Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: (A and B) C57BL/6J mice received an intranigral injection of sterile normal saline (2 μL) or LPS(3 μg in 2 μL of saline; 1.8×10 3 endotoxin unit [EU]). At 24 h after the injection, representative confocal double-labeling fluorescence images showed active morphology of CD11b-IR microglia/macrophages (M) and reduction in nuclear HMGB1 (n-HMGB1) after LPS injection. Arrowheads show strong staining of n-HMGB1 in ramified (resting) microglia/macrophages in saline-injected SN. Arrows show faint n-HMGB1 in active microglia/macrophages with amoeboid-like morphology in LPS-injected SN. H, HMGB1 (A). Quantification of n-HMGB1 staining of the maximum intensity projection of a z stack of 30 confocal images from a brain slice taken at 1 mm step size, which displayed 3D structure in a 2D image. We measured 431 and 863 CD11b-IR microglia/macrophages (M) as well as 1,196 and 1,026 CD11b-negative and DAPI-positive non-microglia/macrophage cells (non-M) in saline- and LPS-injected SN, respectively. Fluorescence intensity of n-HMGB1 staining per cell was calculated and normalized to each respective saline-injected control. n = 3 mice for each group. *p < 0.05; unpaired two-tailed Student’s t test (B). (C) Microglia-enriched cultures were treated with LPS (15 ng/mL), NO donor SNP (20 μM), or vehicle with or without 30-min pretreatment with the iNOS inhibitor 1400W (10 μM). Immunoblotting and densitometry analysis showed the levels of HMGB1 and iNOS in whole-cell lysates and the levels of extracellular HMGB1 in the concentrated culture medium 24 h after the treatment. n = 4. (D and E) Nuclear fractionation and immunoblotting analysis showed blockage of LPS-elicited HMGB1 secretion by 1400W at 24 h after LPS treatment of BV2 microglial cells. The nuclear marker histone H3 was examined to monitor loading errors of nuclear proteins (D). Densitometry quantification of HMGB1 level (E). n = 3. (F) The level of nitrite (an indicator of NO production) in the culture medium was measured at 24 h after microglia-enriched cultures were treated with LPS, poly(I:C), or SNP with or without 1400W pretreatment for 30 min. n = 3. (G) MTT assay revealed no cytotoxicity after microglial cultures were treated with LPS, SNP, and/or 1400W for 24 h. n = 3. *p < 0.05 compared with the corresponding control. # p < 0.05 compared with LPS-treated cultures; one-way ANOVA with Sidak’s multiple comparisons (C, E, F) and one-way ANOVA with Dunnett’s multiple comparisons test (G). Ctrl, control; ns, not significant.

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Injection, Labeling, Fluorescence, Staining, Slice Preparation, Two Tailed Test, Western Blot, Fractionation, Marker, MTT Assay

Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: Mac1 −/− and WT (C57BL/6) mice received an intranigral injection of endotoxin-free HMGB1 (2 μg) or BSA (2 μg; as a control). One month later, PD-like phenotypes were examined. (A and B) HMGB1-elicited nigral microglia/macrophage activation was shown by elevated immunoreactivity of Iba1, CD11b, and TMEM119; enlarged size; and irregular shape in WT mice but not Mac1 −/− mice. Decreased number of TH-IR neurons and damaged integrity of TH-IR fibers indicated HMGB1-elicited dopaminergic neurodegeneration only in WT mice (A). Eight and four evenly spaced brain sections from a series of 24 sections that covered the entire SN were used for the count of TH-IR neurons and the measurement of the optical density of Iba1 immunoreactivity in nigral microglia/macrophages, respectively, by two individuals blind to the treatment (B). n = 3 mice for each group. (C) The rotarod behavior test showed HMGB1-elicited impairment of locomotor activity in WT mice but not Mac1 −/− mice. n = 5 mice for each group. (D) The open field test did not show alteration in exploratory activity or anxiety-related activities in HMGB1- or BSA-injected WT or Mac1−/− mice. n = 5 mice for each group. *p < 0.05 compared with BSA-injected controls; two-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Injection, Activation Assay, Activity Assay

Journal: Cell reports

Article Title: Posttranslational S-nitrosylation modification regulates HMGB1 secretion and promotes its proinflammatory and neurodegenerative effects

doi: 10.1016/j.celrep.2022.111330

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Paraformaldehyde-fixed mouse brain sections and primary microglial cultures grown in glass-bottom microwell dishes (MatTek Corp, MA) or Lab-Tek II chamber slides (Nalge Nunc, Rochester, NY) were immunostained using rabbit polyclonal HMGB1 antibody (1:2000; ChIP Grade; Abcam; Cat# ab18256; RRID:AB_444360), in combination with rat polyclonal CD11b antibody (1:500; Bio-Rad Laboratories; Cat# MCA711G; RRID:AB_323167) or mouse monoclonal S-nitroso-cysteine (SNO-C) antibody (1:1000; Abcam; Cat# ab94930; RRID:AB_10697568).

Techniques: Recombinant, Protease Inhibitor, Western Blot, Transfection, Modification, Plasmid Preparation, Clone Assay, Mutagenesis, Software

Journal: Translational Vision Science & Technology

Article Title: When Is a Control Not a Control? Reactive Microglia Occur Throughout the Control Contralateral Pathway of Retinal Ganglion Cell Projections in Experimental Glaucoma

doi: 10.1167/tvst.10.1.22

Figure Lengend Snippet: Antibody Details

Article Snippet: PE CD11b/c , CD11b (clone OX-42) , Mouse , 1:400 , Nordic BioSite cat # 201807 , FC .

Techniques:

Journal: Neural Regeneration Research

Article Title: Argon reduces microglial activation and inflammatory cytokine expression in retinal ischemia/reperfusion injury

doi: 10.4103/1673-5374.290098

Figure Lengend Snippet: Microglial activation in whole-mount specimen after immunoperoxidase labeling with CD11b (OX-42). (A) Visualization of the morphology of retinal microglia by optical microscopy: Control eye, after ischemia/reperfusion injury (IRI), after IRI and argon post-conditioning, and after IRI treatment after application of the TLR2 and TLR4 inhibitor oxidized phospholipid 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine (OxPAPC, 1.5 mg/kg). Typical changes such as increase of microglial cell bodies, thickening and asymmetric distribution of extensions showing activation after ischemia/reperfusion, which is attenuated by argon (arrows). (B) Statistical analysis of microglial density. Argon significantly reduced the density increase due to IRI. Argon alone had no effect in the absence of IRI treatment. The previous application of OxPAPC prior to IRI neutralized the effect of argon. n = 6. Data are expressed as the mean ± SD. * P < 0.05, *** P < 0.001 (one-way analysis of variance with post-hoc Holm Sidak test).

Article Snippet: After blocking in 10% normal goat serum for 1 hour, the retinas were incubated in PBS plus 0.5% Triton X-100 for 3 days at 4°C under stirring with the primary antibody mouse-anti-rat integrin-alpha M (CD11b) clone OX-42 (1:500; Cat# 1512; Chemicon, Temecula, CA, USA).

Techniques: Activation Assay, Labeling, Microscopy