brefeldin a  (Cayman Chemical)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Brefeldin A
    Description:
    Brefeldin A BFA is a natural fungal metabolite which has been used extensively to study intracellular transport by vesicles or endosomes Early studies demonstrated that BFA reversibly interferes with protein trafficking and secretion mediated by the Golgi apparatus and endoplasmic reticulum BFA directly and reversibly inhibits Sec7 domain containing guanine exchange factors which are necessary for ADP ribosylation factor activation associated with vesicular transport IC 10 μM BFA is used to study endosomal trafficking and function in cells of plants as well as those of fungi invertebrates and vertebrates
    Catalog Number:
    11861
    Product Aliases:
    Ascotoxin,BFA,Cyanein,Decumbin,Nectrolide,NSC 56310,NSC 89671,NSC 107456,NSC 244390,Synergisidin
    Price:
    $35
    Purity:
    ≥98%
    Size:
    5 mg
    Formula:
    A crystalline solid
    Buy from Supplier


    Structured Review

    Cayman Chemical brefeldin a
    Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and <t>Brefeldin</t> A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p
    Brefeldin A BFA is a natural fungal metabolite which has been used extensively to study intracellular transport by vesicles or endosomes Early studies demonstrated that BFA reversibly interferes with protein trafficking and secretion mediated by the Golgi apparatus and endoplasmic reticulum BFA directly and reversibly inhibits Sec7 domain containing guanine exchange factors which are necessary for ADP ribosylation factor activation associated with vesicular transport IC 10 μM BFA is used to study endosomal trafficking and function in cells of plants as well as those of fungi invertebrates and vertebrates
    https://www.bioz.com/result/brefeldin a/product/Cayman Chemical
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "Long-term exposure to “low-dose” bisphenol A decreases mitochondrial DNA copy number, and accelerates telomere shortening in human CD8 + T cells"

    Article Title: Long-term exposure to “low-dose” bisphenol A decreases mitochondrial DNA copy number, and accelerates telomere shortening in human CD8 + T cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-72546-x

    Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and Brefeldin A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p
    Figure Legend Snippet: Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and Brefeldin A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p

    Techniques Used: Expressing

    2) Product Images from "VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation"

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12918

    Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of  Slc17a9  in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m  ionomycin, 10 μ m  brefeldin A, or 10 μ m  clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2  N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m  clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m  clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine  Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired  t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P
    Figure Legend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Techniques Used: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation

    3) Product Images from "CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice"

    Article Title: CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00775

    The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P
    Figure Legend Snippet: The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Injection

    4) Product Images from "CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice"

    Article Title: CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2020.00775

    The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P
    Figure Legend Snippet: The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Injection

    5) Product Images from "VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation"

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12918

    Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of  Slc17a9  in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m  ionomycin, 10 μ m  brefeldin A, or 10 μ m  clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2  N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m  clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m  clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine  Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired  t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P
    Figure Legend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Techniques Used: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation

    6) Product Images from "VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation"

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

    Journal: FEBS Open Bio

    doi: 10.1002/2211-5463.12918

    Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of  Slc17a9  in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m  ionomycin, 10 μ m  brefeldin A, or 10 μ m  clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2  N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m  clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m  clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine  Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired  t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P
    Figure Legend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Techniques Used: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation

    7) Product Images from "Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor"

    Article Title: Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065767

    The promotion of DP1 cell surface targeting by MK-0524 is inhibited by Brefeldin A. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 that were pre-incubated with vehicle or 20 µM of Brefeldin A (BFA) for 30 min, and then treated with 1 µM of MK-0524 or its control vehicle for 90 min. The results are shown as the percentage increase of DP1 cell surface expression when compared to control cells treated with vehicle. Results are the mean ± S.E. of three independent experiments. * is P
    Figure Legend Snippet: The promotion of DP1 cell surface targeting by MK-0524 is inhibited by Brefeldin A. Cell surface receptor expression was measured by ELISA as described under “ Materials and Methods ” in HEK293 cells transiently expressing Flag-DP1 that were pre-incubated with vehicle or 20 µM of Brefeldin A (BFA) for 30 min, and then treated with 1 µM of MK-0524 or its control vehicle for 90 min. The results are shown as the percentage increase of DP1 cell surface expression when compared to control cells treated with vehicle. Results are the mean ± S.E. of three independent experiments. * is P

    Techniques Used: Cell Surface Receptor Assay, Expressing, Enzyme-linked Immunosorbent Assay, Incubation

    MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.
    Figure Legend Snippet: MK-0524 induces translocation of DP1 from intracellular compartments to the plasma membrane. The distribution of DP1 in HEK293 cells was determined by immunofluorescence confocal microscopy. HEK293 cells transfected with Flag-DP1 were treated with vehicle or 1 µM of MK-0524 alone or in presence of 20 µM Brefeldin A for 90 min. Cells were labeled with mouse anti-FLAG and either a rabbit anti-calnexin antibody (top and midlle panels) or a rabbit anti-protein disulfide isomerase (PDI) antibody (lower panel) as described under “ Materials and Methods ”. Secondary antibodies were Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 546 goat anti-rabbit IgG. Merge images of the green-labelled DP1 and red-labeled calnexin or PDI are shown. Bars, 10 µM.

    Techniques Used: Translocation Assay, Immunofluorescence, Confocal Microscopy, Transfection, Labeling

    8) Product Images from "TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities"

    Article Title: TMX2 Is a Crucial Regulator of Cellular Redox State, and Its Dysfunction Causes Severe Brain Developmental Abnormalities

    Journal: American Journal of Human Genetics

    doi: 10.1016/j.ajhg.2019.10.009

    Redox State Assays of Wild-Type and Variant TMX2 (A and B) A non-reducing immunoblot of exogenous wild-type TMX2 versus endogenous control PDI (PDIA1) in HEK293T cells, showing that TMX2 occurs in an oxidized and reduced monomeric form, whereas during H 2 O 2  treatment, a dimer is formed (OX dimer). (A) Left panel: the blot after incubation with anti-wild-type TMX2 antibodies. Middle panel: incubation with anti-Myc antibodies. Right panel: anti-PDI control protein antibodies. (B) Control experiment after expression of exogenous control β-lactamase (Lac-Myc); immunoblotting was performed with the same antibodies as in (A). Native = untreated cells; BFA = cells treated with ER stress inducer Brefeldin A; DTT = cells treated with reducing agent DL-Dithiothreitol; and H2O2 = cells treated with hydrogen peroxide. (C) A non-reducing immunoblot with a similar experimental setup as in (A and B), but here there was also the addition of ER stress inducers Tunicamycin (TM) and Thapsigargin (TG). Redox states of TRX domain p.Cys170Gly variant (lanes 7–12) and the affected individual p.Arg53Cys variant (lanes 13–18) and p.Arg231Trp variant (lanes 19–24) were determined simultaneously. Detection was performed with anti-Myc antibody. (D and E) Semiquantitative densitometry calculations of TMX2 dimer/monomer ratios in native, ER stress, oxidative, and reductive environments for wild-type TMX2, n = 4 immunoblots from biological replicates (D). Semiquantitative densitometry calculations of TMX2 dimer/monomer ratios in wild-type TMX2 to p.Cys170Gly, p.Arg53Cys, and p.Arg231Trp variants in native, ER stress, oxidative or reductive environment (E). Data are represented as the mean ± SEM. Statistical two-tailed unpaired t tests were performed with a confidence interval of 95% in Graphpad Prism 8 (∗p
    Figure Legend Snippet: Redox State Assays of Wild-Type and Variant TMX2 (A and B) A non-reducing immunoblot of exogenous wild-type TMX2 versus endogenous control PDI (PDIA1) in HEK293T cells, showing that TMX2 occurs in an oxidized and reduced monomeric form, whereas during H 2 O 2 treatment, a dimer is formed (OX dimer). (A) Left panel: the blot after incubation with anti-wild-type TMX2 antibodies. Middle panel: incubation with anti-Myc antibodies. Right panel: anti-PDI control protein antibodies. (B) Control experiment after expression of exogenous control β-lactamase (Lac-Myc); immunoblotting was performed with the same antibodies as in (A). Native = untreated cells; BFA = cells treated with ER stress inducer Brefeldin A; DTT = cells treated with reducing agent DL-Dithiothreitol; and H2O2 = cells treated with hydrogen peroxide. (C) A non-reducing immunoblot with a similar experimental setup as in (A and B), but here there was also the addition of ER stress inducers Tunicamycin (TM) and Thapsigargin (TG). Redox states of TRX domain p.Cys170Gly variant (lanes 7–12) and the affected individual p.Arg53Cys variant (lanes 13–18) and p.Arg231Trp variant (lanes 19–24) were determined simultaneously. Detection was performed with anti-Myc antibody. (D and E) Semiquantitative densitometry calculations of TMX2 dimer/monomer ratios in native, ER stress, oxidative, and reductive environments for wild-type TMX2, n = 4 immunoblots from biological replicates (D). Semiquantitative densitometry calculations of TMX2 dimer/monomer ratios in wild-type TMX2 to p.Cys170Gly, p.Arg53Cys, and p.Arg231Trp variants in native, ER stress, oxidative or reductive environment (E). Data are represented as the mean ± SEM. Statistical two-tailed unpaired t tests were performed with a confidence interval of 95% in Graphpad Prism 8 (∗p

    Techniques Used: Variant Assay, Incubation, Expressing, Western Blot, Two Tailed Test

    9) Product Images from "Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis"

    Article Title: Autotaxin expression from synovial fibroblasts is essential for the pathogenesis of modeled arthritis

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20112012

    Increased ATX expression in arthritic ( hTNF +/− ) primary mouse SFs.  (a) SFs from WT and  hTNF +/−  littermate mice were cultured and stained for ATX (red) and nuclei/DNA (DAPI; blue). +GB indicates cultures treated with Golgi block (Brefeldin A). Ctrl refers to negative control. Data are representative of three independent experiments. Bars, 20 µm. (b) ATX protein levels in culture supernatants from WT and  hTNF +/−  SFs were analyzed by Western blot. 4F1 refers to a rat monoclonal Ab against ATX; Caym. refers to a commercial (Cayman Chemical) polyclonal Ab. Data are representative of four independent experiments. (c) Densitometric analysis of b. (d) Expression of ATX, vimentin, and VCAM by arthritic SFs was analyzed by flow cytometry. Data are representative of three independent experiments. (e) ATX mRNA in mouse WT SFs cultured for 24 h in the absence (control) or presence of 10 ng/ml TNF was analyzed by Q-RT-PCR ( n  = 4). Data are representative of three independent experiments. (c and e) Mean ± SE is shown. *, P
    Figure Legend Snippet: Increased ATX expression in arthritic ( hTNF +/− ) primary mouse SFs. (a) SFs from WT and hTNF +/− littermate mice were cultured and stained for ATX (red) and nuclei/DNA (DAPI; blue). +GB indicates cultures treated with Golgi block (Brefeldin A). Ctrl refers to negative control. Data are representative of three independent experiments. Bars, 20 µm. (b) ATX protein levels in culture supernatants from WT and hTNF +/− SFs were analyzed by Western blot. 4F1 refers to a rat monoclonal Ab against ATX; Caym. refers to a commercial (Cayman Chemical) polyclonal Ab. Data are representative of four independent experiments. (c) Densitometric analysis of b. (d) Expression of ATX, vimentin, and VCAM by arthritic SFs was analyzed by flow cytometry. Data are representative of three independent experiments. (e) ATX mRNA in mouse WT SFs cultured for 24 h in the absence (control) or presence of 10 ng/ml TNF was analyzed by Q-RT-PCR ( n = 4). Data are representative of three independent experiments. (c and e) Mean ± SE is shown. *, P

    Techniques Used: Expressing, Mouse Assay, Cell Culture, Staining, Blocking Assay, Negative Control, Western Blot, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction

    Related Articles

    other:

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation
    Article Snippet: Brefeldin A is an inhibitor of vesicular exocytosis.

    Article Title: Long-term exposure to “low-dose” bisphenol A decreases mitochondrial DNA copy number, and accelerates telomere shortening in human CD8 + T cells
    Article Snippet: Phorbol 12-myristate 13-acetate (PMA), ionomycin (calcium salt) and brefeldin A were obtained from Cayman (Hamburg, Germany).

    Article Title: Inverse Agonist and Pharmacochaperone Properties of MK-0524 on the Prostanoid DP1 Receptor
    Article Snippet: Brefeldin A was purchased from Cayman Chemical (Ann Arbor, MI).

    Article Title: CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice
    Article Snippet: The naïve and the healed ears that were not treated (no stimulation) or re-challenged at 3.5 h before (1% TNCB) were subjected to enzymatic digestion with brefeldin A for 3.5 h for intracellular cytokine analyses ( and ).

    Activity Assay:

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation
    Article Snippet: .. As shown in Fig. , clodronate and brefeldin A both suppressed release of ATP from MC3T3‐E1 cells suggesting that ATP release is dependent on VNUT activity and exocytosis. .. Clodronate decreased extracellular ATP levels in a concentration‐dependent manner (Fig. ) and increased ALP activity in response to mechanically loading (Fig. ).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cayman Chemical brefeldin a
    Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and <t>Brefeldin</t> A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p
    Brefeldin A, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a/product/Cayman Chemical
    Average 99 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    brefeldin a - by Bioz Stars, 2020-11
    99/100 stars
      Buy from Supplier

    88
    Cayman Chemical brefeldin a monensin cocktail
    ZnPP enhances lysosome membrane permeability. A2780 cells were treated with 5 μM ZnPP ( A ) or 5 μM SnPP ( B ) for 1, 4 or 21 hours. Cells were then incubated with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, excitation at 460 nm, emission at 650 nm for AO red. C . A2780 cells were pretreated with 21.2 μM <t>Brefeldin</t> A and 4 μM <t>Monensin</t> for 4 hours. The cells were then treated with 5 μM ZnPP for 1 hour followed by incubation with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, and excitation at 460 nm, emission at 650 nm for AO red. Shown are representative images of three individual experiments.
    Brefeldin A Monensin Cocktail, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/brefeldin a monensin cocktail/product/Cayman Chemical
    Average 88 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    brefeldin a monensin cocktail - by Bioz Stars, 2020-11
    88/100 stars
      Buy from Supplier

    Image Search Results


    Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and Brefeldin A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p

    Journal: Scientific Reports

    Article Title: Long-term exposure to “low-dose” bisphenol A decreases mitochondrial DNA copy number, and accelerates telomere shortening in human CD8 + T cells

    doi: 10.1038/s41598-020-72546-x

    Figure Lengend Snippet: Long-term impact of BPA on cytokine levels of CD8 + T cells. Long-term culture of CD8 + T was done using anti-CD2/3/28 beads and IL-2. The cells were continuously exposed to BPA or solvent control (SC = 0.01% DMSO). ( A ) On day 14 or 42 of culture, CD8 + cells were stimulated with PMA/ionomycin and Brefeldin A for 4 h. Then, intracellular IFN-γ expression was analysed using anti-CD8-PE coupled with anti-IFN-γ FITC with a FACSCalibur. Representative scattergrams of intracellular IFN-γ expression at 42d from one experiment are shown. ( B ) Supernatants, harvested on day 3 or 45 of long-term culture were used for analysis of IFN-γ release. To correct for inter-individual differences, each value of BPA treated cells was normalized to its respective SC sample. Raw data are given in Supplement Fig. 2 B, C. Bars are means + SD. Significance of difference was calculated relative to the respective control, * p

    Article Snippet: Phorbol 12-myristate 13-acetate (PMA), ionomycin (calcium salt) and brefeldin A were obtained from Cayman (Hamburg, Germany).

    Techniques: Expressing

    Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of  Slc17a9  in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine  Slc17a9  were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m  ionomycin, 10 μ m  brefeldin A, or 10 μ m  clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2  N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m  clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m  clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine  Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired  t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Journal: FEBS Open Bio

    Article Title: VNUT/SLC17A9, a vesicular nucleotide transporter, regulates osteoblast differentiation

    doi: 10.1002/2211-5463.12918

    Figure Lengend Snippet: Knockdown or inhibition of VNUT stimulates osteoblast differentiation. (A) mRNA levels of Slc17a9 in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 . (B) Extracellular ATP in MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 exposed to compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h). (C‐E) MC3T3‐E1 cells stably expressing control scrambled shRNA or shRNA against murine Slc17a9 were treated with osteoblast differentiation medium for 7 days after the application of compressive force by centrifuge (8.8 × 10 −2  N·cm −2 , 12 h) and mRNA levels of the indicated osteoblast marker genes measured by qPCR or (F) ALP activity determined. (G) Extracellular ATP levels in MC3T3‐E1 cells treated with 1 μ m ionomycin, 10 μ m brefeldin A, or 10 μ m clodronate for 30 min. (H) MC3T3‐E1 cells were exposed to compressive force by centrifugation (8.8 × 10 −2 N·cm −2 , 12 h) and treated with 0, 1.0, or 10 μ m clodronate after which extracellular ATP was measured. (I) MC3T3‐E1 cells were treated with 0, 1.0, or 10 μ m clodronate along with osteoblast differentiation medium, and ALP activity was measured 7 days after the application of compressive force by centrifuge (8.8 × 10 −2 N·cm −2 , 12 h). scrambled shRNA, Scr; shRNA against murine Slc17a9 , sh; ionomycin, Ion; brefeldin A, BfA; clodronate, Clo. Data are expressed as the mean ± SD ( n  = 3). Statistical analysis was performed with unpaired t ‐test (A‐F) and one‐way analysis of variance followed by Bonferroni method (G and H) or Kruskal–Wallis method test (I). * P

    Article Snippet: As shown in Fig. , clodronate and brefeldin A both suppressed release of ATP from MC3T3‐E1 cells suggesting that ATP release is dependent on VNUT activity and exocytosis.

    Techniques: Inhibition, Stable Transfection, Expressing, shRNA, Marker, Real-time Polymerase Chain Reaction, Activity Assay, Centrifugation

    The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P

    Journal: Frontiers in Immunology

    Article Title: CD4+ Resident Memory T Cells Mediate Long-Term Local Skin Immune Memory of Contact Hypersensitivity in BALB/c Mice

    doi: 10.3389/fimmu.2020.00775

    Figure Lengend Snippet: The early production of IFNγ and TNF by CD4 + and CD8 + T RM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1 st challenge) as assessed by qRT-PCR ( n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1 st challenge) ( n = 4) were compared with those of BALB/c mice [data at 6 h in (A) ]. (C,D) IFNγ, TNF, and IL-4 production in CD4 + and CD8 + T RM cells (on day 39 after the 1 st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B . (E) 1 st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1 st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1 + cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1 st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge ( n = 4–5 each group). All graphs represent means ± SE. * P

    Article Snippet: In some experiments, 20 μg/ml brefeldin A (Cayman Chemical) (to all samples) and 1.5 μg/ml ionomycin (Cayman Chemical) + 80 ng/ml phorbol 12-myristate 13-acetate (PMA) (AdipoGen Life Sciences, San Diego, CA, USA) (optional) were added to the digestion enzyme solution.

    Techniques: Mouse Assay, Quantitative RT-PCR, Flow Cytometry, Injection

    ZnPP enhances lysosome membrane permeability. A2780 cells were treated with 5 μM ZnPP ( A ) or 5 μM SnPP ( B ) for 1, 4 or 21 hours. Cells were then incubated with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, excitation at 460 nm, emission at 650 nm for AO red. C . A2780 cells were pretreated with 21.2 μM Brefeldin A and 4 μM Monensin for 4 hours. The cells were then treated with 5 μM ZnPP for 1 hour followed by incubation with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, and excitation at 460 nm, emission at 650 nm for AO red. Shown are representative images of three individual experiments.

    Journal: PLoS ONE

    Article Title: Zinc Protoporphyrin Suppresses β-Catenin Protein Expression in Human Cancer Cells: The Potential Involvement of Lysosome-Mediated Degradation

    doi: 10.1371/journal.pone.0127413

    Figure Lengend Snippet: ZnPP enhances lysosome membrane permeability. A2780 cells were treated with 5 μM ZnPP ( A ) or 5 μM SnPP ( B ) for 1, 4 or 21 hours. Cells were then incubated with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, excitation at 460 nm, emission at 650 nm for AO red. C . A2780 cells were pretreated with 21.2 μM Brefeldin A and 4 μM Monensin for 4 hours. The cells were then treated with 5 μM ZnPP for 1 hour followed by incubation with 2.5 μM AO for 30 minutes at 37°C. After incubation, cells were washed twice with HBSS. Images were captured using a fluorescent microscope (60X) with excitation at 500 nm, emission at 526 nm for AO green, and excitation at 460 nm, emission at 650 nm for AO red. Shown are representative images of three individual experiments.

    Article Snippet: MG132 and Brefeldin A/Monensin cocktail were from Cayman Chemical (Ann Arbor, MI).

    Techniques: Permeability, Incubation, Microscopy