Journal: Frontiers in Molecular Neuroscience

Article Title: The Alternative Complement System Mediates Cell Death in Retinal Ischemia Reperfusion Injury

doi: 10.3389/fnmol.2018.00278

Figure Lengend Snippet: Complement inhibitors gene expression was down-regulated in the IR retina. The mRNA levels of complement inhibitors, Crry (A,D) , Cd55 (B,E) , and Cd59a (C,F) in the retina of wild type control mice (WT), complement factor b-deficient mice ( Fb −/− ) and complement component 3-deficient mice ( C3 −/− ) at 6 h after IR injury were quantified by RT-PCR, and were normalized to sham surgery. N = 4 eyes/group. Data are presented as the mean ± SEM. * P < 0.05 vs. sham, ** P < 0.01 vs. sham, *** P < 0.001 vs. sham.

Article Snippet: Primers to Crry (Mm00787529_s1, ThermoFischer Scientific), Cd55 (Mm00438377_m1, ThermoFischer Scientific), Cd59a (Mm00483149_m1, ThermoFischer Scientific) were used for mouse retinas, and primers to Cd46 (Hs00611257_m1, ThermoFischer Scientific), Cd55 (Hs00892618_m1, ThermoFischer Scientific), and Cd59 (Hs00174141_m1; ThermoFischer Scientific) were used for HRECs with TaqMan Universal PCR Master Mix (4304437, ThermoFischer Scientific).

Techniques: Gene Expression, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a The cartoon representations of the substrates and products of the GWT1 enzyme are displayed. The chemical structure of manogepix, an inhibitor of GWT1 protein, is demonstrated. The hydroxyl group that accepts the palmitoyl group (yellow) is highlighted in the red circle . b Topology of fGWT1 presents the specific sites of GFP insertion. Through screening, A fusion of GFP at the Y422 position construct exhibited the most homogeneous protein properties. c FACS (fluorescence-activated cell sorting) of HEK293T △PIGW cells expressing fGWT1 (light green) using CD59 as the marker with gating strategies. HEK293T WT cells are highlighted in red, while individual HEK293T △PIGW cells are marked in blue. Those HEK293T △PIGW cells responding to fGWT1 are labeled in light green. The background served as a blank control, referring to HEK293T △PIGW cells not stained with CD59 antibodies. d Fluorescent confocal microscopy analyses were conducted to assess fGWT1 activity, with the construct and cell line indicated alongside the images. The first, second, and third columns illustrate confocal fluorescence microscopy images of CD59, DAPI, and merged views, respectively. A scale bar of 10 μm is included. The confocal microscopy experiment was repeated at least three times.

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Construct, Fluorescence, FACS, Expressing, Marker, Labeling, Control, Staining, Confocal Microscopy, Activity Assay, Microscopy

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a , d The cryo-EM map of GWT1 is displayed from both side ( a ) and top ( d ) perspectives, with the luminal access cavity highlighted by a blue dashed oval. b , e An additional density within the highly conserved luminal access cavity. c Molecular lipophilicity potential (MLP) of the luminal access cavity. The surfaces are colored by lipophilicity potential calculated by Chimera X. The putative entry route for GlcN-PI is indicated by a dashed blue path. f Comparison of catalytic sites of GWT1 and HGSNAT. The predicted catalytic sites of HGSNAT are N286 and H297, while the putative catalytic sites of GWT1 are D145 and K155. g Expressing fGWT1 in HEK293T △PIGW cells restores CD59 staining in the FACS assay. HEK293T △PIGW cells, where GPI-AP biosynthesis is blocked, serve as the staining control (gray). Loss-of-function mutants are highlighted in brilliant blue. Coordination residue mutants of the luminal access cavity are highlighted in purple.

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Cryo-EM Sample Prep, Comparison, Expressing, Staining, Control, Residue

Journal: Nature Communications

Article Title: Structural insights into the inhibition mechanism of fungal GWT1 by manogepix

doi: 10.1038/s41467-024-53512-x

Figure Lengend Snippet: a Previously reported drug resistance sites for GWT1 are shown as spheres and colored in gray-blue. b All drug resistance sites, including those previously reported and newly identified, are shown. c Evaluating the sensitivity of manogepix by staining CD59 in the FACS assay, expressing fGWT1 in HEK293T △PIGW cells. Cells treated with manogepix (orange) or without manogepix (blue) were analyzed by flow cytometry using gating strategies, with negative controls marked in red. All experiments were conducted independently three times ( n = 3).

Article Snippet: In experiments using CD59 as the GPI-anchored proteins (GPI-AP) reporter, an allophycocyanin (APC) -conjugated anti-CD59 antibody (Sino Biological) was applied at a 100-fold dilution for incubation with the cells for 1 h in the dark.

Techniques: Staining, Expressing, Flow Cytometry

Journal: PLoS ONE

Article Title: Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration

doi: 10.1371/journal.pone.0166348

Figure Lengend Snippet: Photomicrograph showing CD59a immunolabeling in all layers of mouse retina, RPE and choroid (B), but not in CD59aKO eyes (C). Graph of qPCR results showing that mRNA levels of CD59a in NSR were more than 60-fold higher than that in isolated RPE cells in WT eyes (** P <0.001) (D). Data are expressed as means ± SD. N = 4. RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: The primary antibodies were mouse anti-mouse CD59a (HM1116, Hycult, PA, USA) at 1:500 dilution and a mouse-on-mouse polymer IHC kit (ab127055, Abcam, MA, USA) was used to optimize signal to background staining ratio.

Techniques: Immunolabeling, Isolation

Journal: PLoS ONE

Article Title: Retinal Pre-Conditioning by CD59a Knockout Protects against Light-Induced Photoreceptor Degeneration

doi: 10.1371/journal.pone.0166348

Figure Lengend Snippet: Photomicrograph showing CD59a immunolabeling in all layers of mouse retina, RPE and choroid (B), but not in CD59aKO eyes (C). Graph of qPCR results showing that mRNA levels of CD59a in NSR were more than 60-fold higher than that in isolated RPE cells in WT eyes (** P <0.001) (D). Data are expressed as means ± SD. N = 4. RPE, retinal pigment epithelium; OS, photoreceptor outer segment; IS, photoreceptor inner segment; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer.

Article Snippet: Probes used were cd59a (CD59a, Mm01276239_m1), rhodopsin (Rho, Mm01184405_m1), fibroblast growth factor 2 (Fgf2, Mm00433287_m1), vascular endothelial growth factor (Vegf, Mm01281449_m1), glucose regulated protein 78 (Grp78, Mm00517690_g1), and glucose regulated protein 94 (Grp94, Mm00441926_m1).

Techniques: Immunolabeling, Isolation