced NLRP11 in B Lymphoblasts Suppresses Human CD4+ T Helper Cell Responses"

Article Title: Adenosine-Induced NLRP11 in B Lymphoblasts Suppresses Human CD4+ T Helper Cell Responses

Journal: Journal of Immunology Research

doi: 10.1155/2020/1421795

Adenosine treatment induced NLRP11 expression at both the mRNA and protein levels in B lymphoblasts. (a) NLRP11 mRNA expression after 4 and 10 hours of stimulation with PMA/ionomycin (50 ng/500 ng/ml), CD40L (1 μ /ml), LPS (100 ng/ml), and adenosine (50 μ M) ( ∗ indicates significance at P
Figure Legend Snippet: Adenosine treatment induced NLRP11 expression at both the mRNA and protein levels in B lymphoblasts. (a) NLRP11 mRNA expression after 4 and 10 hours of stimulation with PMA/ionomycin (50 ng/500 ng/ml), CD40L (1 μ /ml), LPS (100 ng/ml), and adenosine (50 μ M) ( ∗ indicates significance at P

Techniques Used: Expressing

3) Product Images from "PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death"

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

Journal: Oncotarget

doi:

Ionomycin enhanced CGK733-induced PERK/CHOP activation and vesicular calcium sequestration A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.
Figure Legend Snippet: Ionomycin enhanced CGK733-induced PERK/CHOP activation and vesicular calcium sequestration A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.

Techniques Used: Activation Assay, Expressing, Western Blot, Microscopy, Fluorescence

4) Product Images from "Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis"

Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Journal: Nature Communications

doi: 10.1038/ncomms15069

Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50. ( a ) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with PMA/Ionomycin from Bcl-3 TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. *** P
Figure Legend Snippet: Bcl-3 inhibits Foxp3 promoter activity via direct binding to p50. ( a ) Reporter assays with constructs containing the endogenous Foxp3 promoter, using in vitro differentiated Tregs restimulated with PMA/Ionomycin from Bcl-3 TOE mice. The Luciferase reads from the pGL4 plasmid that contained the Foxp3 promoter or empty were normalized to a Renilla transfection control plasmid. One representative out of two independent experiments with four parallel transfections is shown. Mean±s.e.m. *** P

Techniques Used: Activity Assay, Binding Assay, Construct, In Vitro, Mouse Assay, Luciferase, Plasmid Preparation, Transfection

Bcl-3 TOE Tregs display diminished suppressive capacity. ( a ) Flow cytometric analysis of MACS-purified CD4 + CD25 + Treg cells of the indicated genotypes restimulated with PMA/Ionomycin and BrefeldinA for 4 h. Cells are gated on Foxp3 + and analysed for IL-10 expression. n =3. ( b ) Quantitative RT–PCR of MACS purified CD4 + CD25 + Treg cells from the indicated mice ( n =3). Shown are transcription counts of the indicated genes normalized to HPRT and littermate controls. * P
Figure Legend Snippet: Bcl-3 TOE Tregs display diminished suppressive capacity. ( a ) Flow cytometric analysis of MACS-purified CD4 + CD25 + Treg cells of the indicated genotypes restimulated with PMA/Ionomycin and BrefeldinA for 4 h. Cells are gated on Foxp3 + and analysed for IL-10 expression. n =3. ( b ) Quantitative RT–PCR of MACS purified CD4 + CD25 + Treg cells from the indicated mice ( n =3). Shown are transcription counts of the indicated genes normalized to HPRT and littermate controls. * P

Techniques Used: Flow Cytometry, Magnetic Cell Separation, Purification, Expressing, Quantitative RT-PCR, Mouse Assay

Bcl-3 prevents p50 binding to the Foxp3 and CTLA-4 promoter. ( a ) Western blot analysis of Bcl-3 and the NF-κB family members p50, p52 and p65 in nuclear versus whole extract of Bcl-3 TOE and littermate controls using Tregs either unstimulated (left) or restimulated for 2 h with PMA/Ionomycin (right). One representative blot from two independent experiments is shown. ( b ) Immunoprecipitation with Bcl-3 antibodies using protein extracts from in vitro differentiated Tregs, restimulated with PMA/Ionomycin for 4 h and followed by immunoblot analysis with antibodies against Bcl-3 or p50. One representative blot from two independent experiments is shown. ( c ) Pull-down experiments with DNA probes from promoters of IL2Ra, Foxp3 CN2 (from top to bottowm) with protein extracts from Tregs to which Bcl-3 or empty vector overexpressing lysates were added. One out of two experiments is demonstrated. ( d ) CHiP assay using p50 and isotope control antibodies antibodies were performed in in vitro differentiated Tregs from Bcl3 TOE mice and littermate controls. Primer were designed as indicated for two sites within the CTLA-4 and Foxp3 Promoter region. One of two representative biological examples is shown.
Figure Legend Snippet: Bcl-3 prevents p50 binding to the Foxp3 and CTLA-4 promoter. ( a ) Western blot analysis of Bcl-3 and the NF-κB family members p50, p52 and p65 in nuclear versus whole extract of Bcl-3 TOE and littermate controls using Tregs either unstimulated (left) or restimulated for 2 h with PMA/Ionomycin (right). One representative blot from two independent experiments is shown. ( b ) Immunoprecipitation with Bcl-3 antibodies using protein extracts from in vitro differentiated Tregs, restimulated with PMA/Ionomycin for 4 h and followed by immunoblot analysis with antibodies against Bcl-3 or p50. One representative blot from two independent experiments is shown. ( c ) Pull-down experiments with DNA probes from promoters of IL2Ra, Foxp3 CN2 (from top to bottowm) with protein extracts from Tregs to which Bcl-3 or empty vector overexpressing lysates were added. One out of two experiments is demonstrated. ( d ) CHiP assay using p50 and isotope control antibodies antibodies were performed in in vitro differentiated Tregs from Bcl3 TOE mice and littermate controls. Primer were designed as indicated for two sites within the CTLA-4 and Foxp3 Promoter region. One of two representative biological examples is shown.

Techniques Used: Binding Assay, Western Blot, Immunoprecipitation, In Vitro, Plasmid Preparation, Chromatin Immunoprecipitation, Mouse Assay

5) Product Images from "Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo"

Article Title: Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0143137

MPs derived from cultured cells and platelets expose PCI on their surface. Jurkat cells and U937 cells were cultured in serum-free media (serum starvation, control) or treated with STS to induce apoptosis. MPs, isolated from the conditioned media of (A) Jurkat cells and (B) U937 cells, were stained with annexin V-EGFP and anti-PCI-IgG-AF633 and subjected to flow cytometry. MPs were defined as events with a size smaller than 1 μm, which show binding of annexin V. Data shown represent means and range of two independent experiments performed in duplicates. Washed platelets (n = 4) were activated with TRAP-6 (20 μM) or ionomycin (40 μM) to induce membrane blebbing and MP release. MP levels obtained from resting platelets served as control. MPs were analyzed by flow cytometry after staining with annexin V-EGFP, anti-PCI-IgG-AF633 and anti-CD62P-IgG-PE. (C) Stimulus dependent numbers of MPs (annexin V positive events). (D) Exposure of PCI on PS/CD62P double positive MPs after platelet activation with TRAP-6 (20 μM) or ionomycin (40 μM) in the absence or presence of pPCI (300 nM).
Figure Legend Snippet: MPs derived from cultured cells and platelets expose PCI on their surface. Jurkat cells and U937 cells were cultured in serum-free media (serum starvation, control) or treated with STS to induce apoptosis. MPs, isolated from the conditioned media of (A) Jurkat cells and (B) U937 cells, were stained with annexin V-EGFP and anti-PCI-IgG-AF633 and subjected to flow cytometry. MPs were defined as events with a size smaller than 1 μm, which show binding of annexin V. Data shown represent means and range of two independent experiments performed in duplicates. Washed platelets (n = 4) were activated with TRAP-6 (20 μM) or ionomycin (40 μM) to induce membrane blebbing and MP release. MP levels obtained from resting platelets served as control. MPs were analyzed by flow cytometry after staining with annexin V-EGFP, anti-PCI-IgG-AF633 and anti-CD62P-IgG-PE. (C) Stimulus dependent numbers of MPs (annexin V positive events). (D) Exposure of PCI on PS/CD62P double positive MPs after platelet activation with TRAP-6 (20 μM) or ionomycin (40 μM) in the absence or presence of pPCI (300 nM).

Techniques Used: Derivative Assay, Cell Culture, Isolation, Staining, Flow Cytometry, Cytometry, Binding Assay, Activation Assay

6) Product Images from "Cyclosporine A Suppressed Glucose Oxidase Induced P53 Mitochondrial Translocation and Hepatic Cell Apoptosis through Blocking Mitochondrial Permeability Transition"

Article Title: Cyclosporine A Suppressed Glucose Oxidase Induced P53 Mitochondrial Translocation and Hepatic Cell Apoptosis through Blocking Mitochondrial Permeability Transition

Journal: International Journal of Biological Sciences

doi: 10.7150/ijbs.13716

CsA was able to block oxidative stress induced p53 mitochondrial translocation due to inhibition of MPTP opening. (A) After exposure to GOX (50 U) for 0-8 hours, HepG 2 cells were stained with Calcein-AM and assessed by flow-cytometry. (B) The ratio of retained and quenched Calcein fluorescence within 8 hours was calculated relative to the retained Calcein fluorescence in control group. (C) Cells were pretreated with or without CsA (10 μM) for 1 hour, followed by GOX (50 U) and ionomycin (5 μM) stimulation for 4 hours. After treatment, cells stained with Calcein-AM and analyzed by confocal laser scanning microscope. (D) The retained Calcein fluorescence was assessed by using Image-Pro plus 6 Software, and the ratio of retained and quenched Calcein fluorescence was calculated. (E) Cells were pretreated with CsA (10 μM) and PFT-μ (5 μM) for 1 hour following by exposure to GOX (50 U) for 4 hours. The expression of p53 protein in mitochondrial lysates was detected by Western blot. (F) The relative protein abundance of mitochondrial p53 was normalized by banding to COX-IV protein. (G) P53 protein sub-cellular location was detected by immunofluorescence method. (H) Cells were exposed to GOX (50 U) for 4 hours in presence or absence of SfA (20μM) for 1 hour. And mitochondrial permeability transition was assessed by flow-cytometry method after staining with Calcein-AM. (I) P53 protein level in mitochondrial lysates was detected by Western blot. Data were expressed as the mean ± SD of triplicate independent experiments (n=3). *P
Figure Legend Snippet: CsA was able to block oxidative stress induced p53 mitochondrial translocation due to inhibition of MPTP opening. (A) After exposure to GOX (50 U) for 0-8 hours, HepG 2 cells were stained with Calcein-AM and assessed by flow-cytometry. (B) The ratio of retained and quenched Calcein fluorescence within 8 hours was calculated relative to the retained Calcein fluorescence in control group. (C) Cells were pretreated with or without CsA (10 μM) for 1 hour, followed by GOX (50 U) and ionomycin (5 μM) stimulation for 4 hours. After treatment, cells stained with Calcein-AM and analyzed by confocal laser scanning microscope. (D) The retained Calcein fluorescence was assessed by using Image-Pro plus 6 Software, and the ratio of retained and quenched Calcein fluorescence was calculated. (E) Cells were pretreated with CsA (10 μM) and PFT-μ (5 μM) for 1 hour following by exposure to GOX (50 U) for 4 hours. The expression of p53 protein in mitochondrial lysates was detected by Western blot. (F) The relative protein abundance of mitochondrial p53 was normalized by banding to COX-IV protein. (G) P53 protein sub-cellular location was detected by immunofluorescence method. (H) Cells were exposed to GOX (50 U) for 4 hours in presence or absence of SfA (20μM) for 1 hour. And mitochondrial permeability transition was assessed by flow-cytometry method after staining with Calcein-AM. (I) P53 protein level in mitochondrial lysates was detected by Western blot. Data were expressed as the mean ± SD of triplicate independent experiments (n=3). *P

Techniques Used: Blocking Assay, Translocation Assay, Inhibition, Staining, Flow Cytometry, Cytometry, Fluorescence, Laser-Scanning Microscopy, Software, Expressing, Western Blot, Immunofluorescence, Permeability

7) Product Images from "A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro"

Article Title: A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060370

PMA increases survival of CLL B-cells in vitro at all doses tested. A. After 72 h, the survival of B-CLL cells in the presence of PMA (1 µg/ml) was significantly greater than that of control cells in complete medium as evaluated by annexin V–PE/7-AAD staining. Exposure to LPS (1 µg/ml) had no effect on cell survival. Right panel: Cytometry plots from a representative patient. Left panel: The presence of PHA (5 µg/ml) or ionomycin (0.1 µg/ml) significantly decreased cell survival. The percentages of CLL B-cells surviving after 72 hrs in 9 independent experiments are presented as box and whisker (minimum to maximum) plots. The significance of differences was calculated with the Wilcoxon test: * p
Figure Legend Snippet: PMA increases survival of CLL B-cells in vitro at all doses tested. A. After 72 h, the survival of B-CLL cells in the presence of PMA (1 µg/ml) was significantly greater than that of control cells in complete medium as evaluated by annexin V–PE/7-AAD staining. Exposure to LPS (1 µg/ml) had no effect on cell survival. Right panel: Cytometry plots from a representative patient. Left panel: The presence of PHA (5 µg/ml) or ionomycin (0.1 µg/ml) significantly decreased cell survival. The percentages of CLL B-cells surviving after 72 hrs in 9 independent experiments are presented as box and whisker (minimum to maximum) plots. The significance of differences was calculated with the Wilcoxon test: * p

Techniques Used: In Vitro, Staining, Cytometry, Whisker Assay

8) Product Images from "Genetic Indicators for Calcium Signaling studies in Toxoplasma gondii"

Article Title: Genetic Indicators for Calcium Signaling studies in Toxoplasma gondii

Journal: Methods in molecular biology (Clifton, N.J.)

doi: 10.1007/978-1-4939-9857-9_11

Characterization of clones in a plate reader: A suspension of 5 × 10 6 ). A, Fluorescence changes before and after the addition of the indicated reagents with two Toxoplasma clones (3 and 5). DMSO is used as a control. B, kinetic measurements for clones 3 and 5 showing changes in the fluorescence of GCamP6f in function of time. IO, ionomycin; TG, thapsigargin, Zap, zaprinast, Ca 2+ , extracellular Ca 2+, 1.8 mM. C, same experiment to the one presented in A with clones 7 and 9 that express GCamP6s and compare with the reference cell line expressing GCamp6f. D, kinetic measurements with clones 7 and 9. Same conditions as B.
Figure Legend Snippet: Characterization of clones in a plate reader: A suspension of 5 × 10 6 ). A, Fluorescence changes before and after the addition of the indicated reagents with two Toxoplasma clones (3 and 5). DMSO is used as a control. B, kinetic measurements for clones 3 and 5 showing changes in the fluorescence of GCamP6f in function of time. IO, ionomycin; TG, thapsigargin, Zap, zaprinast, Ca 2+ , extracellular Ca 2+, 1.8 mM. C, same experiment to the one presented in A with clones 7 and 9 that express GCamP6s and compare with the reference cell line expressing GCamp6f. D, kinetic measurements with clones 7 and 9. Same conditions as B.

Techniques Used: Clone Assay, Fluorescence, Expressing

9) Product Images from "Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii"

Article Title: Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii

Journal: Scientific Reports

doi: 10.1038/srep42746

Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
Figure Legend Snippet: Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

Techniques Used: Fluorescence, Standard Deviation, Microscopy

10) Product Images from "Tissue-resident Eomes+ NK cells are the major innate lymphoid cell population in human infant intestine"

Article Title: Tissue-resident Eomes+ NK cells are the major innate lymphoid cell population in human infant intestine

Journal: Nature Communications

doi: 10.1038/s41467-018-08267-7

Infant intestinal NK cells contain high levels of cytotoxic granules. a viSNE plots of combined flow cytometric data visualizing Eomes, perforin, granzyme B, and KIR expression by epithelial (EP) and lamina propria-derived (LP) infant and adult NK cells. Expression of Eomes, KIR, perforin, and granzyme B (GrzB) is shown by color coding in relative intensity. viSNE plots have been calculated from concatenated FCS files gated on NK cells (infant samples N = 7, adult samples N = 5, iterations = 7500 perplexity = 100, KL divergence = 2.62). b Frequencies of perforin + and GrzB + NK cells in infants (white circles) and adults (dark circles) (EP infant samples ( N = 8), LP infant samples ( N = 7), EP adult samples perforin expression ( N = 9), GrzB expression ( N = 8), LP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8)). c Frequencies of perforin + and granzyme B + cells within CD103 + , CD49a + , or CD69 + NK cell populations in infant (white circles) and adult intestines (dark circles) (EP infant samples ( N = 8), LP infant samples ( N = 7), EP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8), LP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8)). d Frequencies of LP-derived CD107a + , IFN-ɣ + , and TNF-α + NK cells in infant (white circles) and adult intestines (dark circles). Cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin (infant samples N = 5, adult samples N = 7). e Frequencies of LP-derived CD107a + cells within CD103 + , CD49a + , or CD69 + NK cell populations in infant (white circles) and adult intestines (dark circles) after stimulation with PMA and ionomycin for 6 h (infant samples N = 5, adult samples N = 6). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons ( b , d ) and Wilcoxon matched-pairs signed rank tests ( c , e ). Asterisks represent the following p- values: * p
Figure Legend Snippet: Infant intestinal NK cells contain high levels of cytotoxic granules. a viSNE plots of combined flow cytometric data visualizing Eomes, perforin, granzyme B, and KIR expression by epithelial (EP) and lamina propria-derived (LP) infant and adult NK cells. Expression of Eomes, KIR, perforin, and granzyme B (GrzB) is shown by color coding in relative intensity. viSNE plots have been calculated from concatenated FCS files gated on NK cells (infant samples N = 7, adult samples N = 5, iterations = 7500 perplexity = 100, KL divergence = 2.62). b Frequencies of perforin + and GrzB + NK cells in infants (white circles) and adults (dark circles) (EP infant samples ( N = 8), LP infant samples ( N = 7), EP adult samples perforin expression ( N = 9), GrzB expression ( N = 8), LP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8)). c Frequencies of perforin + and granzyme B + cells within CD103 + , CD49a + , or CD69 + NK cell populations in infant (white circles) and adult intestines (dark circles) (EP infant samples ( N = 8), LP infant samples ( N = 7), EP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8), LP adult samples perforin expression ( N = 9), and GrzB expression ( N = 8)). d Frequencies of LP-derived CD107a + , IFN-ɣ + , and TNF-α + NK cells in infant (white circles) and adult intestines (dark circles). Cells were stimulated for 6 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin (infant samples N = 5, adult samples N = 7). e Frequencies of LP-derived CD107a + cells within CD103 + , CD49a + , or CD69 + NK cell populations in infant (white circles) and adult intestines (dark circles) after stimulation with PMA and ionomycin for 6 h (infant samples N = 5, adult samples N = 6). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons ( b , d ) and Wilcoxon matched-pairs signed rank tests ( c , e ). Asterisks represent the following p- values: * p

Techniques Used: Flow Cytometry, Expressing, Derivative Assay, MANN-WHITNEY

11) Product Images from "Overactivation of Mitogen-Activated Protein Kinase and Suppression of Mitofusin-2 Expression Are Two Independent Events in High Mobility Group Box 1 Protein–Mediated T Cell Immune Dysfunction"

Article Title: Overactivation of Mitogen-Activated Protein Kinase and Suppression of Mitofusin-2 Expression Are Two Independent Events in High Mobility Group Box 1 Protein–Mediated T Cell Immune Dysfunction

Journal: Journal of Interferon & Cytokine Research

doi: 10.1089/jir.2012.0054

Effects of MAPK inhibitors on HMGB1-induced suppression of T cell immune response. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then pretreated with ERK1/2 inhibitor (5 μM
Figure Legend Snippet: Effects of MAPK inhibitors on HMGB1-induced suppression of T cell immune response. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then pretreated with ERK1/2 inhibitor (5 μM

Techniques Used: Incubation

HMGB1 induced a transient phosphorylation of ERK1/2 and p38 MAPK. Jurkat cells were cultured in 96-well plates with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with HMGB1 (100 ng/mL) for different
Figure Legend Snippet: HMGB1 induced a transient phosphorylation of ERK1/2 and p38 MAPK. Jurkat cells were cultured in 96-well plates with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with HMGB1 (100 ng/mL) for different

Techniques Used: Cell Culture

Effect of HMGB1 on Mfn-2 expression in response to PMA/ionomycin in Jurkat cells. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with different dosages of HMGB1 (10,
Figure Legend Snippet: Effect of HMGB1 on Mfn-2 expression in response to PMA/ionomycin in Jurkat cells. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with different dosages of HMGB1 (10,

Techniques Used: Expressing, Incubation

NFAT served as a potential target of MAPK on HMGB1-mediated immunosuppression of T lymphocytes. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then pretreated with ERK1/2 inhibitor (5 μM
Figure Legend Snippet: NFAT served as a potential target of MAPK on HMGB1-mediated immunosuppression of T lymphocytes. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then pretreated with ERK1/2 inhibitor (5 μM

Techniques Used: Incubation

The effect of HMGB1 on immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with different dosages of HMGB1 (10,
Figure Legend Snippet: The effect of HMGB1 on immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then stimulated with different dosages of HMGB1 (10,

Techniques Used: Incubation

Overexpression of Mfn-2 attenuated HMGB1-induced immune dysfunction of Jurkat cells. Jurkat cells transfected with Lv-Mfn-2 or Lv-GFP (MOI=50) were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then
Figure Legend Snippet: Overexpression of Mfn-2 attenuated HMGB1-induced immune dysfunction of Jurkat cells. Jurkat cells transfected with Lv-Mfn-2 or Lv-GFP (MOI=50) were incubated with PMA (50 ng/mL) plus ionomycin (1 μM) for 12 h, and then

Techniques Used: Over Expression, Transfection, Incubation

MAPK inhibitors exerted protective effects on HMGB1-induced suppression of T cell immune response through moderately diminishing phosphorylation of ERK1/2 and p38 MAPK. Jurkat cells were stimulated with PMA (50 ng/mL) plus ionomycin (1 μM)
Figure Legend Snippet: MAPK inhibitors exerted protective effects on HMGB1-induced suppression of T cell immune response through moderately diminishing phosphorylation of ERK1/2 and p38 MAPK. Jurkat cells were stimulated with PMA (50 ng/mL) plus ionomycin (1 μM)

Techniques Used:

12) Product Images from "Optical determination of intracellular water in apoptotic cells"

Article Title: Optical determination of intracellular water in apoptotic cells

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2013.263228

Qualitative refractive index map of ionomycin-treated HeLa cells
Figure Legend Snippet: Qualitative refractive index map of ionomycin-treated HeLa cells

Techniques Used:

13) Product Images from "NFAT and IRF Proteins Regulate Transcription of the Anti-HIV Gene, APOBEC3G *"

Article Title: NFAT and IRF Proteins Regulate Transcription of the Anti-HIV Gene, APOBEC3G *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.154377

Enrichment of nuclear NFAT induces transactivation of the A3G promoter in T cells. A , antibodies against NFATc1, NFATc2, IRF-4, and an Ig control were used in a ChIP assay. Immunoprecipitated samples from CEMss and CEM were analyzed in a TaqMan real-time PCR assay using primers directed to the A3G minimal promoter region. The average cycle threshold of GAPDH for input samples was used as an internal control for quality of input DNA, CEMss Ct 27.4 ± .87, CEM Ct 27.3 ± .78. B , Mock-treated (−) and ionomycin ( Iono )-stimulated (+) CEMss and CEM cell lysates were subcellularly fractionated and probed for NFATc1, NFATc2, or IRF-4 expression in the nuclear fraction. A3G expression was assayed in the cytoplasmic fraction. Actin expression is shown as a loading control. C , CEMss and ionomycin stimulated CEMss (CEMss+) cells were assayed for NFATc1, NFATc2, or IRF-4 occupation of the A3G promoter as in A . Average cycle threshold of GAPDH for input samples was used as an internal control for quality of input DNA; CEMss Ct 27.4 ± .87, CEMss+ Ct 28.2 ± .78. Ct , threshold cycle.
Figure Legend Snippet: Enrichment of nuclear NFAT induces transactivation of the A3G promoter in T cells. A , antibodies against NFATc1, NFATc2, IRF-4, and an Ig control were used in a ChIP assay. Immunoprecipitated samples from CEMss and CEM were analyzed in a TaqMan real-time PCR assay using primers directed to the A3G minimal promoter region. The average cycle threshold of GAPDH for input samples was used as an internal control for quality of input DNA, CEMss Ct 27.4 ± .87, CEM Ct 27.3 ± .78. B , Mock-treated (−) and ionomycin ( Iono )-stimulated (+) CEMss and CEM cell lysates were subcellularly fractionated and probed for NFATc1, NFATc2, or IRF-4 expression in the nuclear fraction. A3G expression was assayed in the cytoplasmic fraction. Actin expression is shown as a loading control. C , CEMss and ionomycin stimulated CEMss (CEMss+) cells were assayed for NFATc1, NFATc2, or IRF-4 occupation of the A3G promoter as in A . Average cycle threshold of GAPDH for input samples was used as an internal control for quality of input DNA; CEMss Ct 27.4 ± .87, CEMss+ Ct 28.2 ± .78. Ct , threshold cycle.

Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

Induced A3G protein is stable and exerts an antiviral effect. A. The stability of induced A3G expression in CEMss cells was determined by treating cells with ionomycin ( Iono ) over a 14-day time course. The kinetics of A3G expression induction were examined by immunoblotting for expression on days 4, 6, 10, and 14. B , mock and ionomycin-stimulated ( Stim ) CEMss (CEMss++) cells were treated for 5 days prior to viral challenge with wild-type HIV-1 ( filled symbols ) or HIV-1Δ vif ( open symbols ). Subsequent viral replication was monitored by HIV-1 p24 Gag ELISA. C , CEMss cells were mock or ionomycin-stimulated for 5 days prior to infection. Ionomycin- treated cells then either continued to receive stimulation (++) or ionomycin was washed out of the medium after a 24-h incubation with HIV-1Δ vif virus (+). Spreading infection was assayed by p24 Gag ELISA. D , whole cell lysates were prepared from mock, transient ionomycin-, and chronic ionomycin-stimulated cultures on d1 of the replication curve, corresponding to day 6 of the stimulation time course. Additional whole cell lysates were prepared on day 10 of the replication curve, corresponding to day 16 of the stimulation protocol. Whole cell lysates from both days were probed for A3G and actin expression by Western blot. Unstim , unstimulated.
Figure Legend Snippet: Induced A3G protein is stable and exerts an antiviral effect. A. The stability of induced A3G expression in CEMss cells was determined by treating cells with ionomycin ( Iono ) over a 14-day time course. The kinetics of A3G expression induction were examined by immunoblotting for expression on days 4, 6, 10, and 14. B , mock and ionomycin-stimulated ( Stim ) CEMss (CEMss++) cells were treated for 5 days prior to viral challenge with wild-type HIV-1 ( filled symbols ) or HIV-1Δ vif ( open symbols ). Subsequent viral replication was monitored by HIV-1 p24 Gag ELISA. C , CEMss cells were mock or ionomycin-stimulated for 5 days prior to infection. Ionomycin- treated cells then either continued to receive stimulation (++) or ionomycin was washed out of the medium after a 24-h incubation with HIV-1Δ vif virus (+). Spreading infection was assayed by p24 Gag ELISA. D , whole cell lysates were prepared from mock, transient ionomycin-, and chronic ionomycin-stimulated cultures on d1 of the replication curve, corresponding to day 6 of the stimulation time course. Additional whole cell lysates were prepared on day 10 of the replication curve, corresponding to day 16 of the stimulation protocol. Whole cell lysates from both days were probed for A3G and actin expression by Western blot. Unstim , unstimulated.

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Infection, Incubation, Western Blot

Related Articles

Electroporation:

Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis
Article Snippet: .. Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml−1 , Santa Cruz) and Ionomycin (1 μg ml−1 , Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910). .. Renilla activity was used to normalize transfection efficiency and Luciferase activity.

Luciferase:

Article Title: Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis
Article Snippet: .. Sixteen hours after electroporation, T cells were re-stimulated for 6 h with PMA (25 ng ml−1 , Santa Cruz) and Ionomycin (1 μg ml−1 , Santa Cruz) and then harvested and measured with the Dual Luciferase reporter system (Promega, catalogue number: E1910). .. Renilla activity was used to normalize transfection efficiency and Luciferase activity.

Isolation:

Article Title: Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo
Article Snippet: .. MP generation was induced by addition of thrombin receptor activating peptide-6 (TRAP–6, 20 μM, AnaSpec, Belgium) or ionomycin (40 μM, Santa Cruz Biotechnology, Inc., Germany) in the presence and absence of pPCI (300 nM) to isolated platelets (300,000/μl) in PBS. ..

Incubation:

Article Title: A Combination of Cytokines Rescues Highly Purified Leukemic CLL B-Cells from Spontaneous Apoptosis In Vitro
Article Snippet: .. The cells were incubated, from the initiation of cultures, with various molecules: IL-2 (50 ng/ml), IL-4 (50 ng/ml), IL-6 (50 ng/ml), IL-10 (10 ng/ml), IL-12 (10 ng/ml), IL-15 (10 ng/ml), IL-21(10 ng/ml), BAFF (10 ng/ml), APRIL (10 ng/ml), phorbol myristate acetate (PMA, 1 µg/ml) (Santa Cruz Biotechnology), phytohemagglutinin (PHA, 5 µg/ml) (Sigma-Aldrich), ionomycin (0.1 µg/ml) (Santa Cruz Biotechnology), lipopolysaccharide (LPS, 1 µg/ml), autologous patient serum (AS), heterologous serum (HS) from healthy donors, and a cocktail of cytokines (Cc) which included IL-2, IL-6, IL-10, IL-12, IL-15, IL-21, BAFF and APRIL. .. All cytokines were purchased from PeproTech EC.

Cell Culture:

Article Title: Adenosine-Induced NLRP11 in B Lymphoblasts Suppresses Human CD4+ T Helper Cell Responses
Article Snippet: .. Cells were cultured at an initial density of 2 × 106 cells/ml into 25 cm2 tissue flasks and kept overnight in the incubator, then stimulated with the following: 50 ng/ml lipopolysaccharide (LPS) from Salmonella enteritidis (InvivoGen); 0, 25, 50, 75, and 100 μ M adenosine (Sigma-Aldrich); 1 μ g/ml CD40L (CST); 50 ng/ml PMA (Santa Cruz); and 500 ng/ml ionomycin (Santa Cruz) or 50 μ M caffeine. ..

Calcium Assay:

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death
Article Snippet: .. Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany. .. EGTA (342-01314) was purchased from Dojindo, Kumamoto, Japan.

other:

Article Title: Genetic Indicators for Calcium Signaling studies in Toxoplasma gondii
Article Snippet: Ionomycin (Santa Cruz Biotechnology sc-3592).

Ethidium Homodimer Assay:

Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death
Article Snippet: .. Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany. .. EGTA (342-01314) was purchased from Dojindo, Kumamoto, Japan.

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    Santa Cruz Biotechnology pma ionomycin stimulation
    NFAT1 regulates chromatin architecture at the IL-9 promoter. A , nuclear extracts were prepared from Th9 cells differentiated from WT and NFAT1 −/− ( KO ) mice and stimulated with <t>PMA/ionomycin</t> for the indicated time. The nuclear levels of
    Pma Ionomycin Stimulation, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology ionomycin
    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM <t>ionomycin</t> (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
    Ionomycin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Santa Cruz Biotechnology
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    ionomycin - by Bioz Stars, 2020-09
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    Image Search Results


    NFAT1 regulates chromatin architecture at the IL-9 promoter. A , nuclear extracts were prepared from Th9 cells differentiated from WT and NFAT1 −/− ( KO ) mice and stimulated with PMA/ionomycin for the indicated time. The nuclear levels of

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor of Activated T Cells 1 (NFAT1)-induced Permissive Chromatin Modification Facilitates Nuclear Factor-?B (NF-?B)-mediated Interleukin-9 (IL-9) Transactivation *

    doi: 10.1074/jbc.M112.340356

    Figure Lengend Snippet: NFAT1 regulates chromatin architecture at the IL-9 promoter. A , nuclear extracts were prepared from Th9 cells differentiated from WT and NFAT1 −/− ( KO ) mice and stimulated with PMA/ionomycin for the indicated time. The nuclear levels of

    Article Snippet: Chromatins prepared from Th9 cells after PMA/ionomycin stimulation were immunoprecipitated using antibodies against RNA Pol II (Santa Cruz Biotechnology, Inc.), acetyl histone H3 (AcH3), acetyl histone H4 (AcH4), dimethyl lysine histone H3 (H3K4me2) (Upstate, Lake Placid, NY), p300 (Millipore, Billerica, MA), NFAT1 (Santa Cruz Biotechnology, Inc.), p65 (Abcam, Cambridge, MA), and rabbit IgG (Sigma-Aldrich).

    Techniques: Mouse Assay

    Physical association of NFAT1 with the IL-9 promoter. A , ChIP assay was performed with PMA/ionomycin-stimulated Th1 and Th9 cells using control IgG and NFAT1 antibody. The amounts of precipitated DNA were measured by quantitative PCR with primers specific

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor of Activated T Cells 1 (NFAT1)-induced Permissive Chromatin Modification Facilitates Nuclear Factor-?B (NF-?B)-mediated Interleukin-9 (IL-9) Transactivation *

    doi: 10.1074/jbc.M112.340356

    Figure Lengend Snippet: Physical association of NFAT1 with the IL-9 promoter. A , ChIP assay was performed with PMA/ionomycin-stimulated Th1 and Th9 cells using control IgG and NFAT1 antibody. The amounts of precipitated DNA were measured by quantitative PCR with primers specific

    Article Snippet: Chromatins prepared from Th9 cells after PMA/ionomycin stimulation were immunoprecipitated using antibodies against RNA Pol II (Santa Cruz Biotechnology, Inc.), acetyl histone H3 (AcH3), acetyl histone H4 (AcH4), dimethyl lysine histone H3 (H3K4me2) (Upstate, Lake Placid, NY), p300 (Millipore, Billerica, MA), NFAT1 (Santa Cruz Biotechnology, Inc.), p65 (Abcam, Cambridge, MA), and rabbit IgG (Sigma-Aldrich).

    Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    In vivo and in vitro binding of NF-κB (p65) to the IL-9 promoter. A , ChIP assay was performed with in vitro differentiated and PMA/ionomycin-stimulated Th1 and Th9 cells using control IgG and NF-κB (p65) antibodies. The amounts of precipitated

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Factor of Activated T Cells 1 (NFAT1)-induced Permissive Chromatin Modification Facilitates Nuclear Factor-?B (NF-?B)-mediated Interleukin-9 (IL-9) Transactivation *

    doi: 10.1074/jbc.M112.340356

    Figure Lengend Snippet: In vivo and in vitro binding of NF-κB (p65) to the IL-9 promoter. A , ChIP assay was performed with in vitro differentiated and PMA/ionomycin-stimulated Th1 and Th9 cells using control IgG and NF-κB (p65) antibodies. The amounts of precipitated

    Article Snippet: Chromatins prepared from Th9 cells after PMA/ionomycin stimulation were immunoprecipitated using antibodies against RNA Pol II (Santa Cruz Biotechnology, Inc.), acetyl histone H3 (AcH3), acetyl histone H4 (AcH4), dimethyl lysine histone H3 (H3K4me2) (Upstate, Lake Placid, NY), p300 (Millipore, Billerica, MA), NFAT1 (Santa Cruz Biotechnology, Inc.), p65 (Abcam, Cambridge, MA), and rabbit IgG (Sigma-Aldrich).

    Techniques: In Vivo, In Vitro, Binding Assay, Chromatin Immunoprecipitation

    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Journal: Scientific Reports

    Article Title: Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii

    doi: 10.1038/srep42746

    Figure Lengend Snippet: Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Article Snippet: Images were taken every 10 seconds for a total of 5 minutes and 2 μM ionomycin (Santa Cruz Biotechnology) was added after 2–4 time points were imaged.

    Techniques: Fluorescence, Standard Deviation, Microscopy

    Adenosine treatment induced NLRP11 expression at both the mRNA and protein levels in B lymphoblasts. (a) NLRP11 mRNA expression after 4 and 10 hours of stimulation with PMA/ionomycin (50 ng/500 ng/ml), CD40L (1 μ /ml), LPS (100 ng/ml), and adenosine (50 μ M) ( ∗ indicates significance at P

    Journal: Journal of Immunology Research

    Article Title: Adenosine-Induced NLRP11 in B Lymphoblasts Suppresses Human CD4+ T Helper Cell Responses

    doi: 10.1155/2020/1421795

    Figure Lengend Snippet: Adenosine treatment induced NLRP11 expression at both the mRNA and protein levels in B lymphoblasts. (a) NLRP11 mRNA expression after 4 and 10 hours of stimulation with PMA/ionomycin (50 ng/500 ng/ml), CD40L (1 μ /ml), LPS (100 ng/ml), and adenosine (50 μ M) ( ∗ indicates significance at P

    Article Snippet: Cells were cultured at an initial density of 2 × 106 cells/ml into 25 cm2 tissue flasks and kept overnight in the incubator, then stimulated with the following: 50 ng/ml lipopolysaccharide (LPS) from Salmonella enteritidis (InvivoGen); 0, 25, 50, 75, and 100 μ M adenosine (Sigma-Aldrich); 1 μ g/ml CD40L (CST); 50 ng/ml PMA (Santa Cruz); and 500 ng/ml ionomycin (Santa Cruz) or 50 μ M caffeine.

    Techniques: Expressing

    Ionomycin enhanced CGK733-induced PERK/CHOP activation and vesicular calcium sequestration A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.

    Journal: Oncotarget

    Article Title: PERK/CHOP contributes to the CGK733-induced vesicular calcium sequestration which is accompanied by non-apoptotic cell death

    doi:

    Figure Lengend Snippet: Ionomycin enhanced CGK733-induced PERK/CHOP activation and vesicular calcium sequestration A. The expression of PERK and CHOP were detected by Western blotting after cells were treated with CGK733 for 6 h in the presence or absence of 1 μM of ionomycin. B. MTS viability assays were performed after cells were treated with CGK733 for 24 h in the presence or absence of 1 μM of ionomycin. C. The vesicles were observed under a microscope after PK45-p and PK59 cells were treated with 10 μM of CGK733 in the presence or absence of 1 μM of ionomycin for 12 h and 9 h, respectively. D. Cal-520 fluorescence combined with bright field microscopy was performed after cells were exposed to 20 μM of CGK733 for 12 h in the presence or absence of 1 μM of ionomycin. Bars, SD; Black arrows indicate the observed vesicles; red arrows indicate the co-localization of calcium ions with the vesicles.

    Article Snippet: Anti-p-IREα (NB-100-2323) antibody was purchased from Novus Biologicals Inc, Littleton, CO. Control (sc-37007) and CHOP (sc-35437) siRNA were purchased from Santa Cruz Biotechnology Inc. CGK733 (sc202964), Ionomycin (sc-300835), z-VAD-fmk (sc-3067) and Necrostatin-1 (sc-200142) were purchased from Santa Cruz Biotechnology Inc. Cal-520 No wash Calcium Assay Kit was purchased from Abcam Inc. Apoptosis and Necrosis Detection Kit (EthD III) (PK-CA707-30018) was purchased from Promokine Inc, Heidelberg, Germany.

    Techniques: Activation Assay, Expressing, Western Blot, Microscopy, Fluorescence