Journal: Cell reports

Article Title: LARP7 ameliorates cellular senescence and aging by allosterically enhancing SIRT1 deacetylase activity.

doi: 10.1016/j.celrep.2021.110038

Figure Lengend Snippet: Figure 2. The declined SIRT1 deacetylase activity accounted for LARP7-depletion-induced senescence (A) Western blotting assessing the senescent regulators in IMR90 receiving LARP7 shRNA knockdown for 3 days. (B) Western blotting assessing the senescent regulators in IMR90 receiving LARP7 shRNA knockdown for 2 weeks. (C) LARP7 knockdown for 3 and 14 days in IMR90 cells increased the p53 K382Ac and p65 K310Ac level. The control immunoglobulin G (IgG) (rabbit for p65 and mouse for p53) didn’t pull down either p53 or p65 indicating the specificity of immunoprecipitation. (D) Knocking down LARP7 suppressed the nuclear SIRT activity on days 3 and 14. 10 mM EX527 was used as a positive control. n = 3. (E) The increased p53 and p65 acetylation upon LARP7 depletion was blocked by SRT1720. shCtrl- or shLARP7-transfected IMR90 cells were treated with 1 mM SRT1720 for 12 h and then subjected to immunoprecipitation. (F) qRT-PCR showing SRT1720 abrogated the SASP genes activation. IMR90 cells were treated with 2 mM JSH-23 for 48 h as a positive control. n = 3.

Article Snippet: N/A Bacterial and virus strains BL21/DE3 E. coli NEB Cat #C2527 Biological samples Human coronary artery samples The University of Pennsylvania Human Heart Tissue Bank IRB protocol number: 802781 Chemicals, peptides, and recombinant proteins Tamoxifen Sigma Cat #T5648; CAS:10540-29-1 SRT1720 Selleck Cat #S1129; CAS: 1001645-58-4 KU60019 Selleck Cat #S1570; CAS: 925701-49-1 Oil Red Powder Sigma Cat #O0625; CAS: 1320-06-5 EX527 Selleck Cat #S1541; CAS: 49843-98-3 Fetal bovine serum ExCell Bio Cat #FSP500 Acetyl-CoA Sigma Cat #A2181; CAS: 32140-51-5 NAD+ Selleck Cat #S2518; CAS: 53-84-9 Critical commercial assays Fast Mutagenesis System kit TransGen Biotech Cat #FM111S Senescence b-Galactosidase Staining Kit Cell Signaling Technology Cat #9860S NAD/NADH Quantification Kit Sigma Cat #MAK037 Universal SIRT Activity Assay Kit Abcam Cat #ab156915 Deposited data RNA-Seq This paper GSE160279 Processed RNA-Seq data This paper Table S1 (Continued on next page) e1 Cell Reports 37, 110038, November 23, 2021

Techniques: Histone Deacetylase Assay, Activity Assay, Western Blot, shRNA, Knockdown, Control, Immunoprecipitation, Positive Control, Transfection, Quantitative RT-PCR, Activation Assay

Journal: Parasite immunology

Article Title: SEA Alleviates Hepatic Ischaemia-Reperfusion Injury by Promoting M2 Macrophage Polarisation.

doi: 10.1111/pim.13061

Figure Lengend Snippet: FIGURE 4 | SEA pretreatment promoted the polarisation of M2 macrophages. Mice were subjected to 60 min of hepatic ischaemia, followed by 6 h of reperfusion. (A) Representatives of immunofluorescence staining of CD68 (n = 5 per group, five biological replicates from five individual animals; original magnification 100× and 200×, scale bar: 100 μm). (B) Representatives of immunofluorescence staining of CD163 (n = 5 per group, five biological replicates from five individual animals; original magnification 100× and 200×, scale bar: 100 μm). (C, D) The expression of M2 macrophage marker (CD206 and Arg1) in the liver was detected via qRT-PCR (n = 5 per group, five biological replicates from five individual animals). These results were obtained from at least three independent experiments. Values are presented as mean ± SEM. **p < 0.01, *p < 0.05, ns > 0.05.

Article Snippet: Then the sections were incubated with primary antibodies dissolved in 5% donkey serum solution containing 0.3% Triton at 4°C overnight (anti- MPO, Cat# ab208670, Abcam, 1:100; anti- CD68, Cat# ab53444, Abcam, 1:100; anti- CD163, Cat# 16646- 1- AP, Proteintech, 1:100).

Techniques: Immunofluorescence, Staining, Expressing, Marker, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Alpha-tocopherol inhibits ferroptosis and promotes neural function recovery in rats with spinal cord injury via downregulating Alox15.

doi: 10.1016/j.biopha.2024.116734

Figure Lengend Snippet: Fig. 3. Alpha-tocopherol alleviates ferroptosis after SCI. (A-B) Prussian Blue Iron Staining (Enhance With DAB) and quantitation. The positive stained tissue was observed in SCI group. The positive cells (iron overloaded cells) were colored by yellow-brown, the negative cells (normal cells) were colored by purple or blue. (C-F) WB and quantitation of Alox15, Ptgs2 and 4Hne in vivo. (G-J) IF and quantitation of Alox15 and Ptgs2. NeuN was colored by red, Alox15 and Ptgs2 were colored by green. n = 5 for each group. Data are represented as the means ± SD. ***p < 0.001.

Article Snippet: Slices were incubated overnight at 4°C with the primary antibodies against Alox15 (rabbit, 1:200, Affinity, Cat# DF13494, RRID: AB_2846513), Ptgs2 (rabbit, 1:200, Affinity, Cat# AF7003, RRID: AB_2835311), Gapdh (mouse, 1:200, Proteintech, Wuhan, China, Cat# 60004–1-Ig, RRID: AB_2107436) and neuronal nuclei (NeuN, mouse, 1:200, Proteintech, Cat# 66836–1-Ig, RRID: AB_2882179).

Techniques: Staining, Quantitation Assay, In Vivo

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Alpha-tocopherol inhibits ferroptosis and promotes neural function recovery in rats with spinal cord injury via downregulating Alox15.

doi: 10.1016/j.biopha.2024.116734

Figure Lengend Snippet: Fig. 6. Alpha-tocopherol downregulates expression of Alox15 and Ptgs2 in PC12 cells. (A-B) QPCR for mRNA of Alox15 and Ptgs2. (C-F) WB and quantitation of Alox15, Ptgs2 and 4Hne in vitro. (G-J) IF and quantitation of Alox15 and Ptgs2. Alox15 and Ptgs2 were colored by green. n = 3 for each group. Data are represented as the means ± SD. **p < 0.01, ***p < 0.001.

Article Snippet: Slices were incubated overnight at 4°C with the primary antibodies against Alox15 (rabbit, 1:200, Affinity, Cat# DF13494, RRID: AB_2846513), Ptgs2 (rabbit, 1:200, Affinity, Cat# AF7003, RRID: AB_2835311), Gapdh (mouse, 1:200, Proteintech, Wuhan, China, Cat# 60004–1-Ig, RRID: AB_2107436) and neuronal nuclei (NeuN, mouse, 1:200, Proteintech, Cat# 66836–1-Ig, RRID: AB_2882179).

Techniques: Expressing, Quantitation Assay, In Vitro

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Alpha-tocopherol inhibits ferroptosis and promotes neural function recovery in rats with spinal cord injury via downregulating Alox15.

doi: 10.1016/j.biopha.2024.116734

Figure Lengend Snippet: Fig. 7. Site-directed mutagenesis on Alox15 reverses protective effects of alpha-tocopherol. (A-B) WB validation of Alox15 knockout. (C) Erastin IC50 for Alox15-KO cells after transfection with different plasmids. (D) 48 hours after transfection, cells were treated with 5 μM erastin for 12 hours, with or without treatment of alpha- tocopherol or liproxstatin-1. Cell viabilities were detected by cck-8 assays. (E) 48 hours after transfection, cells were treated with 5 μM erastin for 12 hours, with or without treatment of alpha-tocopherol or liproxstatin-1. Alox15, Ptgs2 and 4Hne expressions were detected by WB. (F-H) Quantitation of WB. (I) Lipid oxidation detected by C11-BODIPY (581/591) probe. Green indicates oxidized lipids and red indicates non-oxidized lipids. (J) Quantitation of C11. n = 3 for each group. Data are represented as the means ± SD. ns: no significance, ***p < 0.001.

Article Snippet: Slices were incubated overnight at 4°C with the primary antibodies against Alox15 (rabbit, 1:200, Affinity, Cat# DF13494, RRID: AB_2846513), Ptgs2 (rabbit, 1:200, Affinity, Cat# AF7003, RRID: AB_2835311), Gapdh (mouse, 1:200, Proteintech, Wuhan, China, Cat# 60004–1-Ig, RRID: AB_2107436) and neuronal nuclei (NeuN, mouse, 1:200, Proteintech, Cat# 66836–1-Ig, RRID: AB_2882179).

Techniques: Mutagenesis, Biomarker Discovery, Knock-Out, Transfection, CCK-8 Assay, Quantitation Assay