Structured Review

Merck & Co ionomycin
NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or <t>ionomycin</t> as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.
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Images

1) Product Images from "Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4"

Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01573

NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.
Figure Legend Snippet: NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.

Techniques Used: Fluorescence, Staining, Immunofluorescence, Microscopy

Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.
Figure Legend Snippet: Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.

Techniques Used: Mouse Assay, Staining, Confocal Microscopy, Infection

Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.
Figure Legend Snippet: Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.

Techniques Used: Isolation, Mouse Assay, Staining, Confocal Microscopy, Electron Microscopy

2) Product Images from "Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect, et al. Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect"

Article Title: Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect, et al. Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect

Journal: CNS Neuroscience & Therapeutics

doi: 10.1111/cns.13062

TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P
Figure Legend Snippet: TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

Techniques Used: Transfection, Western Blot, Cell Culture, Expressing

3) Product Images from "Allergy Testing and Drug Screening on an ITO-Coated Lab-on-a-Disc"

Article Title: Allergy Testing and Drug Screening on an ITO-Coated Lab-on-a-Disc

Journal: Micromachines

doi: 10.3390/mi7030038

AO was released from the granules of live KU-812 cells by NH 4 Cl and ionomycin with PMA. KU-812 cells (1 × 10 6 /mL) were loaded with AO (0.5 μg/mL) overnight at 37 °C, 5% CO 2 . During incubation, AO was actively accumulated in the acidic granules. After loading, cells in suspension were placed in a confocal dish coated with polylysine. After stabilization, cells were activated with NH 4 Cl (10 mM) ( a ) or ionomycin (1.0 μM) with PMA (20 nM) ( b ) at time zero. Bright field (BF), AO red fluorescence, AO green fluorescence images were acquired from a confocal microscope at the time as indicated. Scale bar represents the cell dimension; after stimulation, AO released in the supernatant was transferred to another Eppendorff to label DNA in the chemically fixed HepG2 cells. Histograms from left to right ( x -axis in log scale): fixed HepG2 cells in PBS; fixed HepG2 with the supernatant from untreated KU-812 cells; fixed HepG2 with the supernatant from KU-812 cells treated with ionomycin (1.0 μM) and PMA (20 nM) for 20 min at 37 °C, 5% CO 2 ( c ).
Figure Legend Snippet: AO was released from the granules of live KU-812 cells by NH 4 Cl and ionomycin with PMA. KU-812 cells (1 × 10 6 /mL) were loaded with AO (0.5 μg/mL) overnight at 37 °C, 5% CO 2 . During incubation, AO was actively accumulated in the acidic granules. After loading, cells in suspension were placed in a confocal dish coated with polylysine. After stabilization, cells were activated with NH 4 Cl (10 mM) ( a ) or ionomycin (1.0 μM) with PMA (20 nM) ( b ) at time zero. Bright field (BF), AO red fluorescence, AO green fluorescence images were acquired from a confocal microscope at the time as indicated. Scale bar represents the cell dimension; after stimulation, AO released in the supernatant was transferred to another Eppendorff to label DNA in the chemically fixed HepG2 cells. Histograms from left to right ( x -axis in log scale): fixed HepG2 cells in PBS; fixed HepG2 with the supernatant from untreated KU-812 cells; fixed HepG2 with the supernatant from KU-812 cells treated with ionomycin (1.0 μM) and PMA (20 nM) for 20 min at 37 °C, 5% CO 2 ( c ).

Techniques Used: Incubation, Fluorescence, Microscopy

AO are trapped in the granules of basophils and exhibit red shift at high concentrations. Weakly basic AO molecules are selectively accumulated within granules as a result of the intragranular acidic pH. Upon entering the basophilic granules, AO dyes become protonated and are trapped inside the organelles ( a , right). The resulting high concentrations of AO cause a red shift, thereby giving red fluorescence after excitation at 488 nm. When challenged with NH 4 Cl, NH 4 Cl decomposes into ammonia in solution that passes through membranes easily, accumulating in the basophilic granules and promoting granular alkalinization to release AO ( a , left). When challenged with ionomycin, the intracellular Ca 2+ ion level ([Ca 2+ ] i ) increases, which triggers degranulation and releases AO externally ( a , right). The released AO dyes then bind to the nuclear DNA and generate strong green fluorescence ( a ). To demonstrate the phenomenon of red shift, AO was dissolved at the concentration (mM) as indicated (0.1 (open circle), 1 (solid circle), 10 (open square), 100 (solid square)). Emission spectrum of AO at each concentration was scanned from 500 to 700 nm with excitation at 488 nm; emission peak from the solutions of various AO concentrations was normalized to the same level for comparison because the quantum efficiency of the AO red fluorescence is much weaker than that of the green one. High concentrations of AO (≥10 mM) show a red shift ( b ).
Figure Legend Snippet: AO are trapped in the granules of basophils and exhibit red shift at high concentrations. Weakly basic AO molecules are selectively accumulated within granules as a result of the intragranular acidic pH. Upon entering the basophilic granules, AO dyes become protonated and are trapped inside the organelles ( a , right). The resulting high concentrations of AO cause a red shift, thereby giving red fluorescence after excitation at 488 nm. When challenged with NH 4 Cl, NH 4 Cl decomposes into ammonia in solution that passes through membranes easily, accumulating in the basophilic granules and promoting granular alkalinization to release AO ( a , left). When challenged with ionomycin, the intracellular Ca 2+ ion level ([Ca 2+ ] i ) increases, which triggers degranulation and releases AO externally ( a , right). The released AO dyes then bind to the nuclear DNA and generate strong green fluorescence ( a ). To demonstrate the phenomenon of red shift, AO was dissolved at the concentration (mM) as indicated (0.1 (open circle), 1 (solid circle), 10 (open square), 100 (solid square)). Emission spectrum of AO at each concentration was scanned from 500 to 700 nm with excitation at 488 nm; emission peak from the solutions of various AO concentrations was normalized to the same level for comparison because the quantum efficiency of the AO red fluorescence is much weaker than that of the green one. High concentrations of AO (≥10 mM) show a red shift ( b ).

Techniques Used: Fluorescence, Concentration Assay

4) Product Images from "Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway"

Article Title: Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006819

Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p
Figure Legend Snippet: Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p

Techniques Used: Expressing

Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p
Figure Legend Snippet: Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p

Techniques Used: Infection

5) Product Images from "Serine 165 Phosphorylation of SHARPIN regulates the Activation of NF-κB"

Article Title: Serine 165 Phosphorylation of SHARPIN regulates the Activation of NF-κB

Journal: bioRxiv

doi: 10.1101/2020.07.02.184085

SHARPIN is a Phosphoprotein. A Jurkat cells were treated with or without 50 nM Calyculin A (Cal A) for 30 min prior lysis. Cell lysates from primary human CD4 + or CD8 + T lymphocytes and Jurkat cells were treated with lambda phosphatase (λPP) as indicated, and subjected to Western blotting analysis. Green and white arrowheads indicate phosphorylated and unphosphorylated SHARPIN, respectively. Molecular weight markers ( M r ) are indicated. B Densitometric analysis of the ratio between phosphorylated species of SHARPIN and SHARPIN in primary human T CD4 + cells, primary human T CD8 + T cells, Jurkat T cells, a panel of patient-derived diffuse large B-cell lymphoma cell lines, HeLa and MCF-7 cells. C-D Primary human CD4 + T (E) and CD8 + T lymphocytes (F), and Jurkat cells (G) were stimulated for 30 min or as indicated with 20 ng/mL PMA plus 300 ng/mL ionomycin (PI). Lysates were prepared and treated or not with λPP and Western blotting analysis was performed as indicated. E Jurkat cells were stimulated with 20 ng/mL PMA, 300 ng/mL ionomycin, PMA plus ionomycin (PI), 10 ng/mL TNFα or 12.5 µg/mL Anisomycin, for 30 min. Cell lysates were subjected to Western blotting analysis. F Jurkat cells we pretreated for 1h with 1 µM Trametinib or 500 nM BIMVIII and subsequently stimulated and lysed as in (E). Blue arrowhead indicates HOIL-1 Cter.
Figure Legend Snippet: SHARPIN is a Phosphoprotein. A Jurkat cells were treated with or without 50 nM Calyculin A (Cal A) for 30 min prior lysis. Cell lysates from primary human CD4 + or CD8 + T lymphocytes and Jurkat cells were treated with lambda phosphatase (λPP) as indicated, and subjected to Western blotting analysis. Green and white arrowheads indicate phosphorylated and unphosphorylated SHARPIN, respectively. Molecular weight markers ( M r ) are indicated. B Densitometric analysis of the ratio between phosphorylated species of SHARPIN and SHARPIN in primary human T CD4 + cells, primary human T CD8 + T cells, Jurkat T cells, a panel of patient-derived diffuse large B-cell lymphoma cell lines, HeLa and MCF-7 cells. C-D Primary human CD4 + T (E) and CD8 + T lymphocytes (F), and Jurkat cells (G) were stimulated for 30 min or as indicated with 20 ng/mL PMA plus 300 ng/mL ionomycin (PI). Lysates were prepared and treated or not with λPP and Western blotting analysis was performed as indicated. E Jurkat cells were stimulated with 20 ng/mL PMA, 300 ng/mL ionomycin, PMA plus ionomycin (PI), 10 ng/mL TNFα or 12.5 µg/mL Anisomycin, for 30 min. Cell lysates were subjected to Western blotting analysis. F Jurkat cells we pretreated for 1h with 1 µM Trametinib or 500 nM BIMVIII and subsequently stimulated and lysed as in (E). Blue arrowhead indicates HOIL-1 Cter.

Techniques Used: Lysis, Western Blot, Molecular Weight, Derivative Assay

6) Product Images from "Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway"

Article Title: Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006819

Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p
Figure Legend Snippet: Identification of the CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in human lymphoid organs. ( A ) The representative dot plots show the tissue distribution of CD4 + , CD8 + , and CD4 - CD8 - ILC1 subsets in the peripheral blood, spleen, bone marrow, large intestine, small intestine and liver perfusion. The numbers indicate the percentages of each cell subset. ( B-C ) Summary data of the proportion of total ILC1s in live CD45 + cells ( B ) and the proportion of CD4 + ILC1s in the total ILC1s ( C ) in various lymphoid organs in humans (n = 25 for PBMCs and n = 5 for other organs). ( D ) Representative dot plots depict the expression of transcriptional factor T-bet and Eomes of CD4 + , CD8 + and CD4 - CD8 - ILC1 subsets in peripheral blood of human. The numbers indicate the percentages of transcriptional factors within each ILC1 subset. ( E ) Summary data of the expression of T-bet and Eomes by ILC1 subsets in peripheral blood of human (n = 15). ( F-G ) Summarized data show the production of IFN-γ and TNF-α by peripheral ILC1 subsets from healthy subjects after stimulation with PMA/ionomycin (n = 12, F ) and IL-12 plus IL-18 (n = 12, G ). ( B, C , E-G ) Data represent the mean ± s.e.m. values. Overall, p

Techniques Used: Expressing

Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p
Figure Legend Snippet: Impairment of cytokine production by ILC1 subsets as a result of chronic HIV-1 infection. Summarized data of IFN-γ and TNF-α production from ILC1 subsets in the peripheral blood of HCs (n = 12), HIV-1-infected patients without HAART (n = 18) and with HAART (n = 12) in response to PMA/ionomycin ( A ) or IL-12 plus IL-18 ( B ). Overall, p

Techniques Used: Infection

7) Product Images from "Stat3 Programs Th17-Specific Regulatory T Cells to Control GN"

Article Title: Stat3 Programs Th17-Specific Regulatory T Cells to Control GN

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2013080904

Foxp3 Cre ×Stat3 fl/fl mice show enhanced Th17 responses without developing a spontaneous renal phenotype. (A) FACS analysis of IL-17 + and ROR γ t + Th17 cells in peripheral blood after stimulation with phorbol 12-myristate 13-acetate/ionomycin.
Figure Legend Snippet: Foxp3 Cre ×Stat3 fl/fl mice show enhanced Th17 responses without developing a spontaneous renal phenotype. (A) FACS analysis of IL-17 + and ROR γ t + Th17 cells in peripheral blood after stimulation with phorbol 12-myristate 13-acetate/ionomycin.

Techniques Used: Mouse Assay, FACS

8) Product Images from "IL-17A Production by Renal ?? T Cells Promotes Kidney Injury in Crescentic GN"

Article Title: IL-17A Production by Renal ?? T Cells Promotes Kidney Injury in Crescentic GN

Journal: Journal of the American Society of Nephrology : JASN

doi: 10.1681/ASN.2012010040

γδ T cells aggravate crescentic GN by stimulating the Th17 response. (A) Quantification of the percentage of IL-17A + and IFN-γ + cells in the renal CD4 + T cell subset isolated from controls ( n =4), nephritic wild-type (WT, n =6), and nephritic δTCR −/− mice ( n =7) 6 days after induction of NTN. For restimulation, cells were cultured with PMA/ionomycin for 4.5 hours before intracellular cytokine staining. (B) Percentage of IL-17A + cells in the double-negative T cell subset. (C) Quantitative RT-PCR measurement of IL-17A and IFN-γ mRNA expression in the renal cortex. (D) ELISA measurement of IL-17A and IFN-γ protein concentrations from supernatants of splenocytes stimulated for 60 hours with sheep IgG. (E) Immunohistochemistry for the neutrophil marker GR-1 (original magnification, ×400) and quantification of GR-1 + cells from renal tissue sections. (F) Periodic acid-Schiff staining of renal tissue sections (original magnification, ×400) and quantification of the percentage of crescentic glomeruli. Group sizes for parts B to E are the same as in part A. Bars represent means ± SEM. * P
Figure Legend Snippet: γδ T cells aggravate crescentic GN by stimulating the Th17 response. (A) Quantification of the percentage of IL-17A + and IFN-γ + cells in the renal CD4 + T cell subset isolated from controls ( n =4), nephritic wild-type (WT, n =6), and nephritic δTCR −/− mice ( n =7) 6 days after induction of NTN. For restimulation, cells were cultured with PMA/ionomycin for 4.5 hours before intracellular cytokine staining. (B) Percentage of IL-17A + cells in the double-negative T cell subset. (C) Quantitative RT-PCR measurement of IL-17A and IFN-γ mRNA expression in the renal cortex. (D) ELISA measurement of IL-17A and IFN-γ protein concentrations from supernatants of splenocytes stimulated for 60 hours with sheep IgG. (E) Immunohistochemistry for the neutrophil marker GR-1 (original magnification, ×400) and quantification of GR-1 + cells from renal tissue sections. (F) Periodic acid-Schiff staining of renal tissue sections (original magnification, ×400) and quantification of the percentage of crescentic glomeruli. Group sizes for parts B to E are the same as in part A. Bars represent means ± SEM. * P

Techniques Used: Isolation, Mouse Assay, Cell Culture, Staining, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Marker

Time course of IL-17A production in experimental GN. (A and B) Flow cytometric analysis of renal leukocytes isolated from nephritic mice at day 10 after induction of NTN. For restimulation, cells were cultured with PMA/ionomycin for 4.5 hours before intracellular staining for IL-17A. Plots are gated for live CD45 + lymphocytes. Numbers indicate events in the quadrants in percentage of all gated events. (C) Quantification of the percentage of IL-17A + cells in the respective T cell subset during the time course of the nephritis ( n =7–10 per group for days 0–10 and n =3–4 per group for days 20 and 30). (D) Relative contribution of the IL-17A–producing T cell subsets (in percentages) to total IL-17A + CD3 + cells (100%) after restimulation with PMA/ionomycin at the given time points after induction of NTN. DN, double negative. Bars represent means ± SEM.
Figure Legend Snippet: Time course of IL-17A production in experimental GN. (A and B) Flow cytometric analysis of renal leukocytes isolated from nephritic mice at day 10 after induction of NTN. For restimulation, cells were cultured with PMA/ionomycin for 4.5 hours before intracellular staining for IL-17A. Plots are gated for live CD45 + lymphocytes. Numbers indicate events in the quadrants in percentage of all gated events. (C) Quantification of the percentage of IL-17A + cells in the respective T cell subset during the time course of the nephritis ( n =7–10 per group for days 0–10 and n =3–4 per group for days 20 and 30). (D) Relative contribution of the IL-17A–producing T cell subsets (in percentages) to total IL-17A + CD3 + cells (100%) after restimulation with PMA/ionomycin at the given time points after induction of NTN. DN, double negative. Bars represent means ± SEM.

Techniques Used: Flow Cytometry, Isolation, Mouse Assay, Cell Culture, Staining

9) Product Images from "Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates"

Article Title: Neuropilin-1 Is Expressed on Lymphoid Tissue Residing LTi-like Group 3 Innate Lymphoid Cells and Associated with Ectopic Lymphoid Aggregates

Journal: Cell Reports

doi: 10.1016/j.celrep.2017.01.063

NRP1 + ILC3s Express CD45RO and Produce Higher Amounts of Cytokines Than NRP1 − ILC3s (A) Freshly isolated ILC3s were in vitro stimulated with PMA plus ionomycin and analyzed for intracellular IL-22 (n = 4, ∗ p
Figure Legend Snippet: NRP1 + ILC3s Express CD45RO and Produce Higher Amounts of Cytokines Than NRP1 − ILC3s (A) Freshly isolated ILC3s were in vitro stimulated with PMA plus ionomycin and analyzed for intracellular IL-22 (n = 4, ∗ p

Techniques Used: Isolation, In Vitro

10) Product Images from "Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4"

Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01573

NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.
Figure Legend Snippet: NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.

Techniques Used: Fluorescence, Staining, Immunofluorescence, Microscopy

Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.
Figure Legend Snippet: Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.

Techniques Used: Mouse Assay, Staining, Confocal Microscopy, Infection

Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.
Figure Legend Snippet: Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.

Techniques Used: Isolation, Mouse Assay, Staining, Confocal Microscopy, Electron Microscopy

11) Product Images from "AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells"

Article Title: AMP-activated protein kinase (AMPK)-dependent and -independent pathways regulate hypoxic inhibition of transepithelial Na+ transport across human airway epithelial cells

Journal: British Journal of Pharmacology

doi: 10.1111/j.1476-5381.2012.01993.x

Effect of ionomycin on intracellular Ca 2+ and activation of AMPK in H441 cells. (A) Representative fluorescent confocal x – y images of H441 cells taken before, during and after exposure to 1 µM ionomycin. (B) Plot of the time course of the normalized fluorescence averaged from a group of ∼20 H441 cells briefly exposed to ionomycin (1 µM). (C) H441 cells grown at the air–liquid interface were treated with ionomycin (15 min) with or without pretreatment (30 min) with the CaMKK inhibitor STO609 (STO609). Typical Western blots of cell extracts immunoblotted for phospho-Ser 79 of ACC (pACC), total ACC (tACC), phospho-Thr 172 of AMPKα (pAMPK) or total AMPKα (tAMPK). These blots were repeated with similar results.
Figure Legend Snippet: Effect of ionomycin on intracellular Ca 2+ and activation of AMPK in H441 cells. (A) Representative fluorescent confocal x – y images of H441 cells taken before, during and after exposure to 1 µM ionomycin. (B) Plot of the time course of the normalized fluorescence averaged from a group of ∼20 H441 cells briefly exposed to ionomycin (1 µM). (C) H441 cells grown at the air–liquid interface were treated with ionomycin (15 min) with or without pretreatment (30 min) with the CaMKK inhibitor STO609 (STO609). Typical Western blots of cell extracts immunoblotted for phospho-Ser 79 of ACC (pACC), total ACC (tACC), phospho-Thr 172 of AMPKα (pAMPK) or total AMPKα (tAMPK). These blots were repeated with similar results.

Techniques Used: Activation Assay, Fluorescence, Western Blot

12) Product Images from "Deficits in the IgG+ memory B‐cell recovery after anthracycline treatment is confined to the spleen of rhesus macaques"

Article Title: Deficits in the IgG+ memory B‐cell recovery after anthracycline treatment is confined to the spleen of rhesus macaques

Journal: Clinical & Translational Immunology

doi: 10.1002/cti2.1150

Immunophenotype and functional analysis of spleen and LN‐derived CXCR5 + Th and T FH cells from Doxo‐ and saline‐treated macaques. Representative gating strategy for (a) spleen and (b) LN from rhesus macaques ( n = 10) 5 months postchemotherapy completion ( n = 5 Doxo‐ and 5 saline‐treated, 2 technical replicates per condition). Compiled data from FACS analyses of spleen (c, e) and LN (d, f) CXCR5 + Th and T FH cells from Doxo (open circles)‐ and saline (filled circles)‐treated macaques. IL‐21 production in spleen (g) and LN (h) . IL‐21‐producing MC numbers (left‐hand side in g, h ) determined by ELISPOT ( n = 4 or 5 animals per group, 2–4 replicates per condition), and IL‐21 in MC cultures (right‐hand side in g, h ) quantified by ELISA ( n = 3 animals per group, 2 replicates per condition). Ctrl, cell culture medium; P + I, PMA + ionomycin.
Figure Legend Snippet: Immunophenotype and functional analysis of spleen and LN‐derived CXCR5 + Th and T FH cells from Doxo‐ and saline‐treated macaques. Representative gating strategy for (a) spleen and (b) LN from rhesus macaques ( n = 10) 5 months postchemotherapy completion ( n = 5 Doxo‐ and 5 saline‐treated, 2 technical replicates per condition). Compiled data from FACS analyses of spleen (c, e) and LN (d, f) CXCR5 + Th and T FH cells from Doxo (open circles)‐ and saline (filled circles)‐treated macaques. IL‐21 production in spleen (g) and LN (h) . IL‐21‐producing MC numbers (left‐hand side in g, h ) determined by ELISPOT ( n = 4 or 5 animals per group, 2–4 replicates per condition), and IL‐21 in MC cultures (right‐hand side in g, h ) quantified by ELISA ( n = 3 animals per group, 2 replicates per condition). Ctrl, cell culture medium; P + I, PMA + ionomycin.

Techniques Used: Functional Assay, Derivative Assay, FACS, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Cell Culture

Immunophenotype and functional analysis of spleen and LN‐derived T cells following cessation of anthracycline therapy. Cytotoxic (CD8 + , blue) and helper (CD4 + , red) T cells from Doxo (open circles)‐ and saline (filled circles)‐treated macaques ( n = 5 animals per group, 2 technical replicates for each condition). Representative FACS gating strategy for spleen (a, c, e, g) and LN (b, d, f, h) from the same animal 5 months postchemotherapy. Compiled data from FACS analyses of spleen (box plot in c ) and LN (box plot in d ) IFN‐γ + CD8 and CD4 T cells in Doxo‐ and saline‐treated macaques. Compiled data of spleen (box plots in g ) and LN (box plots in h ) IFN‐γ + T‐cell phenotypes containing enriched T CM (CD28 + CD95 + ) and T EM (CD28 – CD95 + ) cells in Doxo (open circles)‐ and saline (filled circles)‐treated macaques. Ctrl, cell culture medium; P + I, PMA + ionomycin.
Figure Legend Snippet: Immunophenotype and functional analysis of spleen and LN‐derived T cells following cessation of anthracycline therapy. Cytotoxic (CD8 + , blue) and helper (CD4 + , red) T cells from Doxo (open circles)‐ and saline (filled circles)‐treated macaques ( n = 5 animals per group, 2 technical replicates for each condition). Representative FACS gating strategy for spleen (a, c, e, g) and LN (b, d, f, h) from the same animal 5 months postchemotherapy. Compiled data from FACS analyses of spleen (box plot in c ) and LN (box plot in d ) IFN‐γ + CD8 and CD4 T cells in Doxo‐ and saline‐treated macaques. Compiled data of spleen (box plots in g ) and LN (box plots in h ) IFN‐γ + T‐cell phenotypes containing enriched T CM (CD28 + CD95 + ) and T EM (CD28 – CD95 + ) cells in Doxo (open circles)‐ and saline (filled circles)‐treated macaques. Ctrl, cell culture medium; P + I, PMA + ionomycin.

Techniques Used: Functional Assay, Derivative Assay, FACS, Cell Culture

13) Product Images from "Regulation of the terminal maturation of iNKT cells by mediator complex subunit 23"

Article Title: Regulation of the terminal maturation of iNKT cells by mediator complex subunit 23

Journal: Nature Communications

doi: 10.1038/s41467-018-06372-1

Med23 deficiency impairs the anti-tumor function of iNKT cells. a – c WT and Med23 −/− mice were inoculated with 2 × 10 5 B16F10 cells by i.v. injection. On the same day and again on days 4 and 8, WT and Med23 −/− mice received 2 μg of α-GalCer or mock by i.v. injection. Fourteen days after tumor inoculation, the lungs were harvested ( a ), B16F10 colonies were counted ( b ) and the anti-tumor effects of α-GalCer were assessed ( c ) ( n = 7). Scale bar: 0.5 cm. The anti-tumor effect (%) = 1-B16F10 colonies (α-GalCer administration)/B16F10 colonies (mock administration). d , e Flow cytometry data ( d ) and the percentage ( e ) of iNKT cells in the lungs of WT and Med23 −/− mice with post-B16F10 inoculation and α-GalCer or mock administration on days 0 and 4 (WT + Mock, KO + Mock and KO + α-GalCer, n = 8; WT + α-GalCer, n = 7). f , g After in vitro stimulation with PMA and ionomycin for 2 h, flow cytometric analysis ( f ) was performed, and the percentage ( g ) of IFN-γ + and CCL5 + cells among iNKT cells in the lungs of WT and Med23 −/− mice with post-B16F10 inoculation and α-GalCer administration on days 0 and 4 was determined (IFN-γ + cell frequency, n = 7; CCL5 + cell frequency, n = 5). h , i Jα18 −/− mice were inoculated with 2 × 10 5 B16F10 cells by i.v. injection. On the same day and again on days 4 and 8, the Jα18 −/− mice received 2 × 10 5 liver-derived iNKT cells from WT or Med23 −/− mice by i.v. injection with 2 μg of α-GalCer. Fourteen days after tumor inoculation, the lungs were harvested ( h ), and B16F10 colonies were counted ( i ) ( n = 4, paired t -test). Scale bar: 0.5 cm. The data are presented as the mean ± s.d. For all panels: * P
Figure Legend Snippet: Med23 deficiency impairs the anti-tumor function of iNKT cells. a – c WT and Med23 −/− mice were inoculated with 2 × 10 5 B16F10 cells by i.v. injection. On the same day and again on days 4 and 8, WT and Med23 −/− mice received 2 μg of α-GalCer or mock by i.v. injection. Fourteen days after tumor inoculation, the lungs were harvested ( a ), B16F10 colonies were counted ( b ) and the anti-tumor effects of α-GalCer were assessed ( c ) ( n = 7). Scale bar: 0.5 cm. The anti-tumor effect (%) = 1-B16F10 colonies (α-GalCer administration)/B16F10 colonies (mock administration). d , e Flow cytometry data ( d ) and the percentage ( e ) of iNKT cells in the lungs of WT and Med23 −/− mice with post-B16F10 inoculation and α-GalCer or mock administration on days 0 and 4 (WT + Mock, KO + Mock and KO + α-GalCer, n = 8; WT + α-GalCer, n = 7). f , g After in vitro stimulation with PMA and ionomycin for 2 h, flow cytometric analysis ( f ) was performed, and the percentage ( g ) of IFN-γ + and CCL5 + cells among iNKT cells in the lungs of WT and Med23 −/− mice with post-B16F10 inoculation and α-GalCer administration on days 0 and 4 was determined (IFN-γ + cell frequency, n = 7; CCL5 + cell frequency, n = 5). h , i Jα18 −/− mice were inoculated with 2 × 10 5 B16F10 cells by i.v. injection. On the same day and again on days 4 and 8, the Jα18 −/− mice received 2 × 10 5 liver-derived iNKT cells from WT or Med23 −/− mice by i.v. injection with 2 μg of α-GalCer. Fourteen days after tumor inoculation, the lungs were harvested ( h ), and B16F10 colonies were counted ( i ) ( n = 4, paired t -test). Scale bar: 0.5 cm. The data are presented as the mean ± s.d. For all panels: * P

Techniques Used: Mouse Assay, Injection, Flow Cytometry, Cytometry, In Vitro, Derivative Assay

14) Product Images from "Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons"

Article Title: Detrimental effect of zwitterionic buffers on lysosomal homeostasis in cell lines and iPSC-derived neurons

Journal: AMRC open research

doi: 10.12688/amrcopenres.12903.1

Growth of iPSC-derived neurons in HEPES containing media results in altered lysosomal Ca 2+ and causes lysosomal expansion. ( A ) Representative images of iPSC-derived neurons treated for 7 days in media containing 10 mM HEPES. Phase contrast microscopy images show location of neuronal cell bodies. Scale bar = 10 µm, N=3. ( B ) Following 7-day treatment in HEPES, lysosomal Ca 2+ release, triggered by addition of 500 µM GPN, to induce osmotic lysis, after ionomycin to clamp other intracellular Ca 2+ stores, was measured in iPSC-derived neurons, N=4 (7–14 cells analysed per repeat). ( C ) i and ii are Representative traces of Ca 2+ release quantified in ( B ). (*p
Figure Legend Snippet: Growth of iPSC-derived neurons in HEPES containing media results in altered lysosomal Ca 2+ and causes lysosomal expansion. ( A ) Representative images of iPSC-derived neurons treated for 7 days in media containing 10 mM HEPES. Phase contrast microscopy images show location of neuronal cell bodies. Scale bar = 10 µm, N=3. ( B ) Following 7-day treatment in HEPES, lysosomal Ca 2+ release, triggered by addition of 500 µM GPN, to induce osmotic lysis, after ionomycin to clamp other intracellular Ca 2+ stores, was measured in iPSC-derived neurons, N=4 (7–14 cells analysed per repeat). ( C ) i and ii are Representative traces of Ca 2+ release quantified in ( B ). (*p

Techniques Used: Derivative Assay, Microscopy, Lysis

Related Articles

Isolation:

Article Title: Infection and depletion of CD4+ group-1 innate lymphoid cells by HIV-1 via type-I interferon pathway
Article Snippet: .. For intracellular cytokine detection, freshly isolated cells were stimulated for 6 h by culturing with PMA (50 ng/ml, Sigma) and ionomycin (1 μM, Merck) in the presence of BFA (1 μM). .. Alternatively, the cells were incubated with IL-12 (20 ng/ml) plus IL-18 (20 ng/ml) for 12 h, followed by Golgi-stop for an additional 6 h. The cells were then collected for surface marker staining; this was followed by cell permeabilization and intracellular cytokine staining.

Cell Culture:

Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4
Article Snippet: .. Freshly purified murine bone marrow neutrophils were stimulated on glass objective slides in a 24-well cell culture plate with preformed C. albicans hyphae, 100 ng/ml PMA, and 1 µM ionomycin in HBSS for 2.5 h at 37°C and 5% CO2 , then, cells were fixed with 0.1 M Cacodylate buffer (Merck) containing 2.5% glutaraldehyde. .. After washing three times with PBS, the samples were treated with 1% Osmiumtetroxide in PBS for 30 min, washed again three times with PBS, dehydrated by incubation in 70% EtOH in H2 O for 30 min followed by 100% EtOH for 30 min and an incubation with hexamethyldisilazane (Sigma) for 2 h prior to air drying overnight.

Purification:

Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4
Article Snippet: .. Freshly purified murine bone marrow neutrophils were stimulated on glass objective slides in a 24-well cell culture plate with preformed C. albicans hyphae, 100 ng/ml PMA, and 1 µM ionomycin in HBSS for 2.5 h at 37°C and 5% CO2 , then, cells were fixed with 0.1 M Cacodylate buffer (Merck) containing 2.5% glutaraldehyde. .. After washing three times with PBS, the samples were treated with 1% Osmiumtetroxide in PBS for 30 min, washed again three times with PBS, dehydrated by incubation in 70% EtOH in H2 O for 30 min followed by 100% EtOH for 30 min and an incubation with hexamethyldisilazane (Sigma) for 2 h prior to air drying overnight.

Incubation:

Article Title: IL-17A Production by Renal ?? T Cells Promotes Kidney Injury in Crescentic GN
Article Snippet: .. In brief, cells were activated by incubation at 37°C, 5% CO2 for 4 hours with PMA (50 ng/ml; Sigma) and ionomycin (1 µg/ml; Calbiochem-Merck, Darmstadt, Germany) in X-VIVO medium (Lonza AG, Walkersville, MD). .. After 30 minutes of incubation, Brefeldin A (10 µg/ml; Sigma) was added.

other:

Article Title: Allergy Testing and Drug Screening on an ITO-Coated Lab-on-a-Disc
Article Snippet: Acridine Orange (AO), calf thymus DNA, triptolide, phorbol 12-myristate 13-acetate (PMA) and N -formyl-methionine-leucine-phenylalanine (fMLP) were purchased from Sigma-Aldrich, St. Louis, MO, USA, while ionomycin was obtained from Merck, Boston, MA, USA.

Staining:

Article Title: Stat3 Programs Th17-Specific Regulatory T Cells to Control GN
Article Snippet: .. Fluorochrome-labeled antibodies against IL-17, IFN- γ , Foxp3, ROR γ t, Ki67, and T-Bet (Ebioscience) were used as recently published., For intracellular cytokine staining, cells were activated with phorbol 12-myristate 13-acetate (50 ng/ml; Sigma-Aldrich) and ionomycin (1 µ g/ml; Calbiochem-Merck) for 5 hours. .. After 30 minutes of incubation, brefeldin A (10 µ g/ml; Sigma-Aldrich) was added.

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    NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or <t>ionomycin</t> as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.
    Ionomycin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Merck & Co calcium ionophore ionomycin
    Gene expression and COX activity in RO82 W-1 cells after <t>ionomycin</t> or BAPTA/AM treatments. RO82 W-1 cells were incubated in culture medium (containing 1.8 m m Ca 2+ ) with either 2 μ m ionomycin, 2–8 μ m BAPTA/AM (to avoid cell death
    Calcium Ionophore Ionomycin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calcium ionophore ionomycin/product/Merck & Co
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    Image Search Results


    IL-10 is produced by CD4 + T cells during allergen-induced airway inflammation and is increased by transfer of CD4 + CD25 + regulatory T cells. Lungs were digested with collagenase and DNase as described in Materials and methods. Digest cells were stimulated by PMA/Ionomycin in the presence of Brefeldin A for 6 h. Cells were phenotyped by staining for CD4, CD8, CD11b (macrophages), CD11c (dendritic cells), and B220 (B cells). Granulocytes were defined by forward and side scatter. (A) Data are expressed as median cell types producing IL-10 with interquartile range ( n = 6–14 mice/group in three separate experiments). *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Resolution of airway inflammation and hyperreactivity after in vivo transfer of CD4+CD25+ regulatory T cells is interleukin 10 dependent

    doi: 10.1084/jem.20051166

    Figure Lengend Snippet: IL-10 is produced by CD4 + T cells during allergen-induced airway inflammation and is increased by transfer of CD4 + CD25 + regulatory T cells. Lungs were digested with collagenase and DNase as described in Materials and methods. Digest cells were stimulated by PMA/Ionomycin in the presence of Brefeldin A for 6 h. Cells were phenotyped by staining for CD4, CD8, CD11b (macrophages), CD11c (dendritic cells), and B220 (B cells). Granulocytes were defined by forward and side scatter. (A) Data are expressed as median cell types producing IL-10 with interquartile range ( n = 6–14 mice/group in three separate experiments). *, P

    Article Snippet: For intracellular cytokine staining, cells were stimulated with PMA/ionomycin (Merck) in the presence of Brefeldin A (Sigma-Aldrich) for 6 h before extracellular staining.

    Techniques: Produced, Staining, Mouse Assay

    NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.

    Journal: Frontiers in Immunology

    Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

    doi: 10.3389/fimmu.2018.01573

    Figure Lengend Snippet: NETosis in response to Candida albicans hyphae is independent of Peptidylarginine deiminase 4 (PAD4). (A) The release of extracellular DNA from murine bone marrow neutrophils was detected by Sytox green after stimulation for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or ionomycin as indicated. The increase in fluorescence intensity from stimulated relative to unstimulated neutrophils is shown. Each bar represents the mean with standard error of the mean (SEM) of each group ( n = 3) with data pooled from three independent experiments. (B) Bone marrow neutrophils were stimulated with C. albicans yeast cells or preformed hyphae of the highly virulent lab strain SC5413 and the low-virulent strain 101 as indicated. The release of extracellular DNA was detected by Sytox green as in (A) . Data are the mean with SEM of each group ( n = 3) with data pooled from three independent experiments. (C,D) The release of DNA from WT and PAD4 −/− neutrophils that were stimulated and stained with Sytox as described in (A) was visualized by immunofluorescence microscopy. Representative images for each condition are shown in (C) . Scale bar = 20 µm. The frequency of Sytox + neutrophils among all C. albicans hyphae-associated neutrophils is shown in (D) . The bars are the mean with SEM of 15 images analyzed per condition. (E) Human peripheral blood neutrophils from healthy donors that were treated with the PAD inhibitor Cl-amidine or left untreated and from patients with acquired myeloperoxidase-deficiency were stimulated for 2.5 h with the yeast-locked strain Δ hgc1 ( C.a . yeast), preformed hyphae of the control strain Δ hgc1 + HGC1 ( C.a . hyphae), or phorbol 12-myristate 13-acetate. Sytox was detected as described in (A) . Each bar represents the mean with SEM of three technical replicates of each group. Data are representative of one out of two independent experiments.

    Article Snippet: Freshly purified murine bone marrow neutrophils were stimulated on glass objective slides in a 24-well cell culture plate with preformed C. albicans hyphae, 100 ng/ml PMA, and 1 µM ionomycin in HBSS for 2.5 h at 37°C and 5% CO2 , then, cells were fixed with 0.1 M Cacodylate buffer (Merck) containing 2.5% glutaraldehyde.

    Techniques: Fluorescence, Staining, Immunofluorescence, Microscopy

    Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.

    Journal: Frontiers in Immunology

    Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

    doi: 10.3389/fimmu.2018.01573

    Figure Lengend Snippet: Candida albicans induces peptidylarginine deiminase 4 (PAD4)-mediated citrullination of histone H3. (A,B) Bone marrow neutrophils from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae or ionomycin or left unstimulated and then stained for DNA (DAPI; blue) and citrullinated histone H3 (magenta); brightfield. Images were acquired by confocal microscopy (A) and the fraction of CitH3 + neutrophils was determined (B) . In (B) , the bars are the mean with standard error of the mean (SEM) of 10–30 cells analyzed per condition. (C,D) WT and PAD4 −/− mice were infected with C. albicans via the tail vein (C) or sublingually (D) . Consecutive kidney (C) or tongue (D) sections were stained with periodic-acid schiff reagent (C) , α-Ly6G or with α-CitH3 on day 3 postinfection (C) or on day 1 postinfection (D) , respectively. Representative images are shown. Scale bar = 50 µm [in (A) ], 1 mm [in (C) , left panel], 250 µm [in (C) middle and right panel and in (D) ]. See also Figure S1 in Supplementary Material.

    Article Snippet: Freshly purified murine bone marrow neutrophils were stimulated on glass objective slides in a 24-well cell culture plate with preformed C. albicans hyphae, 100 ng/ml PMA, and 1 µM ionomycin in HBSS for 2.5 h at 37°C and 5% CO2 , then, cells were fixed with 0.1 M Cacodylate buffer (Merck) containing 2.5% glutaraldehyde.

    Techniques: Mouse Assay, Staining, Confocal Microscopy, Infection

    Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.

    Journal: Frontiers in Immunology

    Article Title: Candida albicans-Induced NETosis Is Independent of Peptidylarginine Deiminase 4

    doi: 10.3389/fimmu.2018.01573

    Figure Lengend Snippet: Characterization of neutrophil extracellular traps released from WT and PAD4 −/− neutrophils. (A) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with Candida albicans hyphae or left unstimulated and then stained for membrane lipids (PKH26; red), DNA (DAPI; blue), calprotectin (anti-S100A8 and anti-S100A9, green), and myeloperoxidase (yellow). Images were acquired by confocal microscopy. Representative images of each group are shown. Scale bar = 20 µm. (B) Bone marrow neutrophils isolated from WT and PAD4 −/− mice were stimulated for 2.5 h with C. albicans hyphae and phorbol 12-myristate 13-acetate/ionomycin. Cells were fixed and imaged by scanning electron microscopy. Representative images of each group are shown. Scale bar = 1 µm. Red arrowheads show C. albicans hyphae.

    Article Snippet: Freshly purified murine bone marrow neutrophils were stimulated on glass objective slides in a 24-well cell culture plate with preformed C. albicans hyphae, 100 ng/ml PMA, and 1 µM ionomycin in HBSS for 2.5 h at 37°C and 5% CO2 , then, cells were fixed with 0.1 M Cacodylate buffer (Merck) containing 2.5% glutaraldehyde.

    Techniques: Isolation, Mouse Assay, Staining, Confocal Microscopy, Electron Microscopy

    TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect, et al. Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect

    doi: 10.1111/cns.13062

    Figure Lengend Snippet: TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Article Snippet: Ionomycin was purchased from Merck.

    Techniques: Transfection, Western Blot, Cell Culture, Expressing

    Gene expression and COX activity in RO82 W-1 cells after ionomycin or BAPTA/AM treatments. RO82 W-1 cells were incubated in culture medium (containing 1.8 m m Ca 2+ ) with either 2 μ m ionomycin, 2–8 μ m BAPTA/AM (to avoid cell death

    Journal: The Journal of Biological Chemistry

    Article Title: Nitric Oxide and Calcium Participate in the Fine Regulation of Mitochondrial Biogenesis in Follicular Thyroid Carcinoma Cells *

    doi: 10.1074/jbc.M110.217521

    Figure Lengend Snippet: Gene expression and COX activity in RO82 W-1 cells after ionomycin or BAPTA/AM treatments. RO82 W-1 cells were incubated in culture medium (containing 1.8 m m Ca 2+ ) with either 2 μ m ionomycin, 2–8 μ m BAPTA/AM (to avoid cell death

    Article Snippet: We measured the intracellular calcium response to the calcium ionophore ionomycin, in FTC-133 and RO82 W-1 cells ( ).

    Techniques: Expressing, Activity Assay, Incubation

    Dynamics of mitochondrial and endoplasmic reticulum networks in RO82 W-1 cells after ionomycin or BAPTA/AM treatments. A , expression level of eight of the most differential genes observed after ionomycin or BAPTA/AM treatments on microarray analysis compared

    Journal: The Journal of Biological Chemistry

    Article Title: Nitric Oxide and Calcium Participate in the Fine Regulation of Mitochondrial Biogenesis in Follicular Thyroid Carcinoma Cells *

    doi: 10.1074/jbc.M110.217521

    Figure Lengend Snippet: Dynamics of mitochondrial and endoplasmic reticulum networks in RO82 W-1 cells after ionomycin or BAPTA/AM treatments. A , expression level of eight of the most differential genes observed after ionomycin or BAPTA/AM treatments on microarray analysis compared

    Article Snippet: We measured the intracellular calcium response to the calcium ionophore ionomycin, in FTC-133 and RO82 W-1 cells ( ).

    Techniques: Expressing, Microarray