Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

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Article Snippet: Receptor plasmid, human HCA2 (GPR109A) , Origene , N/A.

Techniques: RNAscope, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Reporter Gene Assay, Plasmid Preparation, Software

Journal: Cell Reports Medicine

Article Title: Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell

doi: 10.1016/j.xcrm.2024.101718

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Article Snippet: HM74A APC , R&D Systems , Cat#FAB2760A; RRID: AB_573115.

Techniques: Control, Staining, Recombinant, Liposomes, Cell Isolation, CCK-8 Assay, TUNEL Assay, Apoptosis Assay, Activation Assay, Software

Journal: JBMR Plus

Article Title: 3‐(3‐Hydroxyphenyl)‐Propionic Acid (PPA) Suppresses Osteoblastic Cell Senescence to Promote Bone Accretion in Mice

doi: 10.1002/jbm4.10201

Figure Lengend Snippet: HA and PPA stimulate 35 S‐GTPγS binding to membranes from mouse osteoblastic calvarial cells transfected with GPR109A but not GPR109B. Ligand‐induced 35 S‐GTPγS binding was studied using membranes prepared from mouse fetal calvarial cells expressing mouse GPR109A ( A ) or GPR109B ( B ). Binding of 35 S‐GTPγS was determined in the presence of increased concentration of nicotinic acid, acifran, HA, or PPA. HA = hippuric acid; PPA = 3‐(3‐hydroxyphenyl)‐propionic acid.

Article Snippet: Ligand‐induced 35 S‐GTPγS binding was studied using membrane fractions prepared from mouse fetal calvarial cells stably transfected with mouse GPR109A overexpression plasmid (OriGene, Rockville, MD, USA; catalog number RG206527) or GPR109B (OriGen Technologies; catalog number RG208523).

Techniques: Binding Assay, Transfection, Expressing, Concentration Assay

Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet: 3-Hydroxydecanoate signals through Gα i , Gα q , and GPR84 (A) Baseline normalized cell index (CI) of cells stimulated with 3-hydroxydecanoate (3-OH-C10) or the positive control compound, zaprinast, in the xCELLigence assay (N = 3). (B–D) Maximum baseline normalized CI of cells stimulated with 3-hydroxydecanoate after pre-incubation with (B) the Gα i inhibitor PTX (200 ng/mL) for 16 h (N = 3–4), (C) the Gα q inhibitor YM-254890 (2 μM) for 15 min (N = 5–7), or (D) the β-arrestin inhibitor barbadin (1 μM) for 15 min (N = 3–4). (E and F) PRESTO-Tango luciferase assay in HTLA cells transfected with human GPR84. (E) Transfected cells were stimulated with 3-hydroxydecanoate, the endogenous GPR84 agonist decanoate (capric acid, C10), the GPR84 reference agonist embelin or the GPR84 antagonist AR505962 (N = 4–5). (F) Transfected cells were pre-incubated with the GPR84 antagonist AR505962, followed by stimulation with sub-maximal concentrations of 3-hydroxydecanoate, decanoate, or embelin (N = 3–4). (G) PRESTO-Tango assay in HTLA cells transfected with human GPR109B (HCA3). The cells were stimulated with 3-hydroxydecanoate, the endogenous GPR109B agonist 2-hydroxyoctanoate (2-OH-C8), or the GPR109B reference agonist AR231783. N = 3. Data are shown as mean ± SEM The logEC50 values were calculated in GraphPad Prism using nonlinear regression.∗p < 0.05, ∗∗p < 0.01. p values were determined by one-way ANOVA followed by Tukey’s multiple comparison test (B–D). See also Figure S3 .

Article Snippet: Receptor plasmid, human GPR109B (HCA3) , Origene , N/A.

Techniques: Positive Control, Incubation, Luciferase, Transfection, Comparison

Journal: iScience

Article Title: Type 2 diabetes is associated with increased circulating levels of 3-hydroxydecanoate activating GPR84 and neutrophil migration

doi: 10.1016/j.isci.2022.105683

Figure Lengend Snippet:

Article Snippet: Receptor plasmid, human GPR109B (HCA3) , Origene , N/A.

Techniques: RNAscope, Recombinant, Enzyme-linked Immunosorbent Assay, Isolation, Reporter Gene Assay, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins *

doi: 10.1074/jbc.M114.569632

Figure Lengend Snippet: BAX and BID/BIM synergize to promote LUV permeabilization. A, LUVs were combined with indicated concentrations of BAX for 30 min at 37 °C. B and C, BAX was combined with indicated concentrations of BID or BIM for 30 min at 37 °C. D, BAX (25 nm) was combined with doses of BID (50 nm) or BIM (50 nm) for 30 min at 37 °C. All data are representative of at least triplicate experiments and reported as ± S.D. as required.

Article Snippet: The human BIM and PUMA BH3 domain peptides (>98% purity, Abgent), BID TAMRA and BIM TAMRA peptides (>90% purity, Anaspec), and BID/BID FAM /BIM FAM peptides (Hartwell Center, St. Jude Children's Research Hospital) were solubilized in anhydrous dimethyl sulfoxide in a N 2 environment, stored at −80 °C, and thawed only once.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins *

doi: 10.1074/jbc.M114.569632

Figure Lengend Snippet: BH3 mimetics display derepression activity for BAX from anti-apoptotic BCL-2 proteins. A, BAX (100 nm) was combined with anti-apoptotic proteins (A1, BCL-xL, or MCL-1; 100 and 250 nm) for 30 min at 37 °C. B, relative Ki for indicated BH3 mimetics and anti-apoptotic BCL-2 proteins. Ki values are summarized from the literature (19, 20, 23, 39,–43). ND, not determined. C and D, LUVs were combined with BAX (100 nm) or BH3 mimetics (5 μm) or BAX and BH3 mimetics for 30 min at 37 °C. BIM and PUMA BH3 domain peptides represents positive and negative controls for direct activation. E–G, LUVs were combined with BAX (100 nm), anti-apoptotic BCL-2 proteins (A1, BCL-xL, or MCL-1; 250 nm), and BH3 mimetics (5 μm) for 30 min at 37 °C. The PUMA BH3 domain peptide is a positive control for derepression (5 μm). All data are representative of at least triplicate experiments and are reported as ± S.D. as required.

Article Snippet: The human BIM and PUMA BH3 domain peptides (>98% purity, Abgent), BID TAMRA and BIM TAMRA peptides (>90% purity, Anaspec), and BID/BID FAM /BIM FAM peptides (Hartwell Center, St. Jude Children's Research Hospital) were solubilized in anhydrous dimethyl sulfoxide in a N 2 environment, stored at −80 °C, and thawed only once.

Techniques: Activity Assay, Activation Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins *

doi: 10.1074/jbc.M114.569632

Figure Lengend Snippet: BH3 mimetics display a derepression activity profile that differs for BIM-regulated BAX activation in the presence of A1, BCL-xL, and MCL-1. A, BAX (25 nm) and BIM (50 nm) were combined with anti-apoptotic proteins (A1, BCL-xL, or MCL-1; 50, 100, 175, 250 nm) for 30 min at 37 °C. B–D, BAX (25 nm), BIM (50 nm), anti-apoptotic BCL-2 proteins (A1, BCL-xL, or MCL-1; 250 nm) and BH3 mimetics (5 μm) for 30 min at 37 °C. The PUMA BH3 domain peptide is a positive control for derepression (5 μm). All data are representative of at least triplicate experiments and are reported as ± S.D. as required.

Article Snippet: The human BIM and PUMA BH3 domain peptides (>98% purity, Abgent), BID TAMRA and BIM TAMRA peptides (>90% purity, Anaspec), and BID/BID FAM /BIM FAM peptides (Hartwell Center, St. Jude Children's Research Hospital) were solubilized in anhydrous dimethyl sulfoxide in a N 2 environment, stored at −80 °C, and thawed only once.

Techniques: Activity Assay, Activation Assay, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins *

doi: 10.1074/jbc.M114.569632

Figure Lengend Snippet: BIM is differentially regulated by BH3 mimetics compared with BID. A, primary liver mitochondria were incubated BIMFAM (low, 50 nm; high, 100 nm) for 30 min at 37 °C, before determining the amount of mitochondria-associated fluorescence. B, primary liver mitochondria were loaded with BIMFAM (low, 50 nm; high, 100 nm) before treatment with indicated unlabeled BH3 peptides or drugs (5 μm) for 30 min at 37 °C. Mitochondria-associated fluorescence was adjusted to 100% with DMSO treatments. All data are representative of at least triplicate experiments and are reported as ± S.D. as required. C, same as B, but cytochrome c release was determined and reported as % intact mitochondria. D and E, HeLa cells were treated with indicated endoplasmic reticulum stress agents ± BH3 mimetics (5 μm) for 18 h before annexin V staining and flow cytometry analyses. F, heavy membranes isolated from HeLa cells treated with DTT (0, 2.5, 5 mm) or thapsigargin (Tg; 0, 4, 8 μm) for 12 h were subjected to SDS-PAGE and Western blot analyses for BIM. HSP60 is shown as a loading control. G and H, HeLa cells were treated with indicated endoplasmic reticulum stress agents ± BH3 mimetics (5 μm) for 18 h before annexin V staining and flow cytometry analyses. I, heavy membranes isolated from HeLa cells treated with DTT (5 mm) or thapsigargin (8 μm) ± BH3 mimetics (5 μm) for 18 h were subjected to SDS-PAGE and Western blot analyses for BIM. HSP60 is shown as a loading control. All data are representative of at least triplicate experiments and are reported as ± S.D. as required.

Article Snippet: The human BIM and PUMA BH3 domain peptides (>98% purity, Abgent), BID TAMRA and BIM TAMRA peptides (>90% purity, Anaspec), and BID/BID FAM /BIM FAM peptides (Hartwell Center, St. Jude Children's Research Hospital) were solubilized in anhydrous dimethyl sulfoxide in a N 2 environment, stored at −80 °C, and thawed only once.

Techniques: Incubation, Fluorescence, Staining, Flow Cytometry, Isolation, SDS Page, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: B Cell Lymphoma-2 (BCL-2) Homology Domain 3 (BH3) Mimetics Demonstrate Differential Activities Dependent upon the Functional Repertoire of Pro- and Anti-apoptotic BCL-2 Family Proteins *

doi: 10.1074/jbc.M114.569632

Figure Lengend Snippet: Fluorescence polarization reveals that BID and BIM BH3 domains are differentially derepressed from anti-apoptotic proteins by ABT-737, and a representation of how proapoptotic BCL-2 proteins influence the activity of BH3 mimetics is also shown. A, A1, BCL-xL, or MCL-1 (indicated concentrations) were incubated with BIDTAMRA or BIM TAMRA (50 nm) for 5 min at 25 °C before determining fluorescence polarization. B, polarization of BIDTAMRA or BIM TAMRA (50 nm) was induced by A1, BCL-xL, or MCL-1 (200 nm) coincubation, ABT-737 (5 μm) was added for 60 min, and the % depolarization was determined (upper panel). The % depolarization was normalized to the BCL-xL values and indicated in the lower panel. C, Ki values in Fig. 2B were converted to asterisks: 0–10 nm (*****), 11–100 nm (****), 101–500 nm (***), 501–1000 nm (**), and >1000 (*). D–F, maximal derepression activities by the PUMA BH3 domain peptide were normalized to 100%, and the resulting BH3 mimetic panel activities adjusted and divided into 20% increments: 0–20% (*), 21–40% (**), 41–60% (***), 61–80% (****), and 80–100% (*****) activity compared with the PUMA BH3. The different proapoptotic BCL-2 members investigated are represented as BAX (purple), BID (green), and BIM (blue) tables; this color scheme is consistent throughout the figures. Values presented in C are based on the literature, whereas the relationships in D–F are derived from the current investigation.

Article Snippet: The human BIM and PUMA BH3 domain peptides (>98% purity, Abgent), BID TAMRA and BIM TAMRA peptides (>90% purity, Anaspec), and BID/BID FAM /BIM FAM peptides (Hartwell Center, St. Jude Children's Research Hospital) were solubilized in anhydrous dimethyl sulfoxide in a N 2 environment, stored at −80 °C, and thawed only once.

Techniques: Fluorescence, Activity Assay, Incubation, Derivative Assay