Journal: Cell metabolism
Article Title: Macrophage Metabolism of Apoptotic Cell-Derived Arginine Promotes Continual Efferocytosis and Resolution of Injury.
doi: 10.1016/j.cmet.2020.01.001
Figure Lengend Snippet: Figure 5. The Arg1-ODC-Putrescine Pathway Drives the Expression of the Rac-GEF Dbl (A) Double-cell efferocytosis by ScrRNA-, siArg1-, or siOdc1-transfected macrophages that were treated with vehicle or IL-4, with some of the macrophages receiving 5 mM cytochalasin D 15 min before the addition of the second AC (n = 3–7 biological replicates). (B) First AC and second AC phagosomal Rac1 activity (phagosome YFP:CFP fluorescence ratio [YFP-FRET]—20 min relative 0 min) in macrophages from Raichu- Rac FRET transgenic mice that were transfected with ScrRNA, siArg1, or siOdc1; treated with vehicle or IL-4; incubated with a first set of ACs for 45 min; and then, 2 h later, incubated with a second set of ACs (n = 11–17 cells per group, 2 plates of macrophages per condition). (C) Second AC phagosomal Rac1 activity as in (B), except that some of the cells were treated with 100 mM putrescine with the first AC (n = 8–15 macrophages per group, 2 plates of macrophage per condition). (D and E) (D) Mcf2 mRNA and (E) Dbl expression in control or IL-4-treated macrophages that were incubated ± ACs for 45 min and then 2 h later harvested (n = 3 biological replicates). (F) Mcf2 mRNA in IL-4-treated macrophages that were pre-treated with bafilomycin A1 and incubated ± ACs for 45 min and then 2 h later harvested (n = 4 biological replicates). (G and H) (G) Mcf2 mRNA and (H) Dbl expression in IL-4-treated macrophages that were incubated ± 6-mm phosphatidylserine-coated latex beads or ACs for 45 min and then 2 h later harvested (n = 3 biological replicates). (I) Mcf2 mRNA in ScrRNA- or siOdc1-transfected macrophages that were treated with vehicle or IL-4, incubated ± ACs for 45 min with or without 100 mM pu- trescine, and then harvested 2 h later (n = 3 biological replicates).
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-Arg1 Cell Signaling Technology Cat# 93668; RRID:AB_2800207 (1:1000 dilution) Rabbit anti-Arg1 Sigma Aldrich Cat# HPA024006; RRID:AB_1844987 (1:200 dilution) Rabbit anti-iNOS Abcam Cat# ab213987 (1:200 dilution) Rat anti-Mac2 Cederlane Cat# CL8942AP; RRID:AB_10060357 (1:10,000 dilution) Rabbit anti-Dbl Cell Signaling Technology Cat# 2089; RRID:AB_2143816 (1:50 dilution) Mouse anti-b-actin Santa Cruz Biotechnology Cat# SC-47778; RRID:AB_626632 (1:5000 dilution) Rabbit anti-IL-1b Abcam Cat# ab9722; RRID:AB_308765 (1:200 dilution) Rabbit anti-TNFa Abcam Cat# ab9739; RRID:AB_308774 (1:200 dilution) Rabbit anti-HuR Sigma-Aldrich Cat# 03-102; RRID:AB_11211202 (1:1000 dilution) Rabbit anti-Otc Abcam Cat# ab203859 (1:500 dilution) Rabbit anti-Odc Abcam Cat# ab97395 RRID: AB_10679334 (1:200 dilution) Rabbit anti-Srm Abcam Cat# ab241496 (1:250 dilution) Rabbit anti-Pqlc2 Thermo Scientific Cat# PA5-48512; RRID:AB_2633969 (1:1000 dilution) Mouse anti-CD68 DAKO Cat# M0814; RRID:AB_2314148 (1:400 dilution) Rabbit IgG Sigma-Aldrich Cat# PP64B; RRID:AB_97852 Goat anti-mouse (HRP) Invitrogen Cat# 32230 (1:2000 dilution) Goat anti-rabbit (HRP) Invitrogen Cat# 32260 (1:2000 dilution) Goat anti-mouse (AF488) Invitrogen Cat# A11001; RRID:AB_2534069 (1:200 dilution) Goat anti-mouse (AF555) Invitrogen Cat# A28180; RRID:AB_2536164 (1:200 dilution) Goat anti-rat (AF488) Invitrogen Cat# A11006; RRID:AB_141373 (1:200 dilution) Goat anti-rabbit (AF488) Invitrogen Cat# A11034 (1:200 dilution) Goat anti-rabbit (AF555) Invitrogen Cat# A21428; RRID:AB_141784 (1:200 dilution) Goat anti-Rat (AF647) Invitrogen Cat# A21247; RRID:AB_141778 (1:200 dilution) Rat anti-CD16/CD32 BD Biosciences Cat# 553142; RRID:AB_394657 (1:100 dilution) (Continued on next page) e1 Cell Metabolism 31, 518–533.e1–e10, March 3, 2020
Techniques: Expressing, Transfection, Activity Assay, Transgenic Assay, Incubation, Control
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![Figure 5. The Arg1-ODC-Putrescine Pathway Drives the Expression of the Rac-GEF Dbl (A) Double-cell efferocytosis by ScrRNA-, siArg1-, or siOdc1-transfected macrophages that were treated with vehicle or IL-4, with some of the macrophages receiving 5 mM cytochalasin D 15 min before the addition of the second AC (n = 3–7 biological replicates). (B) First AC and second AC phagosomal Rac1 activity (phagosome YFP:CFP fluorescence ratio [YFP-FRET]—20 min relative 0 min) in macrophages from Raichu- Rac FRET transgenic mice that were transfected with ScrRNA, siArg1, or siOdc1; treated with vehicle or IL-4; incubated with a first set of ACs for 45 min; and then, 2 h later, incubated with a second set of ACs (n = 11–17 cells per group, 2 plates of macrophages per condition). (C) Second AC phagosomal Rac1 activity as in (B), except that some of the cells were treated with 100 mM putrescine with the first AC (n = 8–15 macrophages per group, 2 plates of macrophage per condition). (D and E) (D) Mcf2 mRNA and (E) Dbl expression in control or IL-4-treated macrophages that were incubated ± ACs for 45 min and then 2 h later harvested (n = 3 biological replicates). (F) Mcf2 mRNA in IL-4-treated macrophages that were pre-treated with bafilomycin A1 and incubated ± ACs for 45 min and then 2 h later harvested (n = 4 biological replicates). (G and H) (G) Mcf2 mRNA and (H) Dbl expression in IL-4-treated macrophages that were incubated ± 6-mm phosphatidylserine-coated latex beads or ACs for 45 min and then 2 h later harvested (n = 3 biological replicates). (I) Mcf2 mRNA in ScrRNA- or siOdc1-transfected macrophages that were treated with vehicle or IL-4, incubated ± ACs for 45 min with or without 100 mM pu- trescine, and then harvested 2 h later (n = 3 biological replicates).](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_4476/pm32004476/pm32004476__page10_image1.jpg)