plv plxnb2 mrbd  (Addgene inc)

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 A schematic of an integrated ranking, Rscore, of proteins in breast tumors, cancer cells, and CTCs with tumor specificity and clinical association, using multiple MS proteomic databases The mathematical model of Rscore integrates individual ranks ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i ) of each protein in (1) relative protein abundance, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (pc), in multiple datasets (patient tumors, CTCs, and cell lines), (2) significance changes, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (sc) in tumor specificity, including p-value, ratio or fold change, and absolute change, comparing TNBC voxels (laser capture microdissection) to normal adjacent tissues, and (3) clinical association, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i (ca) including p value and hazard ratio, with OS and DMFS among multiple datasets. The significance of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${r}_{i}$$\end{document} r i is multiplied by its constant weight factor ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${c}_{i}$$\end{document} c i ) with the sum divided by n for an integrated Rscore and final top three candidates. Created in BioRender. Tong, F. (2025) https://BioRender.com/nea43wo . b Representative IHC images of PLXNB2 high TNBC tumor and PLXNB2 low normal breast tissue (adjacent to tumors) from a TNBC patient. c KM plot for OS of patients with all breast cancer in the Tang_2018 data set ( n = 108) via Kaplan-Meier plotter, separated by the best cut-off value of PLXNB2 protein expression (4) in primary tumors to define high vs. low within the expression range (0-11). P values were calculated via the Cox-Mantel (log-rank) test. d KM plot for DMFS of patients with ER − breast cancer, divided by median cut-off of PLXNB2 mRNA expression using data from GEO, EGA, and TCGA, n = 218. P values were calculated using a log-rank test. e Schematic depicting the patient blood sample workflow for CTC analysis on CellSearch. f Representative CellSearch images of a homotypic PLXNB2 + CTC-CTC (CD45 − CK + DAPI + ) cluster, a heterotypic PLXNB2 + CTC-WBC (CD45 + CK - DAPI + ) cluster, and a single PLXNB2 - CTC. Scale bar = 5 µm. g Portion (%) of PLXNB2 + CTCs in single CTCs in comparison with homotypic CTC clusters and heterotypic CTC-WBC clusters), respectively, analyzed via CellSearch as shown in ( f ), n = 41 patients. Data are presented as mean values +/- SD, P values reported are from two-sided unpaired t-tests unless specified. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Quantitative Proteomics, Laser Capture Microdissection, Expressing, Comparison

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 Schematic showing the experimental workflow of orthotopic implantation and analyses of lung metastases and CTCs. b , c Representative images ( b ) and weight quantification of PLXNB2 Con and KO tumors ( c ) from mice at 10 weeks, n = 8 mice/group. d , e Bioluminescence images (BLI) of mouse lungs ex vivo ( d ) and quantified lung metastasis ( e ) at 10 weeks, n = 8 mice/group. f L2G + CTC counts (single and clusters) detected in PLXNB2 Con and KO mouse blood at 10 weeks of spontaneous metastasis, n = 4 mice/group. g , h Representative images ( g ) and metastatic burden ( h ) in PLXNB2 Con and KO metastatic cells of mouse lungs with H&E staining, scale bar = 250 µm; experiments were repeated with the PB2 KO clone, n = 4 lungs/group. Yellow arrows point to the micro metastases in the lungs. i Schematic representing experimental workflow of dual color implantations of L2T + (red) or L2G + (green) MDA-MB-231 tumor cells with PLXNB2 control (ConT, ConG) or PLXNB2 KO (KOT, KOG); mice were sacrificed after 6 weeks for analyses, n = 4 mice/group. j Representative images of green (ConG/KOG) and red (ConT/KOT) MDA-MB-231 colonies in the lungs of mice, n = 4 mice/group. k Counts of L2T + or L2G + colonies in the lungs of mice after 6 weeks orthopedic, n = 4 mice/group. l Counts of dual color colonies with red and green tumor cells in the lungs, n = 4 lungs/group. m CTC clusters in red/green count as analyzed via flow cytometry in the mice bearing the PLXNB2 Control and KO tumors, n = 8 mice/group. n Top left: Schematic of orthotopic implantation of PLXNB2 Con and KO tumor cells into of NSG mice (200,000 cells/site). Bottom left: KM plot of the mice shows Supplementary survival (2 weeks) in the mice bearing Con vs. KO tumors. Right panels: Photos (top) and bar graphs (bottom) of relatively comparable tumor weight between Con (8-week) and KO groups (10-week), n = 5 mice/group. o – q Bar graphs of blood CTC clusters ( o ), cell cycle phases ( p ), and spontaneous lung metastasis ( q ) between the Con and KO tumors, n = 5 mice/group.Data are presented as mean values +/-SD, with P values reported from two-sided unpaired t-testsunless specified. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Ex Vivo, Staining, Control, Flow Cytometry

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: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

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 Schematic of PLXNB2-SEMA4C intercellular interactions in homotypic tumor cell clustering between two cancer cells. b Immunoblots of PLXNB2 (PB2) and SEMA4C expression in different breast cancer cell lines and PDX models using antibodies that are specific to detect both human and mouse isoforms. c Immunoblots of SEMA4C, CDC42, and PLXNB2 in the protein complex immunoprecipitated by anti-PLXNB2 antibody compared to IgG. d Immunoblot images showing reduced SEMA4C levels after SEMA4C KD in MDA-MB-231 tumor cells (left) and depleted PLXNB2 in PLXNB2 KO1 and KO2 cells (right). e Schematic illustration and immunoblotting of MDA-MB-231 control and PLXNB2 -KO cells transfected with siRNA control or siSEMA4C knockdown, followed by tumor cell clustering assessment in suspension. f , g Representative images ( f ) and average cluster area/size curves ( g ) of MDA-MB-231 control (Con) and PB2 KO (KO) cells transfected with control siRNA (siCon) or SEMA4C siRNAs ( si4C ) for KD, measured by IncuCyte, n = 5 technical replicates examined over 5 independent experiments. For cluster size (area) comparisons with the group of Con+ siCon , Student’s t test p = 0.033 for Con+ si4C , p = 0.021 for KO+ si4C , and p = 0.016 for KO+ siCon . For the cluster number comparisons with the Control group, p < 0.001 for all three pairs above. P value were calculated using one-sided ANOVA. h Principal component analysis clusters of MDA-MB-231 single cells, siCon clusters, and si PLXNB2 clusters analyzed from global proteomics analysis with n = 3 technical replicates/ group over three independent experiments. i Heat map of differentially expressed proteins from global proteomics analysis of siCon single cells (blue), siCon clusters (green), and si PLXNB2 clusters (red), with n = 3 technical replicates/ group over three independent experiments. j Gene ontology biological processes analysis of significantly up-regulated and down-regulated proteins in PLXNB2 control vs. PLXNB2 KD clusters in breast cancer cells analyzed by mass spectrometry. Data are presented as mean values ± SD. P values were calculated using one-sided ANOVA. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Western Blot, Expressing, Immunoprecipitation, Control, Transfection, Knockdown, Suspension, Mass Spectrometry

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 Schematic of the interactions between breast cancer cell PLXNB2 and monocyte SEMA4A for heterotypic cluster formation. b Representative images of heterotypic clusters of L2G + MDA-MB-231 control or PLXNB2 KO cells with WBCs from breast cancer patients (Pt). Top: fluorescent images with WBCs from Pt 396 (minimal Cytotox red-stained dead cells). Bottom: brightfield images with WBCs from Pt 431. An average of 13581 and 11376 WBCs is associated with Con and KO clusters, respectively. c Heterotypic cluster area curves as shown in ( b ) ( n = 5 replicates examined over 4 individual experiments). P-value was calculated using two-sided unpaired t-test. d SEMA4A expression on granulocytes, monocytes, and lymphocytes from four patients with advanced breast cancer. P-values were calculated using one-sided ANOVA. e Percent of heterotypic CTC-WBC clusters vs. WBC clusters that express both PB2 and SEMA4A, n = 4 patient samples. P-value was calculated using two-sided unpaired t-test. f , g Immunoblots of SEMA4A ( f ) and CDC42 ( g ) with cell lysates immunoprecipitated with anti-PlexinB2 (αPB2) or IgG isotype. h , i Representative images ( h ) and cluster area curves ( i ) of heterotypic clustering of MDA-MB-231 cells (green) and THP1 monocytes (yellow) mixed at a 1:4 ratio, n = 3 technical replicates examined over 3 individual experiments. P-value was calculated using two-sided unpaired t-test. j , k Representative images ( j ) and cluster size curves ( k ) of heterotypic clustering with L2G + TN1 PDX tumor cells (green, sorted by PLXNB2 high and low) and THP1 (yellow), n = 3 technical replicates examined over 3 individual experiments. P-value was calculated using two-sided unpaired t-test. l-m) Representative images ( l ) and cluster size curves ( m ) of MDA-MB-231 Con or KO cells clustering with THP1 (transfected with siCon or siSEMA4A ), n = 3 technical replicates examined over 3 individual experiments. P values were calculated using one-sided ANOVA.n) BLI images of NSG mice after tail vein injection of pre-clustered MDA-MB-231 cells alone ( PLXNB2 Con or KO), and with THP1 cells ( PLXNB2 Con/THP1, KO/THP1), n = 3 mice/group. P values were calculated using one-sided ANOVA. o BLI flux of lungs, n = 3 mice/group at each time point. Data are presented as mean values ± SD. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Control, Staining, Expressing, Western Blot, Immunoprecipitation, Transfection, Injection

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 , b Representative images at 6 h clustering ( a ) and quantification ( b ) of 4T1 cells transfected with siCon and siPlxnb2 during co-clustering with mouse white blood cells over 6 h; n = 5 technical replicates examined over 3 individual experiments, p = 0.008. c Schematic and quantification of % of heterotypic mouse 4T1 CTC clusters with monocytes at 6 h after tail vein injection of the tumor cells transfected with siRNA control (Con) and si Plxnb2 (>80% knockdown efficiencies by flow). 5 × 10 5 4T1 tumor cells were injected into Balb-c mice via the tail vein and cardiac blood was collected at 6 h for CTC analysis via flow cytometry ( n = 5 mice/group). d – i Schematic of orthotopic 4T1 breast tumor implantations (1.5 × 10 6 cells) at the L4/R4 mammary fat pads and following metastasis and CTC analyses ( d ), representative photos of 4T1 tumors (Control and Plxnb2 KO) and ex vivo lung bioluminescence images on Day 9 ( e ), and quantified tumor weight and volume ( f ), lung metastasis (total flux of bioluminescence) ( g ) and CTCs, including single CTCs in live blood cells ( h ) as well as 4T1-WBC heterotypic clusters and CTC-myeloid clusters ( i ) among all white blood cells (WBCs), shown as # of events in 10,000 WBCs, n = 5 mice/group. Tumor burden includes both L4/R4 tumors in each mouse and is used to normalize lung metastasis signals in ( g ). Data are presented as mean values +/-SD, with P values reported from two-sided unpaired t-tests. Source data are provided as a Source Data file.

Article Snippet: The following overexpression vectors were used: pLV- PLXNB2 - mRBD (Addgene #86240), pLV- PLXNB2-dVTDL (Addgene 86239), pLV- PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1( + ) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1 + /C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Transfection, Injection, Control, Knockdown, Flow Cytometry, Ex Vivo

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A) Flow panel showing the gating of PB2 + and PB2 - cells sorted from dissociated primary TN3 L2G + PDX cells for clustering in (B-C). B & C) Representative images at 0 and 6 h of clustering (B) and cluster count curves of sorted PB2 high and PB2 low TN3 PDX cells (C), measured by IncuCyte imaging system; N=3 experiments with at least 5 technical replicates each. D & E) Representative images (D) and cluster area growth curves (E) of MDA-MB-231 cells treated with control (siCon), PB2 KD via SmartPool siRNA (siPB2), and single target siRNA (siPB2-10), N=3 experiments with at least 5 technical replicates each. F & G) Representative images (F) and cluster area growth curves (G) of MDA-MB-468 cells treated with siCon, PB2 KD via siPB2 SmartPool (siPB2), and siPB2-10. H ) Schematic of PB2 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 PB2 full-length or mutants (mRBD, dECTO, or dVTDL) over 6 h as measured via IncuCyte; Con vs. KO+PB2 p=0.67, Con vs. KO1 p=0.0002, Con vs. KO2 p=1.43e-6, KO+PB2 vs. KO+mRBD p=0.04, KO+PB2 vs. KO+dECTO p=1.69e-6, KO+PB2 vs. KO+dVTDL p=0.005, KO+PB2 vs. KO1 p=0.0002. L-O) IVIS bioluminescence images (L, N) of NSG mice on days 0 and 4, and quantified signals of metastasis (M, O) of dissected lungs ex vivo on day 4 after tail vein injection of MDA-MB- 231 cells transfected with siCon, siPB2 (L), or siPB-11 (N), N=4 (L-M) and 3 (N-O) biological replicates. Data are presented as mean values +/- SD, p-values reported are from two-sided unpaired t-tests.

Article Snippet: The following overexpression vectors were used: pLV-PLXNB2-mRBD (Addgene #86240), pLV-PLXNB2-dVTDL (Addgene 86239), pLV-PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1(+) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1+/C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

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

Journal: bioRxiv

Article Title: Computational ranking-assisted identification of Plexin-B2 in homotypic and heterotypic clustering of circulating tumor cells in breast cancer metastasis

doi: 10.1101/2023.04.10.536233

Figure Lengend Snippet: A ) Immunoblots of full-length (FL) and truncated (Tr) PB2 in MDA-MB-231 cells with two pooled clones of CRISPR/Cas9-mediated KO, KO1 and KO2, with PB2-specific bands at 250 kDa and 75 kDa, N=3 experiments; B-D) Flow cytometry analysis of MDA-MB-231 Cas9 control cells for PB2, KO1, and KO2. E) Proliferation assay of MDA-MB-231 control and KO cells over 60+ h measured as percent confluence by IncuCyte, N=3 experiments, data reported as mean +/- SD, p-value is from a two-sided unpaired t-test. F-G) Representative images (F) and cluster size quantification (G) of MDA-MB-231 PB2 control, KO1, and KO2 cell clusters after 6 h as taken by the IncuCyte Live Cell Imager; N=3 experiments with at least 3 technical replicates each, data are presented as mean values +/- SD, p-value is from a two-sided unpaired t-test. H ) Flow cytometry analysis of PB2 overexpression in MDA-MB-231 PB2 KO cells. I & J) Representative images (I) of average mammosphere counts of 10 cells or more (J) of MDA-MB-231 WT cells with scramble (siCon), PB2 SmartPool (siPB2), and PB2 single (siPB2-10) siRNA KD after 7 days; N=3 experiments with at least 5 technical replicates each, data are presented as mean values +/- SD, p-values are from a two-sided unpaired t-test. K & L) Mammosphere images (K) and counts (L) of MDA-MB-231 KO2 cells with overexpression of PB2 full-length (PB2 OE) and a PB2 mutant with depleted extracellular domain (dECTO) for 10 days, N=4 replicates (bottom). Mammospheres larger than 100 microns across were counted. Data are presented as mean +/-SD, p-value is from a two-sided unpaired t-test.

Article Snippet: The following overexpression vectors were used: pLV-PLXNB2-mRBD (Addgene #86240), pLV-PLXNB2-dVTDL (Addgene 86239), pLV-PLXNB2-dECTO (Addgene #86238), PLXNB2 OHu01778C_pcDNA3.1(+) N-Terminal Flag-Tag (GenScript #SC1626), PLXNB2 OHu01778D_pcDNA3.1+/C- C-Terminal Flag-Tag (GeneScript #OHu01778D), PLXNB2 Untagged Construct (GeneScript #SC1625).

Techniques: Western Blot, Clone Assay, CRISPR, Flow Cytometry, Control, Proliferation Assay, Over Expression, Mutagenesis