Journal: Frontiers in Molecular Neuroscience

Article Title: Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

doi: 10.3389/fnmol.2020.00100

Figure Lengend Snippet: Effects of hypothermia on the immunoreactivity of anti-induced nitric oxide synthase (iNOS)-positive microglia using immunofluorescence assays. (A) Immunofluorescence images showing the BV2 microglia following OGD/R labeled with the iNOS antibody or secondary antibody (control negative). Red fluorescence indicates iNOS-positive cells, while blue fluorescence indicates 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)-labeled nuclei. Scale bar: 20 μm. (B) Dot plots (upper panel) and a bar graph (lower panel) showing a quantitative analysis of red/blue fluorescence ratios (25 random observations for each group). Values show the mean ± SD, n = 5. * P < 0.05, vs. sham group; θ P < 0.05, vs. OGD group; φ P < 0.05, vs. OGD/R2h-HT groups; # P < 0.05, vs. OGD/R2h-NT groups; ▴ P < 0.05, vs. OGD/R8h-NT groups.

Article Snippet: The materials used in this study are as follows: BV2 microglial cell lines (Procell, Wuhan, China); high glucose Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Logan, UT, USA); glucose-free DMEM (Jinuo Technologies, Hangzhou, China); 10% fetal bovine serum (FBS; Gibco Grand Island, NY, USA); cell counting kit-8 (CCK-8, Dojindo Molecular Technologies, Tokyo, Japan); 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1, Beyontime, Shanghai, China); tumor necrosis factor (TNF) α, interleukin (IL)-1β and (IL)-10 enzyme-linked immunosorbent assay (ELISA) kits (R & D Systems, Minneapolis, MN, USA); rabbit anti-induced nitric oxide synthase (iNOS) antibody (Proteintech, Wuhan, China); rabbit anti-arginase 1 (Arg1) antibody (Proteintech, Wuhan, China); mouse anti-ubiquitin antibody (Thermo Fisher Scientific, Waltham, MA, USA); rabbit anti-VDAC3 antibody (Aviva Systems Biology, San Diego, CA, USA); rabbit anti-caspase3 antibody (Proteintech, Wuhan, China); mouse anti-beta actin antibody (Abcam, Cambridge, UK); CoraLite594-conjugated goat anti-rabbit (red) IgG (Proteintech, Wuhan, China); CoraLite488-conjugated goat anti-mouse (green) IgG (Proteintech, Wuhan, China); horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Abcam, Cambridge, UK); SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories, Hercules, CA, USA); RNAprep Pure Cell kit (Tiangen Biotech, Beijing, China); first strand cDNA synthesis kit (Abm, Richmond, BC, Canada); EvaGreen 2× quantitative polymerase chain reaction (qPCR) master mix kit (Abm, Richmond, BC, Canada).

Techniques: Immunofluorescence, Labeling, Control, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: EphA5 is expressed in human lung cancer cells and in patient-derived lung cancer specimens. A , expression of EphA5 in a panel of human lung cancer cells. B–D , quantitative real time PCR of EPH receptors and ephrins in H460 ( B ), H1299 ( C ), and H522 ( D ) cells. E , overall assessment of EphA5 expression in ACC and SCC samples. High levels of EphA5 expression were predominantly associated with SCC versus ACC. p < 0.0005 (Wilcoxon rank sum test). Means are indicated by ×. F , representative microphotographs of EphA5 staining in ACC and SCC (magnification, ×200). Insets , irrelevant IgG used as negative control. Bottom panels show low expression of EphA5 in normal lung. M.W. , molecular mass.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Derivative Assay, Expressing, Real-time Polymerase Chain Reaction, Staining, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: Evaluation of EphA5 function in human lung cancer. A , expression of EphA5 in H460 human lung cancer cells before and after shRNA silencing. B and C , soft agar assay ( B ) showing a reduced number of colonies formed after EphA5 silencing and tumor intake ( C ) after subcutaneous cell implantation in mice. D–F , cell cycle distribution of H460 lung cancer cells after IR. Fractions of H460 control cells ( D ), H460 EphA5-shRNA ( E ), and H460 EphA5-rescued cell ( F ) in G 1 and S phases. EphA5-silenced cells do not arrest in G 1 upon IR-induced DNA damage, as indicated by a 2-fold decrease in the G 1 to S ratio. *, p < 0.05 (Student's t test). G , surviving fractions of A549 and H226 cells compared with H460 cells. H–J , surviving fractions of control and EphA5-shRNA cells as a function of increasing doses of IR. H460 ( H ), H1299 ( I ), and H522 ( J ). Error bars indicate S.D. Experiments were performed two times with similar results. *, p < 0.05. K–M , surviving fractions of A549 control and EphA5-shRNA cells ( K ), H226 control and EphA5-shRNA cells ( L ), and human pulmonary fibroblasts expressing or not human EphA5 ( M ) as a function of increasing doses of IR. Error bars indicate S.D. N , quantification of pATM foci in H460 control, EphA5-shRNA, and EphA5-rescued cells treated with 3 Gy of IR and evaluated 10 min and 24 h after irradiation. Error bars indicate S.D. Experiments were performed two times with similar results. *, p < 0.05.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Expressing, shRNA, Soft Agar Assay, Control, Irradiation

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: Expression of EphA5 in patients receiving IR treatment prior to surgical resection of lung cancer. A , high levels of EphA5 expression are directly associated with radiotherapy failure. p = 0.0021 (log rank test). B and C , locoregional recurrence ( B ) and cumulative overall patient survival ( C ) rates as a function of EphA5 expression.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Expressing

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: EphA5 and ATM interact at sites of DNA damage repair. A and B , a series of solution binding assays with pATM, ATM, and domains of EphA5 demonstrate that specific binding occurs only between pATM and the complete cytoplasmic domain of EphA5. A , red oval indicates the cytoplasmic domain of EphA5 coimmunoprecipitated ( IP ) with pATM. B , controls to confirm the presence of ATM and pATM in each experimental condition ( left panels ) and to rule out nonspecific interactions between pATM and the GST tag ( right panel ). C , immunoblot of fractionated irradiated cells immunoprecipitated with anti-pATM ( left panel ) or anti-EphA5 ( right panel ) antibodies. Coimmunoprecipitation of EphA5 and pATM is observed in both chromatin-enriched and nuclear soluble fractions. D , control nonirradiated H460 cells or cells treated with 3 Gy of IR were fractionated into membrane/cytosol, nuclear soluble, and DNA-associated proteins (chromatin-enriched). EphA5 expression is shown in the membrane/cytosol of control and treated cells. EphA5 translocation and interaction with DNA is observed only in IR-treated cells. Orc2 and MEK1/2 were used as controls for effective fractionation. E–H , confocal analysis of EphA5 expression and distribution in H460 cells before and after IR; DAPI staining of the nuclei is shown in blue. E , EphA5 and pATM in untreated cells. F , Z-stack of EphA5 expression in untreated H460 cells. White dashed lines point to orthogonal planes, showing EphA5 distribution in the perinuclear region of the cellular cytoplasm. G , coimmunostaining of EphA5 and pATM 10 min after cell irradiation. White arrowheads point to nuclear foci of active DNA repair containing colocalizing pATM and EphA5. H , orthogonal planes of a Z-stack image, confirming colocalization of EphA5 and pATM at nuclear sites of DNA damage repair. Scale bar , 10 μm. I , irradiation-induced phosphorylation of Chk2 is impaired in EphA5-silenced cells. Immunoblotting and quantification of protein bands show reduced phosphorylation of Chk2 in EphA5-shRNA cells compared with control cells (NT-shRNA). K and L , phosphorylation and nuclear import of EphA5. Cytoplasmic ( lanes C ) and nuclear ( lanes N ) proteins were isolated from nonirradiated H460 cells or from cells treated with IR and were subjected to immunoprecipitation with an anti-EphA5 antibody. Detection of phosphorylated and total EphA5 was performed with an anti-phosphotyrosine antibody or an antibody against EphA5 ( K ). Phosphorylated EphA5 was detected primarily in the nuclear fractions of irradiated H460 cells. The cytoplasmic protein MEK1/2 and the nuclear protein Orc2 ( L ) were used as control for fractionation efficiency.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Binding Assay, Western Blot, Irradiation, Immunoprecipitation, Control, Membrane, Expressing, Translocation Assay, Fractionation, Staining, Phospho-proteomics, shRNA, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: EphA5 acts through p53 to control the fate of lung cancer cells. A , percentage of apoptotic H1299 p53-null control and EphA5-shRNA cells before and after IR. Approximately 60% of H1299 EphA5-shRNA p53-null cells die after IR. Error bars indicate S.D. Experiments were performed three times with similar results. B , senescent H1299 control and EphA5-shRNA cells as determined by SA-β-gal staining. Panels show illustrative photomicrographs of cells stained for SA-β-gal activity postirradiation. Error bars indicate S.D. *, p < 0.05 (Student's t test). C , percentage of apoptotic H460 EphA5-shRNA p53-shRNA and control cells before and after IR. An increase of ∼30% in cell death is observed after IR. Error bars indicate S.D. Experiments were performed three times with similar results. D , the role of p53 in an EphA5-dependent cellular senescence pathway was studied in H460 EphA5-shRNA cells with or without p53-shRNA. The concomitant silencing of p53 and EphA5 abrogates the capacity of cells to become senescent. Error bars indicate S.D. *, p < 0.05 (Student's t test). Experiments were performed two times with similar results.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Control, shRNA, Staining, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: Validation of anti-EphA5 monoclonal antibody 11C12. A , ELISA showing binding of 11C12 to human and rat EphA5. The monoclonal antibody 11C12 does not recognize mouse EphA5. B and C , epitope identification was performed by peptide ( Pept ) mapping ( B ) using ELISA ( C ). D and E , receptor-mediated internalization ( D ) and receptor degradation ( E ) were also evaluated.

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Receptor Tyrosine Kinase EphA5 Is a Functional Molecular Target in Human Lung Cancer *

doi: 10.1074/jbc.M114.630525

Figure Lengend Snippet: The monoclonal antibody 11C12 radiosensitizes lung cancer cells in vitro and targets EphA5-expressing tumors in vivo . A , surviving fraction of A549 cells (low EphA5 expression) treated with 11C12 or a control IgG. B and C , surviving fractions of H460 ( B ) and H522 ( C ) human lung cancer cells treated with the mAb 11C12 and subjected to increasing doses of IR. Isotype IgG was used as control. *, p < 0.05 (Student's t test). D , illustrative pictures of colony counts (H460 cells). Experiments were performed two times with similar results. E and F , illustrative SPECT/CT images of the distribution of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG in tumor-bearing rats at 48, 96, and 144 h after antibody injection. Arrows point to the locations of the tumors. G , antibody concentration (n m ) in the tumors as a function of time after injection. H , tumor to heart ratio of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG as an indication of targeting specificity. I , distribution of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG in the brains of rats bearing tumors. J , distribution of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG in the kidneys of rats bearing tumors. K , distribution of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG in the liver of rats bearing tumors. L , tumor-focused images of mice inoculated with 111 In-DTPA-11C12 and 111 In-DTPA-control IgG. Maximum and minimum values of ID/g (injected dose per gram of tissue) are indicated by the color bar , where white represents the highest value, and dark blue represents the lowest value. M , longitudinal radial profiles of the distribution of 111 In-DTPA-11C12 and 111 In-DTPA-control IgG within tumors. Radial profiles of mean uptake were calculated for each tumor at each time point. The surface plots illustrate the tumor activity profiles as a function of time for 11C12 ( red ) and control IgG ( blue ).

Article Snippet: Rescue of EphA5 expression in EphA5-silenced cells was performed with an expression vector containing the human EphA5 receptor cDNA ORF (NM_004439.6), purchased from Origene (RG213206).

Techniques: In Vitro, Expressing, In Vivo, Control, Single Photon Emission Computed Tomography, Injection, Concentration Assay, Activity Assay

Journal: Frontiers in Endocrinology

Article Title: Comparative analysis of co-culture and monoculture models in simulating diabetic neurovascular dysfunction: insights into diabetic retinopathy

doi: 10.3389/fendo.2023.1215218

Figure Lengend Snippet: Morphology of RGCs cells in monoculture and co-culture models. (A) Shows the morphology of RGCs observed with an inverted fluorescent microscope (magnification 200×). (B) Shows the proportion of RGCs with neurite extensions. Green arrow: Rabbit anti-beta-III tubulin-labeled RGCs. Red arrow: Mouse anti-Brn3a-labeled RGCs; Blue arrow: DAPI labeled RGCs nuclei; White arrow: Synthetic plot of cellular immunofluorescence staining of labeled RGCs; RGCs: Rat retinal ganglion cells; Brn3a: Brain-specific homeobox/POU domain protein 3A; DAPI: 4’,6-diamidino-2-phenylindole; Co, Co-culture; Mono, Monoculture; HG, High glucose; NG, Normal glucose. *The difference is statistically significant, P <0.05.

Article Snippet: Other chemicals, including rabbit anti-beta-III tubulin (TUJ1) antibody (Cat#ab18207), goat anti-rabbit (Cat# Cat#ab150116), and goat anti-mouse FITC fluorescent (Cat#ab150077) antibody (Abcam, Cambridge, Britain), mouse anti-brain-specific homeobox/POU domain protein 3A (Brn3a) (Cat#sc-8429, Santa, Cruz, USA), occludin (OCLN), zonula occludens-1 (ZO-1) ELISA kit (Cloud-Clone Crop, Wuhan, China), Trizol solution (Biomiga, San Diego, USA) and cDNA synthesis kit (SYBR qPCR Master Mix, Novoprotein, Shanghai, China) were purchased previously.

Techniques: Co-Culture Assay, Microscopy, Labeling, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) Existing model by which SIRPα suppresses phagocytosis by interacting in trans with CD47 on target cells. See text for details. The 3 Ig-like domains of SIRPα (1 IgV and 2 IgCs) and the single Ig-V domain of CD47 are shown as ellipses. Mβs, macrophages. ( B ) Depiction of SIRPα variants and their functional characteristics. SIRPα FFFF contained substitution of tyrosine (Y)-to-phenylalanine (F) substitution at Y436, 460, 477, and 501; SIRPα ΔIC lacked most of the cytoplasmic domain of SIRPα, ending with arginine 401; SIRPα T96V carried a threonine (T)-to-valine (V) mutation at position 96 (shown by lavender star), which abolishes CD47-binding; SIRPα T96V,FFFF had the T96V and FFFF mutations; SIRPα T96V,ΔIC had the T96V and the ΔIC mutations. KO, knock-out. ITIM, immunoreceptor tyrosine-based inhibitory motif. ( C to G ) SIRPα variants or empty vector were expressed in SIRPα KO BMDMs and tested. Wild-type (WT) BMDMs were used as control. ( C ) Schematic representation of assays performed. Fc, fragment crystallizable. ( D ) Flow cytometry analyses of SIRPα expression and CD47-binding. APC, allophycocyanin. AF647, Alexa fluor 647. ( E and F ) Representative ( E ) and compiled data ( F ) of pHrodo-based phagocytosis assays using L1210 derivatives expressing Tac and opsonized with Tac monoclonal antibody (mAb) 7G7, as targets. Positive cells with percentages are boxed. G , Efficiency of phagocytosis inhibition in SIRPα KO BMDMs expressing or not the indicated SIRPα variants was calculated using the values in ( F ). SIRPα KO expressing WT SIRPα or empty vector displayed 100% and 0% inhibition efficiency, respectively. All data are means ± s.e.m., **** p < 0.0001. Results in ( D and E ) are representative of 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that are representative of 3 experiments. Results in ( F and G ) are pooled from a total of 6 mice studied in 6 independent experiments, except for SIRPα T96V , SIRPα T96V, FFFF and SIRPα T96V, ΔIC that involved 3 mice in 3 experiments. Each symbol in ( F ) represents one mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Functional Assay, Mutagenesis, Binding Assay, Knock-Out, Plasmid Preparation, Control, Flow Cytometry, Expressing, Inhibition

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) Immunoprecipitation followed by mass spectrometry of SIRPα-associated proteins. ( A ) Schematic representation of assay. ( B ) Plasma membrane-associated proteins found in SIRPα immunoprecipitates from WT BMDMs, but not from SIRPα KO BMDMs. c , Co-immunoprecipitation assay of SIRPα, CD18 and CD11b in WT and SIRPα KO BMDMs. IP, immunoprecipitation. Abs, antibodies. ( D to F ) FRET assays. ( D ) Schematic representation of FRET assay in HEK293T cells. ( E and F ) Representative confocal microscopy images ( E ) and compiled data ( F ) of FRET assays with donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of control (Ctrl) IgG, CD18 mAb GAME-46 or CD11b mAb 5C6. Yellow to purple spectrum denotes strong to weak FRET. DIC, differential interference contrast. Scale bars, 5 μm. ( G and H ) LUV-FRET assay. ( G ) Schematic representation of LUV-FRET assay. ( H ), Time-course of donor-labeled SIRPα fluorescence intensity after addition of acceptor-labeled CD18 or CD11b, monitored with a real-time plate reader. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( C , E and H ) are representative of 3 independent experiments. Results in ( B and F ) are pooled from a total of 3 independent experiments. Each symbol in ( F ) represents one cell.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Confocal Microscopy, Labeling, Control, Fluorescence

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A and B ) FRET assays with SIRPα and SIRPβ1a. ( A ) A schematic representation of SIRPα and SIRPβ1a, with their 1 IgV domain and 2 IgC domains, is depicted. ( B ) Compiled data of 3 independent experiments using donor-labeled SIRPα or SIRPβ1a, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( C and D ) FRET assays using SIRPα IgV domain. ( C ) A schematic representation of a SIRPα variant having only the IgV domain is shown. ( D ) Compiled data of 3 independent experiments using donor-labeled SIRPα IgV, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( E - H ) FRET assays using SIRPα variants carrying non-conserved residues from SIRPβ1a. ( E and G ) Schematic representations of SIRPα variants. ( F and H ) Compiled data from 3 independent experiments using donor-labeled SIRPα variants, acceptor-labeled CD18 and unlabeled CD11b, as done for , D to F. ( I to K ) Proximity ligation assay (PLA) of SIRPα and CD18 in BMDMs expressing or not the indicated SIRPα variants. (I) Flow cytometry analyses of SIRPα expression. ( J and K ) Representative confocal microscopy images ( J ) and compiled data from 3 independent experiments ( K ) of PLA for SIRPα and CD18. Scale bar, 10 μm. All data are means ± s.e.m. ns, not significant, **** p < 0.0001. Results in ( I and J ) are representative of 3 independent experiments. Results in ( B , D , F , H and K ) are pooled from 3 independent experiments. Each symbol in ( B , D , F , H and K ) represents one cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Variant Assay, Proximity Ligation Assay, Expressing, Flow Cytometry, Confocal Microscopy

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A to C ) The impact of SIRPα variants defective in CD18-binding, CD47-binding or phosphatase signaling, alone or in combination, expressed in BMDMs, was analyzed. ( A ) Schematic depictions of SIRPα variants, as was done for . SIRPα R91T carried an arginine (R)-to-threonine (T) mutation at position 91 (shown by blue star), which abolished CD18-binding. ( B ) Phagocytosis assays of IgG-opsonized L1210 cells by BMDMs, as was done for . ( C ) Efficiency of phagocytosis inhibition was calculated as for , using values from . ( D and E ) Representative flow cytometry profiles ( D ) and compiled data from 3 independent experiments ( E ) of ICAM-1-binding using SIRPα KO BMDMs expressing WT SIRPα or SIRPα R91T BMDMs, in the presence or absence of FcR triggering using mouse IgG2a. ( F and G ) The impact of a SIRPα variant carrying the isoleucine-to-glycine 332 (I332G) mutation, expressed in SIRPα KO BMDMs, was analyzed. (F) Flow cytometry analyses of CD11b expression. ( G ) Compiled data from 3 independent phagocytosis assays, assessed by microscopy. ( H ) FRET assays of donor-labeled SIRPα, acceptor-labeled CD18 and unlabeled CD11b in the presence of WT CD11b or CD11b I332G , as was done for , D to F. ( I ) FRET assays of donor-labeled human SIRPα version (V) 1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b, in the presence of Ctrl IgG, human CD18 mAbs CBR LFA1/2 or TS1/18, as was done for , D to F. ( J ) Phagocytosis of human lymphoma cells Raji, which were opsonized with CD20 mAbs, by human peripheral blood monocyte (PBMC)-derived macrophages, in the presence of the indicated mAbs, was assessed by microscopy. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01 and **** p < 0.0001. Results in ( D and F ) are representative of 3 independent experiments. Results in ( B , C , E and G to J ) are pooled from 3 independent experiments. Each symbol in ( B , E and G to J ) represents one cell, mouse or healthy donor.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Binding Assay, Mutagenesis, Inhibition, Flow Cytometry, Expressing, Variant Assay, Microscopy, Labeling, Derivative Assay

Journal: bioRxiv

Article Title: Binding of inhibitory checkpoints to CD18 in cis hinders anti-cancer immune responses

doi: 10.1101/2025.09.10.675342

Figure Lengend Snippet: ( A ) FRET assays of donor-labeled mouse SIRPα with acceptor-labeled mouse CD18 and unlabeled mouse CD11b, in the presence of Fc-silent mouse SIRPα mAbs, as was done for , D to F. ( B ) Binding of a soluble CD47-Fc fusion protein to EL-4 cells, expressing or not expressing mouse SIRPα, was studied by flow cytometry. ( C to K ) Generation and impact of bispecific antibody (BsAb) against mouse SIRPα. ( C ) Schematic representation of Fc-silent BsAb combining one arm of mAb #17 with one arm of mAb #27, using the “knob-into-hole” technology. Phagocytosis of IgG-opsonized L1210 cells ( D ) and EL-4 cells ( E ) by WT BMDMs, in the presence of mAbs, was assessed by a microscopy assays. ( F to K ) Schematic depictions of the assays are shown in (F and I). RAG-1 KO mice injected subcutaneously with Tac + L1210 cells ( G and H ), or C57BL/6J mice injected subcutaneously with Tac + EL-4 cells ( J and K ), were treated by intraperitoneal injection of Fc-silent mAbs, alongside Tac mAb 7G7 for opsonization. Tumor volume was measured using a caliper ( G and J ) and survival was recorded ( H and K ). ( L ) FRET assays of donor-labeled human SIRPα V1 or V2 with acceptor-labeled human CD18 and unlabeled human CD11b in the presence of Fc-silent Ctrl IgG and human SIRPα mAbs KWAR23, 40A, 50A, or 18D5, as was done for , D to F. The mAbs were rendered Fc-silent by the LALAPG mutation. ( M ) Phagocytosis of IgG-opsonized Raji cells by human macrophages in the presence of Fc-silent Ctrl IgG and SIRPα mAbs KWAR23, 40A, 50A, or 18D5, was assayed as for . ( N ) FRET assays of donor-labeled human 2B4 (SLAMF4), PD-1 or LILRB1 with acceptor-labeled human CD18, in the presence of Ctrl IgG or human CD18 mAb were done as for , D to F. All data are means ± s.e.m. ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Results are pooled from a total of two ( H and K ), three ( A , D , E , G , J , L and N ) or five ( B and M ) independent experiments. Each symbol in ( A , D , E and L to N ) represents one healthy donor, cell or mouse.

Article Snippet: Human SIRPα V2 cDNA (Cat: SC125649) was from OriGene (Rockville, MD).

Techniques: Labeling, Binding Assay, Expressing, Flow Cytometry, Microscopy, Injection, Mutagenesis