Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Flow panel showing the gating of PLXNB2 + and PLXNB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in ( b , c ). b , c Representative images of clustering ( b ) and average cluster area and cluster count curves of sorted PLXNB2 high and low TN3 PDX cells ( c ); n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. d , e Representative images ( d ) and cluster area ( e ) of MDA-MB-231 cells transfected with siRNA control (siCon), PLXNB2 SmartPool siRNA ( siPLXNB2 ), and single siRNA ( siPLXNB2-10 ), n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. f , g Representative images ( f ) and cluster area ( g ) of MDA-MB-468 cells transfected with siCon, si PLXNB2 , and si PLXNB2 -10, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. h Schematic of PLXNB2 domains: Extracellular (Ecto) domains include SEMA = SEMAphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-SEMAphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). i Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. j , k Representative images ( j ) and cluster area growth curves ( k ) of MDA-MB-231 PB2 KO clusters with overexpression of PLXNB2 full-length or mutants (mRBD, dECTO, or dVTDL); Con vs. KO + PLXNB2 p = 0.67, Con vs. KO1 p = 0.0002, Con vs. KO2 p = 1.43e-6, KO + PLXNB2 vs. KO+mRBD p = 0.04, KO + PLXNB2 vs. KO+dECTO p = 1.69e-6, KO + PB2 vs. KO+dVTDL p = 0.005, KO + PLXNB2 vs. KO1 p = 0.0002, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. l – o Bioluminescence images ( l , n ) and quantified BLI signals ( m , o ) of dissected lungs ex vivo after transfections with siCon, siPLXNB2 ( l ), or si PLXNB2 -11 ( n ), n = 4 mice/group ( l , m ) and n = 3 mice/group ( n , o ). P-value between two groups was calculated using two-sided unpaired t-test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Cells were then stained for CK, DAPI, and CD45 (3 mL for each, Menarini # 7900001) and Plexin B2 (1:1000, Miltenyl Biotec #130-126-566).

Techniques: Transfection, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Flow panel showing the gating of PLXNB2 + and PLXNB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in ( b , c ). b , c Representative images of clustering ( b ) and average cluster area and cluster count curves of sorted PLXNB2 high and low TN3 PDX cells ( c ); n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. d , e Representative images ( d ) and cluster area ( e ) of MDA-MB-231 cells transfected with siRNA control (siCon), PLXNB2 SmartPool siRNA ( siPLXNB2 ), and single siRNA ( siPLXNB2-10 ), n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. f , g Representative images ( f ) and cluster area ( g ) of MDA-MB-468 cells transfected with siCon, si PLXNB2 , and si PLXNB2 -10, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. h Schematic of PLXNB2 domains: Extracellular (Ecto) domains include SEMA = SEMAphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-SEMAphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). i Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. j , k Representative images ( j ) and cluster area growth curves ( k ) of MDA-MB-231 PB2 KO clusters with overexpression of PLXNB2 full-length or mutants (mRBD, dECTO, or dVTDL); Con vs. KO + PLXNB2 p = 0.67, Con vs. KO1 p = 0.0002, Con vs. KO2 p = 1.43e-6, KO + PLXNB2 vs. KO+mRBD p = 0.04, KO + PLXNB2 vs. KO+dECTO p = 1.69e-6, KO + PB2 vs. KO+dVTDL p = 0.005, KO + PLXNB2 vs. KO1 p = 0.0002, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. l – o Bioluminescence images ( l , n ) and quantified BLI signals ( m , o ) of dissected lungs ex vivo after transfections with siCon, siPLXNB2 ( l ), or si PLXNB2 -11 ( n ), n = 4 mice/group ( l , m ) and n = 3 mice/group ( n , o ). P-value between two groups was calculated using two-sided unpaired t-test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: Plexin B2 PE (1:1000, human, R&D Systems #FAB53291P), Plexin B2 APC (1:1000, human, R&D Systems #FAB53291A), Plexin B2 FITC (1:1000, mouse, R&D Systems #FAB6836G), SEMA4A PE (1:1000, human/mouse, BioLegend #148404).

Techniques: Transfection, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Flow panel showing the gating of PLXNB2 + and PLXNB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in ( b , c ). b , c Representative images of clustering ( b ) and average cluster area and cluster count curves of sorted PLXNB2 high and low TN3 PDX cells ( c ); n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. d , e Representative images ( d ) and cluster area ( e ) of MDA-MB-231 cells transfected with siRNA control (siCon), PLXNB2 SmartPool siRNA ( siPLXNB2 ), and single siRNA ( siPLXNB2-10 ), n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. f , g Representative images ( f ) and cluster area ( g ) of MDA-MB-468 cells transfected with siCon, si PLXNB2 , and si PLXNB2 -10, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. h Schematic of PLXNB2 domains: Extracellular (Ecto) domains include SEMA = SEMAphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-SEMAphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). i Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. j , k Representative images ( j ) and cluster area growth curves ( k ) of MDA-MB-231 PB2 KO clusters with overexpression of PLXNB2 full-length or mutants (mRBD, dECTO, or dVTDL); Con vs. KO + PLXNB2 p = 0.67, Con vs. KO1 p = 0.0002, Con vs. KO2 p = 1.43e-6, KO + PLXNB2 vs. KO+mRBD p = 0.04, KO + PLXNB2 vs. KO+dECTO p = 1.69e-6, KO + PB2 vs. KO+dVTDL p = 0.005, KO + PLXNB2 vs. KO1 p = 0.0002, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. l – o Bioluminescence images ( l , n ) and quantified BLI signals ( m , o ) of dissected lungs ex vivo after transfections with siCon, siPLXNB2 ( l ), or si PLXNB2 -11 ( n ), n = 4 mice/group ( l , m ) and n = 3 mice/group ( n , o ). P-value between two groups was calculated using two-sided unpaired t-test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: Cells were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and florescent antibody for 15 minutes on ice: Plexin B2 PE (1:1000, human, R&D Systems #FAB53291P), Plexin B2 APC (1:1000, human, R&D Systems #FAB53291A), Plexin B2 FITC (1:1000, mouse, R&D Systems #FAB6836G), SEMA4A PE (1:1000, human/mouse, BioLegend #148404).

Techniques: Transfection, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo

Journal: Nature Communications

Article Title: Computational ranking identifies Plexin-B2 in circulating tumor cell clustering with monocytes in breast cancer metastasis

doi: 10.1038/s41467-025-62862-z

Figure Lengend Snippet: a Flow panel showing the gating of PLXNB2 + and PLXNB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in ( b , c ). b , c Representative images of clustering ( b ) and average cluster area and cluster count curves of sorted PLXNB2 high and low TN3 PDX cells ( c ); n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. d , e Representative images ( d ) and cluster area ( e ) of MDA-MB-231 cells transfected with siRNA control (siCon), PLXNB2 SmartPool siRNA ( siPLXNB2 ), and single siRNA ( siPLXNB2-10 ), n = 5 technical replicates examined over 3 independent experiments. P-value was calculated using two-sided unpaired t-test. f , g Representative images ( f ) and cluster area ( g ) of MDA-MB-468 cells transfected with siCon, si PLXNB2 , and si PLXNB2 -10, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. h Schematic of PLXNB2 domains: Extracellular (Ecto) domains include SEMA = SEMAphorin domain; IPT = Ig-like fold domain; and PSI = Plexin-SEMAphorin-integrin domain. Intracellular domains include RBD = Rho-binding domain; GAP = GTPase activating protein domain; and VTDL = PDZ-domain binding site (Rho-GEF binding). i Immunoblots of overexpressed PB2 mutants with either full-length (fl) or truncated (tr) depletions: mutated RBD (mRBD) tr, depleted Ecto domain (dECTO) fl and depleted VTDL (dVTDL) tr in MDA-MB-231 PB2 KO cells. j , k Representative images ( j ) and cluster area growth curves ( k ) of MDA-MB-231 PB2 KO clusters with overexpression of PLXNB2 full-length or mutants (mRBD, dECTO, or dVTDL); Con vs. KO + PLXNB2 p = 0.67, Con vs. KO1 p = 0.0002, Con vs. KO2 p = 1.43e-6, KO + PLXNB2 vs. KO+mRBD p = 0.04, KO + PLXNB2 vs. KO+dECTO p = 1.69e-6, KO + PB2 vs. KO+dVTDL p = 0.005, KO + PLXNB2 vs. KO1 p = 0.0002, n = 5 technical replicates examined over 3 independent experiments. P values were calculated using one-sided ANOVA. l – o Bioluminescence images ( l , n ) and quantified BLI signals ( m , o ) of dissected lungs ex vivo after transfections with siCon, siPLXNB2 ( l ), or si PLXNB2 -11 ( n ), n = 4 mice/group ( l , m ) and n = 3 mice/group ( n , o ). P-value between two groups was calculated using two-sided unpaired t-test. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: They were blocked with mouse IgG (Sigma #I5381) for 10 minutes on ice and incubated with fluorescence-conjugated antibodies for 15 minutes on ice: Plexin B2 PE (phycoerythrin) (1:1,000, human, R&D Systems #FAB53291P), Plexin B2 APC (1:1,000, human, R&D Systems #FAB53291A), Plexin B2 FITC (1:1,000, mouse, R&D Systems #FAB6836G), CD45 (1:1,000, human, BD Bioscience #557748), EpCAM FITC (1:1,000, human, BD Bioscience #347197), SEMA4A PE (1:1,000, human/mouse, BioLegend #148404), SEMA4A APC (1:1,000, human/mouse Biolegend #148406).

Techniques: Transfection, Control, Binding Assay, Western Blot, Over Expression, Ex Vivo

Journal: bioRxiv

Article Title: A proteogenomic tool uncovers protein markers for human microglial states

doi: 10.1101/2025.03.31.646212

Figure Lengend Snippet: A. Identification of human microglial cell surface proteins and panel design. For each of the datasets (generated proteomics dataset from freshly isolated human microglia, microglia-specific disease-relevant GWAS hits, Input ), cell surface and cell membrane proteins were identified using the databases Uniprot ( https://www.uniprot.org/ ) and the Surfaceome ( Query ). Proteins/candidates that scored in both approaches - the Uniprot and Surfaceome analysis - for the proteomics as well as the GWAS hits, were subsequently compiled into a list of n=91 initial candidates ( Output ). Candidate expression was then assessed in other available proteomics datasets of HCM3 microglia and iPSC-derived microglia as well as brain RNA-Seq datasets (n=87 candidates), commercially available antibodies were then titrated using flow cytometry (n=32) and selected candidates subsequently added to the production of the customized microglia-specific CITE-Seq panel produced in Total-Seq A (TS-A) format by BioLegend (n=17 microglia-specific antibodies, n=150 Universal cocktail 1.0 TS-A antibodies)( Panel assembly ). B. Examples for titration results for HMC3 microglia. To evaluate all titration results for the antibody candidates titrated in HMC3 microglia, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 ) were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here. C. Examples for titration results for PBMCs. Examples for titration results for PBMCs. To evaluate all titration results for the antibody candidates titrated in PBMCs, the PE-positive population of all samples (unstained 1 , Isotype control 2 , 1:32 3 , 1:16 4 , 1:8 5 , 1:4 6 , 1:2 7 , recommended concentration 8 , doubled recommended concentration (2R) 9 ) gated on lymphocytes and monocytes were concatenated and plotted based on their Comp-PE-A values. For visualization, ADAM10 and PLXNB2 are shown here.

Article Snippet: Antibodies that were successfully titrated in HMC3s included: ADAM10 (BioLegend, Cat#: 352703), CD49 (BioLegend, Cat#: 328009), CD51 (BioLegend, Cat#: 327909), HLA-C (BioLegend, Cat#: 373302), ITGB5 (BioLegend, Cat#: 345203), PLXNB2 (Miltenyi, Cat#: 130-095-212), PTPRJ (BioLegend, Cat#: 328708).

Techniques: Generated, Isolation, Membrane, Expressing, Derivative Assay, RNA Sequencing, Flow Cytometry, Produced, Titration, Control, Concentration Assay