Structured Review

Fisher Scientific ionomycin
CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and <t>ionomycin</t> in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
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Images

1) Product Images from "Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors"

Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

Journal: Scientific Reports

doi: 10.1038/s41598-018-29262-4

CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
Figure Legend Snippet: CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

Techniques Used: Purification, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

2) Product Images from "Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells"

Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

Journal: Biophysical Journal

doi: 10.1016/j.bpj.2018.05.018

Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

Techniques Used:

Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

Techniques Used:

Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

Techniques Used:

Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

Techniques Used:

Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

Techniques Used:

Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.
Figure Legend Snippet: Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

Techniques Used:

3) Product Images from "Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions"

Article Title: Localized Calcium Signaling and the Control of Coupling at Cx36 Gap Junctions

Journal: eNeuro

doi: 10.1523/ENEURO.0445-19.2020

Calcium responses of Cx36-GCaMP. A , Fluorescence response to application of 5 μ M ionomycin for 40 s (black bar). Data shown are means of 15 gap junctions in three experiments ± 95% confidence limits of the mean. Note that one of three experiments ended at 92 s, so the final 28 s show 10 gap junctions. B , Fluorescence response to application of 100 n M thapsigargin (gray bar). Data shown are means of five gap junctions in one experiment ± 95% confidence limits of the mean. C , Fluorescence response to application of 5 μ M ionomycin (black bar) in the presence of 100 n M thapsigargin. Shown are means of 15 gap junctions in three experiments ± 95% confidence limits of the mean. The mean response to ionomycin in control conditions is shown by the black line for reference. D , Fluorescence response to application of 5 μ M ionomycin (black bar) in the presence of 200 n M SEA 0400. Shown are means of 14 gap junctions in three experiments ± 95% confidence limits of the mean. The mean response to ionomycin in control conditions is shown by the black line for reference.
Figure Legend Snippet: Calcium responses of Cx36-GCaMP. A , Fluorescence response to application of 5 μ M ionomycin for 40 s (black bar). Data shown are means of 15 gap junctions in three experiments ± 95% confidence limits of the mean. Note that one of three experiments ended at 92 s, so the final 28 s show 10 gap junctions. B , Fluorescence response to application of 100 n M thapsigargin (gray bar). Data shown are means of five gap junctions in one experiment ± 95% confidence limits of the mean. C , Fluorescence response to application of 5 μ M ionomycin (black bar) in the presence of 100 n M thapsigargin. Shown are means of 15 gap junctions in three experiments ± 95% confidence limits of the mean. The mean response to ionomycin in control conditions is shown by the black line for reference. D , Fluorescence response to application of 5 μ M ionomycin (black bar) in the presence of 200 n M SEA 0400. Shown are means of 14 gap junctions in three experiments ± 95% confidence limits of the mean. The mean response to ionomycin in control conditions is shown by the black line for reference.

Techniques Used: Fluorescence

4) Product Images from "ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation"

Article Title: ATR kinase inhibitor AZD6738 potentiates CD8+ T cell–dependent antitumor activity following radiation

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI96519

AZD6738 attenuates coexpression of CD8 + T cell exhaustion markers and promotes CD8 + T cell effector function in CT26 tumors following radiation. ( A ) Representative contour plots depicting PD-1 and LAG-3 expression on splenic and tumor-infiltrating (TIL) CD8 + T cells for the designated treatment groups at day 12. ( B ) Quantitation of the percentage of TIL CD8 + T cells that coexpress PD-1 and LAG-3 or PD-1 and Tim-3 at day 12. Data from 3 independent experiments per time point, each with 1–3 mice per arm. n at day 12 = 6 per arm (7 IR). ( C ) Representative contour plots depicting IFN-γ and TNF-α expression by splenic and tumor-infiltrating (TIL) CD8 + T cells for the designated treatment groups following stimulation with PMA/ionomycin at day 12. ( D ) Quantitation of the percentage of TIL CD8 + T cells that elicit IFN-γ or IFN-γ and TNF-α following stimulation with PMA and ionomycin at days 9 and 12. Day 9 data from 1 experiment with the IR/AZD6738 + IR arms and vehicle/AZD6738 arms staggered and harvested/stained on separate days. n at day 9 = 5 per arm (4 IR). Day 12 data from 3 independent experiments, each with 1–3 mice per arm, with harvesting/staining for all arms performed on the same day within a given experiment. n at day 12 = 5 vehicle, 6 AZD6738, 6 IR, 7 AZD6738 + IR. ( B and D ) Mean and SD bars shown. * P
Figure Legend Snippet: AZD6738 attenuates coexpression of CD8 + T cell exhaustion markers and promotes CD8 + T cell effector function in CT26 tumors following radiation. ( A ) Representative contour plots depicting PD-1 and LAG-3 expression on splenic and tumor-infiltrating (TIL) CD8 + T cells for the designated treatment groups at day 12. ( B ) Quantitation of the percentage of TIL CD8 + T cells that coexpress PD-1 and LAG-3 or PD-1 and Tim-3 at day 12. Data from 3 independent experiments per time point, each with 1–3 mice per arm. n at day 12 = 6 per arm (7 IR). ( C ) Representative contour plots depicting IFN-γ and TNF-α expression by splenic and tumor-infiltrating (TIL) CD8 + T cells for the designated treatment groups following stimulation with PMA/ionomycin at day 12. ( D ) Quantitation of the percentage of TIL CD8 + T cells that elicit IFN-γ or IFN-γ and TNF-α following stimulation with PMA and ionomycin at days 9 and 12. Day 9 data from 1 experiment with the IR/AZD6738 + IR arms and vehicle/AZD6738 arms staggered and harvested/stained on separate days. n at day 9 = 5 per arm (4 IR). Day 12 data from 3 independent experiments, each with 1–3 mice per arm, with harvesting/staining for all arms performed on the same day within a given experiment. n at day 12 = 5 vehicle, 6 AZD6738, 6 IR, 7 AZD6738 + IR. ( B and D ) Mean and SD bars shown. * P

Techniques Used: Expressing, Quantitation Assay, Mouse Assay, Staining

5) Product Images from "IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell: Tumor promoting activity of IRF1"

Article Title: IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell: Tumor promoting activity of IRF1

Journal: Cancer immunology research

doi: 10.1158/2326-6066.CIR-18-0711

IRF1 deficient tumor cells affect infiltrating T cell functions ex vivo and are more vulnerable to CD8 + T-cell cytotoxicity in vitro . (A) Intracellular cytokine production of infiltrating CD8 + T cells in B16-F10 WT and IRF1-KO tumors (n=9, 8). Single cell suspension was prepared after tumor collection. The mixture of tumor cells and infiltrated lymphocytes was stimulated overnight with PMA and ionomycin, then cytokine secretion was blocked with GolgiPlug™ for 4 hrs. Representative flow-cytogram for intracellular cytokine expression is shown. For each type of cytokine expression, cumulative mean and SEM were plotted from twice repeated experiments, statistical significance was calculated by unpaired student t test, and presented as * P
Figure Legend Snippet: IRF1 deficient tumor cells affect infiltrating T cell functions ex vivo and are more vulnerable to CD8 + T-cell cytotoxicity in vitro . (A) Intracellular cytokine production of infiltrating CD8 + T cells in B16-F10 WT and IRF1-KO tumors (n=9, 8). Single cell suspension was prepared after tumor collection. The mixture of tumor cells and infiltrated lymphocytes was stimulated overnight with PMA and ionomycin, then cytokine secretion was blocked with GolgiPlug™ for 4 hrs. Representative flow-cytogram for intracellular cytokine expression is shown. For each type of cytokine expression, cumulative mean and SEM were plotted from twice repeated experiments, statistical significance was calculated by unpaired student t test, and presented as * P

Techniques Used: Ex Vivo, In Vitro, Flow Cytometry, Expressing

6) Product Images from "Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes"

Article Title: Rhesus rotavirus VP6 regulates ERK-dependent calcium influx in cholangiocytes

Journal: Virology

doi: 10.1016/j.virol.2016.09.014

Role of calcium inhibition on calcium influx (A) Fluo-4 loaded cholangiocytes were treated with increasing doses of Verapamil followed by infection with RRV. This resulted in a dose dependent reduction of calcium influx into the cells. (B) Similar results were observed when cholangiocytes were treated with Diltiazem HCl, a second calcium channel inhibitor. (C) In order to determine fluo-4 loading in the presence of ERK 1/2 inhibitor and Verapamil, cells were treated with 10mM Ionomycin to open calcium channels. No reduction in the ability to fluoresce in the presence of calcium was witnessed with Verapamil or ERK inhibitor. (D) Cholangiocytes were pre-treated with calcium-free media followed by infection with RRV in calcium-free media to determine if the fluo-4 was detecting intracellular calcium release or extracellular influx. No fluorescence was observed following infection with RRV or exposure to VP6 protein in calcium-free media. (E) Cholangiocytes treated with virus-depleted stock displayed no calcium influx, while those treated with the triple-layer particles of RRV reacted similarly to the RRV virus lysate. (* = p
Figure Legend Snippet: Role of calcium inhibition on calcium influx (A) Fluo-4 loaded cholangiocytes were treated with increasing doses of Verapamil followed by infection with RRV. This resulted in a dose dependent reduction of calcium influx into the cells. (B) Similar results were observed when cholangiocytes were treated with Diltiazem HCl, a second calcium channel inhibitor. (C) In order to determine fluo-4 loading in the presence of ERK 1/2 inhibitor and Verapamil, cells were treated with 10mM Ionomycin to open calcium channels. No reduction in the ability to fluoresce in the presence of calcium was witnessed with Verapamil or ERK inhibitor. (D) Cholangiocytes were pre-treated with calcium-free media followed by infection with RRV in calcium-free media to determine if the fluo-4 was detecting intracellular calcium release or extracellular influx. No fluorescence was observed following infection with RRV or exposure to VP6 protein in calcium-free media. (E) Cholangiocytes treated with virus-depleted stock displayed no calcium influx, while those treated with the triple-layer particles of RRV reacted similarly to the RRV virus lysate. (* = p

Techniques Used: Inhibition, Infection, Fluorescence

7) Product Images from "N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection"

Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006995

Increased viral gene expression upon m 6 A writer and reader depletion in TREX-BCBL-1 cells. (A) TREX-BCBL-1 cells were reactivated with dox for 72 hr, then total RNA was isolated and subjected to m 6 A RIP, followed by RT-qPCR for analysis of KSHV ORF50, GAPDH, and DICER. Values are displayed as fold change over input, normalized to the GAPDH negative control. Data are included from 3 biological replicates. (B-D) TREX-BCBL-1 cells were nucleofected with control scramble (scr) siRNAs or siRNAs specific to METTL3, YTHDF2, or YTHDF3, then lytically reactivated by treatment with dox, TPA and ionomycin for 72 hr. (B) Knockdown efficiency of the m 6 A proteins relative to the loading control GAPDH was visualized by western blot. (C) Levels of the KSHV ORF50 and ORF59 proteins were assayed by western blot in the control and m 6 . (D) ORF50 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs and reactivated for 36 hr with dox, TPA and ionomycin. (E) Viral supernatant from the reactivated control or m 6 A protein depleted TREX-BCBL-1 cells was transferred to uninfected HEK293T recipient cells, whereupon transfer of infection was quantified by RT-qPCR for the viral LANA transcript 48 hr post supernatant transfer. Individual data points represent 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values
Figure Legend Snippet: Increased viral gene expression upon m 6 A writer and reader depletion in TREX-BCBL-1 cells. (A) TREX-BCBL-1 cells were reactivated with dox for 72 hr, then total RNA was isolated and subjected to m 6 A RIP, followed by RT-qPCR for analysis of KSHV ORF50, GAPDH, and DICER. Values are displayed as fold change over input, normalized to the GAPDH negative control. Data are included from 3 biological replicates. (B-D) TREX-BCBL-1 cells were nucleofected with control scramble (scr) siRNAs or siRNAs specific to METTL3, YTHDF2, or YTHDF3, then lytically reactivated by treatment with dox, TPA and ionomycin for 72 hr. (B) Knockdown efficiency of the m 6 A proteins relative to the loading control GAPDH was visualized by western blot. (C) Levels of the KSHV ORF50 and ORF59 proteins were assayed by western blot in the control and m 6 . (D) ORF50 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs and reactivated for 36 hr with dox, TPA and ionomycin. (E) Viral supernatant from the reactivated control or m 6 A protein depleted TREX-BCBL-1 cells was transferred to uninfected HEK293T recipient cells, whereupon transfer of infection was quantified by RT-qPCR for the viral LANA transcript 48 hr post supernatant transfer. Individual data points represent 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Negative Control, Western Blot, Infection

8) Product Images from "N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection"

Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006995

Increased viral gene expression upon m 6 A writer and reader depletion in TREX-BCBL-1 cells. (A) TREX-BCBL-1 cells were reactivated with dox for 72 hr, then total RNA was isolated and subjected to m 6 A RIP, followed by RT-qPCR for analysis of KSHV ORF50, GAPDH, and DICER. Values are displayed as fold change over input, normalized to the GAPDH negative control. Data are included from 3 biological replicates. (B-D) TREX-BCBL-1 cells were nucleofected with control scramble (scr) siRNAs or siRNAs specific to METTL3, YTHDF2, or YTHDF3, then lytically reactivated by treatment with dox, TPA and ionomycin for 72 hr. (B) Knockdown efficiency of the m 6 A proteins relative to the loading control GAPDH was visualized by western blot. (C) Levels of the KSHV ORF50 and ORF59 proteins were assayed by western blot in the control and m 6 A protein-depleted samples. Additional replicates are shown in S4 Fig . (D) ORF50 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs and reactivated for 36 hr with dox, TPA and ionomycin. (E) Viral supernatant from the reactivated control or m 6 A protein depleted TREX-BCBL-1 cells was transferred to uninfected HEK293T recipient cells, whereupon transfer of infection was quantified by RT-qPCR for the viral LANA transcript 48 hr post supernatant transfer. Individual data points represent 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values
Figure Legend Snippet: Increased viral gene expression upon m 6 A writer and reader depletion in TREX-BCBL-1 cells. (A) TREX-BCBL-1 cells were reactivated with dox for 72 hr, then total RNA was isolated and subjected to m 6 A RIP, followed by RT-qPCR for analysis of KSHV ORF50, GAPDH, and DICER. Values are displayed as fold change over input, normalized to the GAPDH negative control. Data are included from 3 biological replicates. (B-D) TREX-BCBL-1 cells were nucleofected with control scramble (scr) siRNAs or siRNAs specific to METTL3, YTHDF2, or YTHDF3, then lytically reactivated by treatment with dox, TPA and ionomycin for 72 hr. (B) Knockdown efficiency of the m 6 A proteins relative to the loading control GAPDH was visualized by western blot. (C) Levels of the KSHV ORF50 and ORF59 proteins were assayed by western blot in the control and m 6 A protein-depleted samples. Additional replicates are shown in S4 Fig . (D) ORF50 gene expression was analyzed by RT-qPCR from cells treated with the indicated siRNAs and reactivated for 36 hr with dox, TPA and ionomycin. (E) Viral supernatant from the reactivated control or m 6 A protein depleted TREX-BCBL-1 cells was transferred to uninfected HEK293T recipient cells, whereupon transfer of infection was quantified by RT-qPCR for the viral LANA transcript 48 hr post supernatant transfer. Individual data points represent 3 independent experiments. Unpaired Student’s t test was used to evaluate the statistical difference between samples. Significance is shown for P values

Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Negative Control, Western Blot, Infection

9) Product Images from "MALT1 is required for EGFR induced NF-κB activation and contributes to EGFR-driven lung cancer progression"

Article Title: MALT1 is required for EGFR induced NF-κB activation and contributes to EGFR-driven lung cancer progression

Journal: Oncogene

doi: 10.1038/onc.2015.146

MALT1 serves as a scaffold protein by recruiting TRAF6 to IKK complex (a) MALT1-deficient cells (Malt1 −/− ), wild-type MALT1-reconstituted cells (Malt1 −/−WT ) and protease-deficient mutant reconstituted cells (Malt1 −/−C464A ) were stimulated with PMA and Ionomycin (50ng/ml; 100ng/ml) for indicated periods, respectively. Nuclear lysates were isolated and subjected to gel shift analysis for NF-κB activation. (b) A431 cells with a MALT1 knockdown (shMALT1), wild-type MALT1-reconstituted cells (shMALT1 WT ) and protease-deficient mutant reconstituted cells (shMALT1 C464A ) were stimulated with EGF (100ng/ml) or TNFα (10ng/ml) for indicated periods. Nuclear lysates were isolated and subjected to gel shift analysis for NF-κB activation. (c) MALT1-reconstituted cells were stimulated with EGF (100ng/ml) for indicated time and MALT1-Flag was immunoprecipitated (IP) by anti-Flag conjugated beads. The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (d) MALT1-reconstituted cells were stimulated with EGF (100ng/ml) and TNFa, respectively. MALT1-Flag was immunoprecipitated (IP) by anti-Flag conjugated beads. The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (e) Control (shCtl) or MALT1-silenced (shMALT1) A431 cells were either unstimulated or stimulated with EGF (100ng/ml) for 15 minutes and IKKg was immunoprecipitated (IP). The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (f) A431 cells with a TRAF6 knockdown (shTRAF6) and control cells (shCtl) were stimulated with EGF (100ng/ml), PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated time points, respectively. NF-κB activation and Oct-1 (loading control) levels were determined by the gel shift assay.
Figure Legend Snippet: MALT1 serves as a scaffold protein by recruiting TRAF6 to IKK complex (a) MALT1-deficient cells (Malt1 −/− ), wild-type MALT1-reconstituted cells (Malt1 −/−WT ) and protease-deficient mutant reconstituted cells (Malt1 −/−C464A ) were stimulated with PMA and Ionomycin (50ng/ml; 100ng/ml) for indicated periods, respectively. Nuclear lysates were isolated and subjected to gel shift analysis for NF-κB activation. (b) A431 cells with a MALT1 knockdown (shMALT1), wild-type MALT1-reconstituted cells (shMALT1 WT ) and protease-deficient mutant reconstituted cells (shMALT1 C464A ) were stimulated with EGF (100ng/ml) or TNFα (10ng/ml) for indicated periods. Nuclear lysates were isolated and subjected to gel shift analysis for NF-κB activation. (c) MALT1-reconstituted cells were stimulated with EGF (100ng/ml) for indicated time and MALT1-Flag was immunoprecipitated (IP) by anti-Flag conjugated beads. The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (d) MALT1-reconstituted cells were stimulated with EGF (100ng/ml) and TNFa, respectively. MALT1-Flag was immunoprecipitated (IP) by anti-Flag conjugated beads. The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (e) Control (shCtl) or MALT1-silenced (shMALT1) A431 cells were either unstimulated or stimulated with EGF (100ng/ml) for 15 minutes and IKKg was immunoprecipitated (IP). The IP samples and lysates were analyzed by immunoblotting using the indicated antibodies. (f) A431 cells with a TRAF6 knockdown (shTRAF6) and control cells (shCtl) were stimulated with EGF (100ng/ml), PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated time points, respectively. NF-κB activation and Oct-1 (loading control) levels were determined by the gel shift assay.

Techniques Used: Mutagenesis, Isolation, Electrophoretic Mobility Shift Assay, Activation Assay, Immunoprecipitation

MALT1 is selectively involved in EGFR-induced NF-κB activation (a) A431 cells with a MALT1 knockdown (shMALT1) and control cells (shCtl) were stimulated with EGF (100ng/ml), PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated periods. NF-κB activation and Oct-1 (loading control) levels were determined by the gel shift assay. (b) MEFs from Malt1 +/− and Malt1 −/− embryos were isolated. Early-passage (P1) MEFs were stimulated with PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated periods. NF-κB activation and Oct-1 levels were determined by the gel shift assay. (c) Nuclear lysates from A431 and A431-shMATL1 cells were analyzed by immunoblotting using indicated antibodies. (d) shMALT1 and shGFP (control) A431 cells were stimulated with EGF (100ng/ml) for indicated periods. Cell lysates were analyzed by immunoblotting using indicated antibodies.
Figure Legend Snippet: MALT1 is selectively involved in EGFR-induced NF-κB activation (a) A431 cells with a MALT1 knockdown (shMALT1) and control cells (shCtl) were stimulated with EGF (100ng/ml), PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated periods. NF-κB activation and Oct-1 (loading control) levels were determined by the gel shift assay. (b) MEFs from Malt1 +/− and Malt1 −/− embryos were isolated. Early-passage (P1) MEFs were stimulated with PMA and Ionomycin (50ng/ml; 100ng/ml) or TNFα (10ng/ml) for indicated periods. NF-κB activation and Oct-1 levels were determined by the gel shift assay. (c) Nuclear lysates from A431 and A431-shMATL1 cells were analyzed by immunoblotting using indicated antibodies. (d) shMALT1 and shGFP (control) A431 cells were stimulated with EGF (100ng/ml) for indicated periods. Cell lysates were analyzed by immunoblotting using indicated antibodies.

Techniques Used: Activation Assay, Electrophoretic Mobility Shift Assay, Isolation

10) Product Images from "Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation"

Article Title: Identification of Novel Death-Associated Protein Kinase 2 Interaction Partners by Proteomic Screening Coupled with Bimolecular Fluorescence Complementation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.00515-15

Confirmation of novel DAPK2 interaction partners α-actinin-1 and 14-3-3-β by BiFC. (A) DAPK2 interacts with α-actinin-1 and 14-3-3-β under steady-state conditions. A significant increase in fluorescence could be observed after coexpression of split YFP constructs YFP N -DAPK2 and α-actinin-1–YFP C and of YFP N -DAPK2 and 14-3-3-β–YFP C in HEK-293T cells. Ectopic expression of split YFP constructs YFP N -DAPK2 and G6PD-YFP C or SIPA-YFP C did not increase fluorescence above background levels ( n = 8). (B) Increased intracellular Ca 2+ levels due to ionomycin treatment augments association of DAPK2 with α-actinin-1. Treatment of HEK-293T cells with 1 μM ionomycin for 1 h resulted in increased fluorescence of cells ectopically expressing split YFP construct YFP N -DAPK2 and α-actinin-1–YFP C and also YFP C -DAPK2 and CaM-YFP N , measured by flow cytometry 24 h later ( n ≥ 3). (C) Direct interaction of DAPK2 with α-actinin-1 and 14-3-3-β. Recombinant human α-actinin-1 (35 nM) and 14-3-3-β (35 and 70 nM) were coated onto ELISA plates and incubated with 20 nM recombinant human DAPK2. Absolute absorbance values of noncoated wells were subtracted from absolute absorbance values of coated wells, resulting in relative absorbance, shown in this graph ( n = 3).
Figure Legend Snippet: Confirmation of novel DAPK2 interaction partners α-actinin-1 and 14-3-3-β by BiFC. (A) DAPK2 interacts with α-actinin-1 and 14-3-3-β under steady-state conditions. A significant increase in fluorescence could be observed after coexpression of split YFP constructs YFP N -DAPK2 and α-actinin-1–YFP C and of YFP N -DAPK2 and 14-3-3-β–YFP C in HEK-293T cells. Ectopic expression of split YFP constructs YFP N -DAPK2 and G6PD-YFP C or SIPA-YFP C did not increase fluorescence above background levels ( n = 8). (B) Increased intracellular Ca 2+ levels due to ionomycin treatment augments association of DAPK2 with α-actinin-1. Treatment of HEK-293T cells with 1 μM ionomycin for 1 h resulted in increased fluorescence of cells ectopically expressing split YFP construct YFP N -DAPK2 and α-actinin-1–YFP C and also YFP C -DAPK2 and CaM-YFP N , measured by flow cytometry 24 h later ( n ≥ 3). (C) Direct interaction of DAPK2 with α-actinin-1 and 14-3-3-β. Recombinant human α-actinin-1 (35 nM) and 14-3-3-β (35 and 70 nM) were coated onto ELISA plates and incubated with 20 nM recombinant human DAPK2. Absolute absorbance values of noncoated wells were subtracted from absolute absorbance values of coated wells, resulting in relative absorbance, shown in this graph ( n = 3).

Techniques Used: Bimolecular Fluorescence Complementation Assay, Fluorescence, Construct, Expressing, Chick Chorioallantoic Membrane Assay, Flow Cytometry, Cytometry, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation

Related Articles

Flow Cytometry:

Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors
Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

Isolation:

Article Title: IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell: Tumor promoting activity of IRF1
Article Snippet: .. For intracellular cytokine staining, isolated tumor cells and lymph node cells were cultured in 96-well plates with R10 media containing 100 ng/mL PMA and 500 ng/mL Ionomycin (Fisher Scientific, Hampton, NH) overnight. .. The next day, cells were incubated with BD GolgiPlug Protein Transport Inhibitor (1:1000) for 4 h. After incubation, cells were harvested for intracellular cytokine staining as described above.

Cytometry:

Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors
Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

Cell Culture:

Article Title: IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell: Tumor promoting activity of IRF1
Article Snippet: .. For intracellular cytokine staining, isolated tumor cells and lymph node cells were cultured in 96-well plates with R10 media containing 100 ng/mL PMA and 500 ng/mL Ionomycin (Fisher Scientific, Hampton, NH) overnight. .. The next day, cells were incubated with BD GolgiPlug Protein Transport Inhibitor (1:1000) for 4 h. After incubation, cells were harvested for intracellular cytokine staining as described above.

Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors
Article Snippet: .. Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours. .. Cells were then fixed, permeabilized and stained with fluorescent-labelled antibodies for CD4, FoxP3, IL-17 and IL-22 using FoxP3 Fix/Perm Buffer Set (Biolegend).

Concentration Assay:

Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells
Article Snippet: .. An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific). ..

Expressing:

Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
Article Snippet: .. Lytic reactivation of TREX-BCBL-1 cells was achieved by treatment of 7x105 cells/ml with 20 ng/ml 2-O-tetradecanoylphorbol-13-acetate (TPA, Sigma), 1 μg/ml doxycycline (BD Biosciences), and 500 ng/ml ionomycin (Fisher Scientific) for 72 hr (western Blot blots for viral gene expression), or for 120 hr (supernatant transfer experiments). .. siRNA experiments For iSLK.219 cells, 100 pmol of siRNA was reverse transfected into 5x105 cells plated in a 6-well dish using Lipofectamine RNAimax (Life Technologies).

Staining:

Article Title: IRF1 inhibits antitumor immunity through the upregulation of PD-L1 in the tumor cell: Tumor promoting activity of IRF1
Article Snippet: .. For intracellular cytokine staining, isolated tumor cells and lymph node cells were cultured in 96-well plates with R10 media containing 100 ng/mL PMA and 500 ng/mL Ionomycin (Fisher Scientific, Hampton, NH) overnight. .. The next day, cells were incubated with BD GolgiPlug Protein Transport Inhibitor (1:1000) for 4 h. After incubation, cells were harvested for intracellular cytokine staining as described above.

Western Blot:

Article Title: N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection
Article Snippet: .. Lytic reactivation of TREX-BCBL-1 cells was achieved by treatment of 7x105 cells/ml with 20 ng/ml 2-O-tetradecanoylphorbol-13-acetate (TPA, Sigma), 1 μg/ml doxycycline (BD Biosciences), and 500 ng/ml ionomycin (Fisher Scientific) for 72 hr (western Blot blots for viral gene expression), or for 120 hr (supernatant transfer experiments). .. siRNA experiments For iSLK.219 cells, 100 pmol of siRNA was reverse transfected into 5x105 cells plated in a 6-well dish using Lipofectamine RNAimax (Life Technologies).

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  • 93
    Fisher Scientific ionomycin
    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and <t>ionomycin</t> in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).
    Ionomycin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ionomycin/product/Fisher Scientific
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    Fisher Scientific pma ionomycin ex vivo stimulation
    Combination of ALX148 and anti-PD-1 reduces suppressive tumor microenvironment and activates adaptive immune response in CT26 tumor. (A) Percent FOXP3 + CD25 + in tumor. (B) Percent Ki67 + Tregs in tumor. (C) Percent AH1-tet + CD8 + T cells. Following antibody treatment, ex vivo tumor-derived single cell suspension stimulation with AH1 peptide at 10 μg/mL. (D) Percent intracellular IFNγ expressing CD8 + T cells (left panel) and percent IFNγ + of AH1-tet + CD8 + T cells (right panel). Following antibody treatment, ex vivo tumor-derived single cell suspension were stimulated in the presence of <t>PMA/ionomycin</t> or AH1 peptide at 10 μg/mL. Results are representative of 1–3 independent experiments of n = 5–6 mice/group.
    Pma Ionomycin Ex Vivo Stimulation, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific ionomycin calcium salt
    Extracellular calcium regulates MAPKs and C/EBPβ activity independently of intracellular calcium. ( A ) C/EBPδ and C/EBPβ expression normalized to TBP during the first 48 hours of differentiation in 1.8 mM calcium (control), 10 mM magnesium, 10 mM calcium and calcium free medium (n = 6). ( B ) Western Blot for phospho- (Thr235)/total C/EBPβ as well as phospho-/total ERK and phospho- (Ser473)/total AKT during the first 48 hours of differentiation under 1.8 mM calcium (control), 10 mM magnesium (Mg 2+ ) and 10 mM calcium (Ca 2+ ) conditions with β-Actin as loading control. ( C ) Western Blot for phospho- and total ERK and β-Actin as loading control of the 8 day time course under control (1.8 mM calcium), 10 mM calcium and 10 mM magnesium conditions as well as control and calcium free conditions for the indicated duration. ( D ) Relative increase of intracellular calcium in brown preadipocytes maintained in 1.8 mM calcium (control) or 10 mM calcium directly upon and 10 min after injection of IBMX or induction mix shown as percent increase of fluorescence to basal level. Cells were loaded with the calcium binding fluorophore Fluo-4 (4 µM) and fluorescence was recorded at Ex/Em = 485/520 in orbital averaging (n = 3 with 3–4 replicates each). ( E ) PPARγ expression normalized on TBP in preadipocytes (day 0) and differentiated cells at day 8 treated day 0–8 with 1.8 mM calcium (Control day 0–8) or 10 mM calcium (10 mM Ca 2+ day 0–8) supplemented with DMSO, <t>ionomycin</t> (2 µM) and/or the calcineurin inhibitor FK506 (1 µM) (n = 3). ( F ) Western Blot for phospho-/total ERK in preadipocytes (0 h) and induced preadipocytes (18 h) with different concentrations of the MEK inhibitor PD0325901. ( G ) PPARγ expression as percent of control in differentiated adipocytes treated with 1.8 mM calcium (control) or 10 mM calcium +/− the MEK inhibitor PD0325901 (250 nM) for 0–8 days (n = 5). For ( A,D,E and G ) data are shown as mean ± SEM, *p
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    CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Journal: Scientific Reports

    Article Title: Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

    doi: 10.1038/s41598-018-29262-4

    Figure Lengend Snippet: CD4 T cell phenotype in serum-free RPMI. Human peripheral blood naïve CD4 T cells were purified by negative selection and cultured for five days with plate-bound anti-CD3 and soluble anti-CD28. Additional treatment groups included Th17-inducing cytokines (IL-1β, IL-6, IL-23, TGF-β), AhR agonist (FICZ, 200 nM) or AhR antagonist (CH223191, 4 uM), as indicated. On day 5, T cells were restimulated with PMA and ionomycin in the presence of brefeldin A for 4 hours, stained with fluorescent-labelled antibodies and analyzed by flow cytometry. Shown are representative dot plots of CD4 versus FoxP3 on live cells (top) or IL-22 versus IL-17 on CD4 + FoxP3 − cells (bottom).

    Article Snippet: Flow cytometry Following T cell culture, cells were resuspended in media containing phorbol 12-myristate 13-acetate (PMA, 50 ng/mL, Fisher Scientific), ionomycin (750 ng/mL, Fisher Scientific) and brefeldin A (5 ug/mL, Tocris Bioscience) for 4 hours.

    Techniques: Purification, Selection, Cell Culture, Staining, Flow Cytometry, Cytometry

    Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Combination of ALX148 and anti-PD-1 reduces suppressive tumor microenvironment and activates adaptive immune response in CT26 tumor. (A) Percent FOXP3 + CD25 + in tumor. (B) Percent Ki67 + Tregs in tumor. (C) Percent AH1-tet + CD8 + T cells. Following antibody treatment, ex vivo tumor-derived single cell suspension stimulation with AH1 peptide at 10 μg/mL. (D) Percent intracellular IFNγ expressing CD8 + T cells (left panel) and percent IFNγ + of AH1-tet + CD8 + T cells (right panel). Following antibody treatment, ex vivo tumor-derived single cell suspension were stimulated in the presence of PMA/ionomycin or AH1 peptide at 10 μg/mL. Results are representative of 1–3 independent experiments of n = 5–6 mice/group.

    Journal: PLoS ONE

    Article Title: ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile

    doi: 10.1371/journal.pone.0201832

    Figure Lengend Snippet: Combination of ALX148 and anti-PD-1 reduces suppressive tumor microenvironment and activates adaptive immune response in CT26 tumor. (A) Percent FOXP3 + CD25 + in tumor. (B) Percent Ki67 + Tregs in tumor. (C) Percent AH1-tet + CD8 + T cells. Following antibody treatment, ex vivo tumor-derived single cell suspension stimulation with AH1 peptide at 10 μg/mL. (D) Percent intracellular IFNγ expressing CD8 + T cells (left panel) and percent IFNγ + of AH1-tet + CD8 + T cells (right panel). Following antibody treatment, ex vivo tumor-derived single cell suspension were stimulated in the presence of PMA/ionomycin or AH1 peptide at 10 μg/mL. Results are representative of 1–3 independent experiments of n = 5–6 mice/group.

    Article Snippet: For PMA/ionomycin ex-vivo stimulation, cells were plated at 1–1.5 x 106 cells/well in complete RPMI comprised of 10% heat-inactivated FBS, 2% Pen/Strep, 1% Glutamax, 1% MEAA, 1% sodium pyruvate, 25 mM HEPES and 5μM ß-mercaptoethanol supplemented with 50 ng/mL PMA (Fisher Scientific) and 1μM ionomycin (Sigma) in the presence of Golgi-Stop for at least 4 hours at 37°C, 5% CO2 .

    Techniques: Ex Vivo, Derivative Assay, Expressing, Mouse Assay

    ALX148 activates adaptive immune response in the spleen. Spleen of CT26 tumor-bearing mice, 10 days post single-dose of PBS, ALX148, anti-PD-1 or ALX148 in combination with one dose of anti-PD-1. (A-B) Percent effector memory (CD44 + CD62L - ) and central memory (CD44 + CD62L + ) CD4 + T cells. (C-D) Percent effector memory and central memory CD8 + T cells. (E) KLRG1 expression on CD8 + T cells. (F) Percent intracellular IFNγ expressing CD8 + T cells following ex vivo stimulation of splenocytes with PMA/ionomycin and Golgi-Stop for 4 hours. (G) Percent intracellular granzyme B expressing CD8 + T cells. (H) Percent CD8 + AH1-tet + T cells following ex vivo stimulation of splenocytes with 10 μg/mL AH1 peptide for 4 hours. Results are representative of 1–3 independent experiments of n = 4–6 mice/group. **p

    Journal: PLoS ONE

    Article Title: ALX148 blocks CD47 and enhances innate and adaptive antitumor immunity with a favorable safety profile

    doi: 10.1371/journal.pone.0201832

    Figure Lengend Snippet: ALX148 activates adaptive immune response in the spleen. Spleen of CT26 tumor-bearing mice, 10 days post single-dose of PBS, ALX148, anti-PD-1 or ALX148 in combination with one dose of anti-PD-1. (A-B) Percent effector memory (CD44 + CD62L - ) and central memory (CD44 + CD62L + ) CD4 + T cells. (C-D) Percent effector memory and central memory CD8 + T cells. (E) KLRG1 expression on CD8 + T cells. (F) Percent intracellular IFNγ expressing CD8 + T cells following ex vivo stimulation of splenocytes with PMA/ionomycin and Golgi-Stop for 4 hours. (G) Percent intracellular granzyme B expressing CD8 + T cells. (H) Percent CD8 + AH1-tet + T cells following ex vivo stimulation of splenocytes with 10 μg/mL AH1 peptide for 4 hours. Results are representative of 1–3 independent experiments of n = 4–6 mice/group. **p

    Article Snippet: For PMA/ionomycin ex-vivo stimulation, cells were plated at 1–1.5 x 106 cells/well in complete RPMI comprised of 10% heat-inactivated FBS, 2% Pen/Strep, 1% Glutamax, 1% MEAA, 1% sodium pyruvate, 25 mM HEPES and 5μM ß-mercaptoethanol supplemented with 50 ng/mL PMA (Fisher Scientific) and 1μM ionomycin (Sigma) in the presence of Golgi-Stop for at least 4 hours at 37°C, 5% CO2 .

    Techniques: Mouse Assay, Expressing, Ex Vivo

    Extracellular calcium regulates MAPKs and C/EBPβ activity independently of intracellular calcium. ( A ) C/EBPδ and C/EBPβ expression normalized to TBP during the first 48 hours of differentiation in 1.8 mM calcium (control), 10 mM magnesium, 10 mM calcium and calcium free medium (n = 6). ( B ) Western Blot for phospho- (Thr235)/total C/EBPβ as well as phospho-/total ERK and phospho- (Ser473)/total AKT during the first 48 hours of differentiation under 1.8 mM calcium (control), 10 mM magnesium (Mg 2+ ) and 10 mM calcium (Ca 2+ ) conditions with β-Actin as loading control. ( C ) Western Blot for phospho- and total ERK and β-Actin as loading control of the 8 day time course under control (1.8 mM calcium), 10 mM calcium and 10 mM magnesium conditions as well as control and calcium free conditions for the indicated duration. ( D ) Relative increase of intracellular calcium in brown preadipocytes maintained in 1.8 mM calcium (control) or 10 mM calcium directly upon and 10 min after injection of IBMX or induction mix shown as percent increase of fluorescence to basal level. Cells were loaded with the calcium binding fluorophore Fluo-4 (4 µM) and fluorescence was recorded at Ex/Em = 485/520 in orbital averaging (n = 3 with 3–4 replicates each). ( E ) PPARγ expression normalized on TBP in preadipocytes (day 0) and differentiated cells at day 8 treated day 0–8 with 1.8 mM calcium (Control day 0–8) or 10 mM calcium (10 mM Ca 2+ day 0–8) supplemented with DMSO, ionomycin (2 µM) and/or the calcineurin inhibitor FK506 (1 µM) (n = 3). ( F ) Western Blot for phospho-/total ERK in preadipocytes (0 h) and induced preadipocytes (18 h) with different concentrations of the MEK inhibitor PD0325901. ( G ) PPARγ expression as percent of control in differentiated adipocytes treated with 1.8 mM calcium (control) or 10 mM calcium +/− the MEK inhibitor PD0325901 (250 nM) for 0–8 days (n = 5). For ( A,D,E and G ) data are shown as mean ± SEM, *p

    Journal: Scientific Reports

    Article Title: Extracellular calcium modulates brown adipocyte differentiation and identity

    doi: 10.1038/s41598-017-09025-3

    Figure Lengend Snippet: Extracellular calcium regulates MAPKs and C/EBPβ activity independently of intracellular calcium. ( A ) C/EBPδ and C/EBPβ expression normalized to TBP during the first 48 hours of differentiation in 1.8 mM calcium (control), 10 mM magnesium, 10 mM calcium and calcium free medium (n = 6). ( B ) Western Blot for phospho- (Thr235)/total C/EBPβ as well as phospho-/total ERK and phospho- (Ser473)/total AKT during the first 48 hours of differentiation under 1.8 mM calcium (control), 10 mM magnesium (Mg 2+ ) and 10 mM calcium (Ca 2+ ) conditions with β-Actin as loading control. ( C ) Western Blot for phospho- and total ERK and β-Actin as loading control of the 8 day time course under control (1.8 mM calcium), 10 mM calcium and 10 mM magnesium conditions as well as control and calcium free conditions for the indicated duration. ( D ) Relative increase of intracellular calcium in brown preadipocytes maintained in 1.8 mM calcium (control) or 10 mM calcium directly upon and 10 min after injection of IBMX or induction mix shown as percent increase of fluorescence to basal level. Cells were loaded with the calcium binding fluorophore Fluo-4 (4 µM) and fluorescence was recorded at Ex/Em = 485/520 in orbital averaging (n = 3 with 3–4 replicates each). ( E ) PPARγ expression normalized on TBP in preadipocytes (day 0) and differentiated cells at day 8 treated day 0–8 with 1.8 mM calcium (Control day 0–8) or 10 mM calcium (10 mM Ca 2+ day 0–8) supplemented with DMSO, ionomycin (2 µM) and/or the calcineurin inhibitor FK506 (1 µM) (n = 3). ( F ) Western Blot for phospho-/total ERK in preadipocytes (0 h) and induced preadipocytes (18 h) with different concentrations of the MEK inhibitor PD0325901. ( G ) PPARγ expression as percent of control in differentiated adipocytes treated with 1.8 mM calcium (control) or 10 mM calcium +/− the MEK inhibitor PD0325901 (250 nM) for 0–8 days (n = 5). For ( A,D,E and G ) data are shown as mean ± SEM, *p

    Article Snippet: For inhibitor experiments the compounds were kept as 1 mM (FK506, Ionommycin) and 10 mM (PD325901) stock solutions in DMSO at −20 °C and supplemented to the medium in the appropriate dilution: 2 µM Ionomycin calcium salt (Fisher Scientific), 1 µM calcineurin inhibitor FK506 (Tacrolismus - Abcam), 250 nM MEK inhibitor PD0325901 (Sigma).

    Techniques: Activity Assay, Expressing, Western Blot, Injection, Fluorescence, Binding Assay