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Revvity Signals apc cyanine7 anti human cd46 antibody
Apc Cyanine7 Anti Human Cd46 Antibody, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Becton Dickinson anti cd46 apc
Differential myeloma drug sensitivity profiles of malignant subpopulations at diagnosis. A, Flow cytometry showed a monotypic population by typical markers CD138 and CD38 (left), but showed rarer subpopulations with CD45 expression, with or without CD19 (right) that were not detected by scRNA-seq. B, Of the cell surface proteins measured by flow cytometry, the mRNA for <t>CD46</t> showed the most differential expression (l 2 FC = 0.44, P = 5.76 × 10 −7 ) between the LCE-multiple myeloma (MM) and IGH-MM subpopulations. C, Relatively CD46-high and CD46-low populations were divided to approximate subpopulation drug sensitivity of IGH-MM and LCE-MM, respectively. D, The drug sensitivity profiles implied that the CD46-high/IGH-MM subpopulation had a primary IMiD refractory phenotype and was relatively less bortezomib sensitive compared with LCE-MM ( n = 3, tested by t test, bars depict standard deviation. *, P < 0.05; ***, P < 0.001). Bor, bortezomib; Car, carfilzomib; Dara, daratumumab; Len, lenalidomide; norm %, percent viability normalized to untreated controls; Pom, pomalidomide.
Anti Cd46 Apc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Becton Dickinson anti cd46 fitc
Differential myeloma drug sensitivity profiles of malignant subpopulations at diagnosis. A, Flow cytometry showed a monotypic population by typical markers CD138 and CD38 (left), but showed rarer subpopulations with CD45 expression, with or without CD19 (right) that were not detected by scRNA-seq. B, Of the cell surface proteins measured by flow cytometry, the mRNA for <t>CD46</t> showed the most differential expression (l 2 FC = 0.44, P = 5.76 × 10 −7 ) between the LCE-multiple myeloma (MM) and IGH-MM subpopulations. C, Relatively CD46-high and CD46-low populations were divided to approximate subpopulation drug sensitivity of IGH-MM and LCE-MM, respectively. D, The drug sensitivity profiles implied that the CD46-high/IGH-MM subpopulation had a primary IMiD refractory phenotype and was relatively less bortezomib sensitive compared with LCE-MM ( n = 3, tested by t test, bars depict standard deviation. *, P < 0.05; ***, P < 0.001). Bor, bortezomib; Car, carfilzomib; Dara, daratumumab; Len, lenalidomide; norm %, percent viability normalized to untreated controls; Pom, pomalidomide.
Anti Cd46 Fitc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Thermo Fisher mouse anti human cd46 igg1
(A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an <t>anti-CD46</t> antibody, an anti-CD147 antibody. A mouse <t>IgG</t> isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.
Mouse Anti Human Cd46 Igg1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Revvity Signals anti cd46
A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of <t>CD46</t> negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.
Anti Cd46, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Revvity Signals anti cd46 antibody
A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of <t>CD46</t> negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.
Anti Cd46 Antibody, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Revvity Signals cd46 knockout efficiency
A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of <t>CD46</t> negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.
Cd46 Knockout Efficiency, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Thermo Fisher anti cd46 thermo
Immunofluorescence analysis of the spermatozoa from the control and from the patient. The acrosome, nucleus, and neck of the sperm were stained with <t>CD46</t> (red), DAPI (blue) and PMFBP1 antibodies (green), respectively. The localization of PMFBP1 in sperm from control and patient samples was determined by immunofluorescence staining. The PMFBP1 protein is localized at the head–tail junction of spermatozoa in control samples but is not detected in patient spermatozoa. 40 times oil microscope observation, scale bar = 4 µm
Anti Cd46 Thermo, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Sanquin cd46
Immunofluorescence analysis of the spermatozoa from the control and from the patient. The acrosome, nucleus, and neck of the sperm were stained with <t>CD46</t> (red), DAPI (blue) and PMFBP1 antibodies (green), respectively. The localization of PMFBP1 in sperm from control and patient samples was determined by immunofluorescence staining. The PMFBP1 protein is localized at the head–tail junction of spermatozoa in control samples but is not detected in patient spermatozoa. 40 times oil microscope observation, scale bar = 4 µm
Cd46, supplied by Sanquin, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Revvity Signals cells pe cd46
( A ) Diagram of FCRL3 protein. Ig-like C2 domains are depicted as circles with the color indicating their previously established phylogenetic relationship, while tyrosines involved in intracellular signaling motifs are shown as stars. Red indicates an ITAM, light blue an ITIM, and purple indicates the ITIM-like/HemITAM Y722 motif in FCRL3. ( B ) Flow cytometry of Y. pestis infection of HeLa cells. HeLa cells (transfected with empty vector or FCRL3 plasmid) were infected for 1 hr with KIM6+ +pMMB67GFP, treated with gentamicin to kill extracellular bacteria for 1hr, and then GFP was induced in living, intracellular bacteria for 2 hrs with IPTG. Two populations of GFP+ cells were detected, consisting of HeLa cells infected with living, intracellular bacteria (GFP high , orange) in both conditions and those with attached, dead bacteria (GFP low , light blue) found in the FCRL3 overexpression condition. ( C ) Overexpression of FCRL3 increases attachment (GFP low , light blue) and invasion (GFP high , orange). HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometry as described in B. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. A western blot was performed to confirm the presence of FCRL3 after overexpression but not with empty vector using 1:200 FcRH3 antibody (Santa Cruz, C-2). ( D ) GFP low cells have attached, extracellular Y. pestis. FCRL3 transfected HeLa cells were infected as described in B and fixed with 4% paraformaldehyde (PFA) after 4 hrs. Infected and uninfected cells were blocked with normal goat serum (but not permeabilized) and then incubated with 1:20 polyclonal anti- Yersinia pestis F1-Antigen antibody (BEI Resources, NR-31024) to stain extracellular bacteria. DNA was then stained with 1 drop/10mL of a membrane permeable dye NucBlue for nuclei visualization. To visualize the GFP low bacteria, a high and low exposure GFP panel is shown. GFP low bacteria are indicated with a teal arrowhead and GFP high bacteria with an orange arrowhead. Cells were imaged using a 40× air objective on an EVOS M5000 Microscope. A 20µm scale bar is located on the merged image. ( E ) CRISPR-mediated knockout (KO) of FCRL3 in LCL HG02678 causes significant decrease in attachment and invasion of Y. pestis . Cells were electroporated with either only <t>CD46</t> (control) or FCRL3 + CD46 guides and CRISPR Cas9. Pooled KO cells (>70% FCRL3 -/- ) were sorted for CD46 -/- cells. Cells were assayed for KIM6+ Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three experiments with two biological replicates of each condition were plotted and an unpaired t-test was performed to determine significance. ( F ) FCRL3 overexpression increases attachment and invasion of Y. pestis KIM5. HeLa cells transfected with empty vector or FCRL3 plasmid were assayed for KIM5 Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi, after subculturing bacteria for 2 hrs and 40 min at either 26 or 37 degrees C. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( G ) FCRL3 overexpression has no effect on Y. pseudotuberculosis attachment and invasion. HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( C, E-G ) Experiments were normalized by grand mean.
Cells Pe Cd46, supplied by Revvity Signals, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Differential myeloma drug sensitivity profiles of malignant subpopulations at diagnosis. A, Flow cytometry showed a monotypic population by typical markers CD138 and CD38 (left), but showed rarer subpopulations with CD45 expression, with or without CD19 (right) that were not detected by scRNA-seq. B, Of the cell surface proteins measured by flow cytometry, the mRNA for CD46 showed the most differential expression (l 2 FC = 0.44, P = 5.76 × 10 −7 ) between the LCE-multiple myeloma (MM) and IGH-MM subpopulations. C, Relatively CD46-high and CD46-low populations were divided to approximate subpopulation drug sensitivity of IGH-MM and LCE-MM, respectively. D, The drug sensitivity profiles implied that the CD46-high/IGH-MM subpopulation had a primary IMiD refractory phenotype and was relatively less bortezomib sensitive compared with LCE-MM ( n = 3, tested by t test, bars depict standard deviation. *, P < 0.05; ***, P < 0.001). Bor, bortezomib; Car, carfilzomib; Dara, daratumumab; Len, lenalidomide; norm %, percent viability normalized to untreated controls; Pom, pomalidomide.

Journal: Cancer Research Communications

Article Title: Single-Cell RNA Sequencing before and after Light Chain Escape Reveals Intrapatient Multiple Myeloma Subpopulations with Divergent Osteolytic Gene Expression

doi: 10.1158/2767-9764.CRC-24-0170

Figure Lengend Snippet: Differential myeloma drug sensitivity profiles of malignant subpopulations at diagnosis. A, Flow cytometry showed a monotypic population by typical markers CD138 and CD38 (left), but showed rarer subpopulations with CD45 expression, with or without CD19 (right) that were not detected by scRNA-seq. B, Of the cell surface proteins measured by flow cytometry, the mRNA for CD46 showed the most differential expression (l 2 FC = 0.44, P = 5.76 × 10 −7 ) between the LCE-multiple myeloma (MM) and IGH-MM subpopulations. C, Relatively CD46-high and CD46-low populations were divided to approximate subpopulation drug sensitivity of IGH-MM and LCE-MM, respectively. D, The drug sensitivity profiles implied that the CD46-high/IGH-MM subpopulation had a primary IMiD refractory phenotype and was relatively less bortezomib sensitive compared with LCE-MM ( n = 3, tested by t test, bars depict standard deviation. *, P < 0.05; ***, P < 0.001). Bor, bortezomib; Car, carfilzomib; Dara, daratumumab; Len, lenalidomide; norm %, percent viability normalized to untreated controls; Pom, pomalidomide.

Article Snippet: To identify multiple myeloma cells, samples were stained with anti-CD38-PerCP-Cy5.5, anti-CD138-BV421, anti-CD45-BV510, anti-CD19-BB515, and anti-CD46-APC (BD Biosciences).

Techniques: Flow Cytometry, Expressing, Standard Deviation

(A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Knock-Out, Infection, Bacteria, Derivative Assay, Expressing, Flow Cytometry, Control

WT cells were treated with specific siRNAs 72 hours prior to infection. Cells were treated with 800C or a scrambled control peptide for 60 minutes prior to infection. Cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. (A) Flow cytometry demonstrating efficient knockdown of meningococcal and staphylococcal receptors after siRNA treatment prior to infection. Cells were probed with anti-CD147, anti-CD46 and anti-CD44 antibodies. n=1, mean. (B) CD147 knockdown and CD9-derived peptide treatment demonstrate no additive effects on meningococcal adherence. WT and CD9 -/- siRNA treated cells were infected with meningococci (MC58) for 60 mins at MOI=50. (C) CD44 knockdown and CD9-derived peptide treatment demonstrate reduced additive effects in staphylococcal adherence. WT and CD9 -/- siRNA treated cells were infected with staphylococci (SH1000) for 60 mins at MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by cfu. n=3, mean + SEM, One-Way ANOVA.

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: WT cells were treated with specific siRNAs 72 hours prior to infection. Cells were treated with 800C or a scrambled control peptide for 60 minutes prior to infection. Cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. (A) Flow cytometry demonstrating efficient knockdown of meningococcal and staphylococcal receptors after siRNA treatment prior to infection. Cells were probed with anti-CD147, anti-CD46 and anti-CD44 antibodies. n=1, mean. (B) CD147 knockdown and CD9-derived peptide treatment demonstrate no additive effects on meningococcal adherence. WT and CD9 -/- siRNA treated cells were infected with meningococci (MC58) for 60 mins at MOI=50. (C) CD44 knockdown and CD9-derived peptide treatment demonstrate reduced additive effects in staphylococcal adherence. WT and CD9 -/- siRNA treated cells were infected with staphylococci (SH1000) for 60 mins at MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by cfu. n=3, mean + SEM, One-Way ANOVA.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Infection, Control, Flow Cytometry, Knockdown, Derivative Assay, Bacteria

A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Article Snippet: 7-, 14-, 21-, and 28-days post infection, samples were stained with anti-CD46 (Biolegend, 352409) to quantify outgrowth of edited cells.

Techniques: Isolation, Knock-Out, Generated, Infection, Control, Western Blot, Expressing, Molecular Weight

( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: 7-, 14-, 21-, and 28-days post infection, samples were stained with anti-CD46 (Biolegend, 352409) to quantify outgrowth of edited cells.

Techniques: Infection, Control, Standard Deviation, Transfection, Generated, Knock-Out, Virus

A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Article Snippet: 7-, 14-, 21-, and 28-days post infection, samples were stained with anti-CD46 (Biolegend, 352409) to quantify outgrowth of edited cells.

Techniques: Expressing

( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: 7-, 14-, 21-, and 28-days post infection, samples were stained with anti-CD46 (Biolegend, 352409) to quantify outgrowth of edited cells.

Techniques: Luciferase, Expressing, Infection, Knock-Out, Virus, Control

A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Isolation, Knock-Out, Generated, Infection, Control, Western Blot, Expressing, Molecular Weight

( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Infection, Control, Standard Deviation, Transfection, Generated, Knock-Out, Virus

A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Expressing

( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Luciferase, Expressing, Infection, Knock-Out, Virus, Control

A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. Purity of isolated CD19 positive naïve B cell fractions for donors in screen. B. Knockout score for each LPKO LCL generated by 28 days post infection. C. Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT infected cells 28 days post infection. D. Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons. ** indicates p-values <0.01. E. Knockout score of LPKO LCLs generated after a total of seven weeks in culture, significantly delayed outgrowth compared to rescued LPKO LCLs and WT LCLs. F. Western blot for Sp100 protein expression in LPKO LCLs with SP100 KO or SP140L KO from donor 3. Molecular weight in kDa is indicated.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Isolation, Knock-Out, Generated, Infection, Control, Western Blot, Expressing, Molecular Weight

( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ) Schematic of Cas9/RNP screening approach. ( B ) Log(10) Fold Change from Day 2 of the total number of CD46 negative cells for each condition 2, 7-, 14-, 21-, and 28-days post-infection. Dark turquoise line represents the mean number of CD46 negative cells in CD46 only control WT- or LPKO-infected cells 28 days post infection. SD = standard deviation. ( C ) Total number of CD46 negative cells 28 days post infection for each condition plotted as log fold change from 2 days post infection. P values calculated by one-way ANOVA with multiple comparisons to compare to LPKO infected cells transfected with guide targeting CD46 only as control. *Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001. ( D ) Total number of LPKO LCLs in each donor for each target out of 4 replicates per donor. # Indicates conditions in which at least one LCL was generated after an additional three weeks in culture with variable knockout efficiency. ^Indicates conditions in which at least one LCL was generated in the same time frame at WT virus, but target gene was not knocked out. P values calculated using Fischer’s exact test to compare outcomes to LPKO control condition ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Infection, Control, Standard Deviation, Transfection, Generated, Knock-Out, Virus

A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: A. CD46 negative and positive populations for each RNAseq sample at time of collection. B. ICAM1 expression in each sample from second donor not shown in .

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Expressing

( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Journal: bioRxiv

Article Title: Sp140L Is a Novel Herpesvirus Restriction Factor

doi: 10.1101/2024.12.13.628399

Figure Lengend Snippet: ( A ). Schematic of experiment. Fold change in luciferase expression HFF cells (n=7) ( B ) or MRC5 cells (n=5) ( C ) infected with HVS-ORF3 Knockout or WT virus encoding luciferase with knockout of control CD46 , SP100 , or SP140L . Two virus preparation (A and B) were used. P values calculated by Dunnett’s multiple comparisons test using an ordinary two-way ANOVA. * Indicates p-values <0.05, ** indicates p-values <0.01, **** indicates p value < 0.0001.

Article Snippet: When cell confluency reached 80-90%, a quarter of the well was stained with anti-CD46 antibody (Biolegend, 352401) to measure CD46-knockout efficiency by flow cytometry as described above.

Techniques: Luciferase, Expressing, Infection, Knock-Out, Virus, Control

Immunofluorescence analysis of the spermatozoa from the control and from the patient. The acrosome, nucleus, and neck of the sperm were stained with CD46 (red), DAPI (blue) and PMFBP1 antibodies (green), respectively. The localization of PMFBP1 in sperm from control and patient samples was determined by immunofluorescence staining. The PMFBP1 protein is localized at the head–tail junction of spermatozoa in control samples but is not detected in patient spermatozoa. 40 times oil microscope observation, scale bar = 4 µm

Journal: Basic and Clinical Andrology

Article Title: Pathogenesis of acephalic spermatozoa syndrome caused by PMFBP1 mutation

doi: 10.1186/s12610-024-00240-3

Figure Lengend Snippet: Immunofluorescence analysis of the spermatozoa from the control and from the patient. The acrosome, nucleus, and neck of the sperm were stained with CD46 (red), DAPI (blue) and PMFBP1 antibodies (green), respectively. The localization of PMFBP1 in sperm from control and patient samples was determined by immunofluorescence staining. The PMFBP1 protein is localized at the head–tail junction of spermatozoa in control samples but is not detected in patient spermatozoa. 40 times oil microscope observation, scale bar = 4 µm

Article Snippet: Next, the primary antibodies (4′, 6-diamidino-2-phenylindole(DAPI), anti- PMFBP1 and anti-CD46 Thermo) were applied to the samples at a dilution of 1:200.

Techniques: Immunofluorescence, Control, Staining, Microscopy

( A ) Diagram of FCRL3 protein. Ig-like C2 domains are depicted as circles with the color indicating their previously established phylogenetic relationship, while tyrosines involved in intracellular signaling motifs are shown as stars. Red indicates an ITAM, light blue an ITIM, and purple indicates the ITIM-like/HemITAM Y722 motif in FCRL3. ( B ) Flow cytometry of Y. pestis infection of HeLa cells. HeLa cells (transfected with empty vector or FCRL3 plasmid) were infected for 1 hr with KIM6+ +pMMB67GFP, treated with gentamicin to kill extracellular bacteria for 1hr, and then GFP was induced in living, intracellular bacteria for 2 hrs with IPTG. Two populations of GFP+ cells were detected, consisting of HeLa cells infected with living, intracellular bacteria (GFP high , orange) in both conditions and those with attached, dead bacteria (GFP low , light blue) found in the FCRL3 overexpression condition. ( C ) Overexpression of FCRL3 increases attachment (GFP low , light blue) and invasion (GFP high , orange). HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometry as described in B. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. A western blot was performed to confirm the presence of FCRL3 after overexpression but not with empty vector using 1:200 FcRH3 antibody (Santa Cruz, C-2). ( D ) GFP low cells have attached, extracellular Y. pestis. FCRL3 transfected HeLa cells were infected as described in B and fixed with 4% paraformaldehyde (PFA) after 4 hrs. Infected and uninfected cells were blocked with normal goat serum (but not permeabilized) and then incubated with 1:20 polyclonal anti- Yersinia pestis F1-Antigen antibody (BEI Resources, NR-31024) to stain extracellular bacteria. DNA was then stained with 1 drop/10mL of a membrane permeable dye NucBlue for nuclei visualization. To visualize the GFP low bacteria, a high and low exposure GFP panel is shown. GFP low bacteria are indicated with a teal arrowhead and GFP high bacteria with an orange arrowhead. Cells were imaged using a 40× air objective on an EVOS M5000 Microscope. A 20µm scale bar is located on the merged image. ( E ) CRISPR-mediated knockout (KO) of FCRL3 in LCL HG02678 causes significant decrease in attachment and invasion of Y. pestis . Cells were electroporated with either only CD46 (control) or FCRL3 + CD46 guides and CRISPR Cas9. Pooled KO cells (>70% FCRL3 -/- ) were sorted for CD46 -/- cells. Cells were assayed for KIM6+ Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three experiments with two biological replicates of each condition were plotted and an unpaired t-test was performed to determine significance. ( F ) FCRL3 overexpression increases attachment and invasion of Y. pestis KIM5. HeLa cells transfected with empty vector or FCRL3 plasmid were assayed for KIM5 Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi, after subculturing bacteria for 2 hrs and 40 min at either 26 or 37 degrees C. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( G ) FCRL3 overexpression has no effect on Y. pseudotuberculosis attachment and invasion. HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( C, E-G ) Experiments were normalized by grand mean.

Journal: bioRxiv

Article Title: Human genetic variation reveals FCRL3 is a lymphocyte receptor for Yersinia pestis

doi: 10.1101/2024.12.05.626452

Figure Lengend Snippet: ( A ) Diagram of FCRL3 protein. Ig-like C2 domains are depicted as circles with the color indicating their previously established phylogenetic relationship, while tyrosines involved in intracellular signaling motifs are shown as stars. Red indicates an ITAM, light blue an ITIM, and purple indicates the ITIM-like/HemITAM Y722 motif in FCRL3. ( B ) Flow cytometry of Y. pestis infection of HeLa cells. HeLa cells (transfected with empty vector or FCRL3 plasmid) were infected for 1 hr with KIM6+ +pMMB67GFP, treated with gentamicin to kill extracellular bacteria for 1hr, and then GFP was induced in living, intracellular bacteria for 2 hrs with IPTG. Two populations of GFP+ cells were detected, consisting of HeLa cells infected with living, intracellular bacteria (GFP high , orange) in both conditions and those with attached, dead bacteria (GFP low , light blue) found in the FCRL3 overexpression condition. ( C ) Overexpression of FCRL3 increases attachment (GFP low , light blue) and invasion (GFP high , orange). HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometry as described in B. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. A western blot was performed to confirm the presence of FCRL3 after overexpression but not with empty vector using 1:200 FcRH3 antibody (Santa Cruz, C-2). ( D ) GFP low cells have attached, extracellular Y. pestis. FCRL3 transfected HeLa cells were infected as described in B and fixed with 4% paraformaldehyde (PFA) after 4 hrs. Infected and uninfected cells were blocked with normal goat serum (but not permeabilized) and then incubated with 1:20 polyclonal anti- Yersinia pestis F1-Antigen antibody (BEI Resources, NR-31024) to stain extracellular bacteria. DNA was then stained with 1 drop/10mL of a membrane permeable dye NucBlue for nuclei visualization. To visualize the GFP low bacteria, a high and low exposure GFP panel is shown. GFP low bacteria are indicated with a teal arrowhead and GFP high bacteria with an orange arrowhead. Cells were imaged using a 40× air objective on an EVOS M5000 Microscope. A 20µm scale bar is located on the merged image. ( E ) CRISPR-mediated knockout (KO) of FCRL3 in LCL HG02678 causes significant decrease in attachment and invasion of Y. pestis . Cells were electroporated with either only CD46 (control) or FCRL3 + CD46 guides and CRISPR Cas9. Pooled KO cells (>70% FCRL3 -/- ) were sorted for CD46 -/- cells. Cells were assayed for KIM6+ Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three experiments with two biological replicates of each condition were plotted and an unpaired t-test was performed to determine significance. ( F ) FCRL3 overexpression increases attachment and invasion of Y. pestis KIM5. HeLa cells transfected with empty vector or FCRL3 plasmid were assayed for KIM5 Y. pestis attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi, after subculturing bacteria for 2 hrs and 40 min at either 26 or 37 degrees C. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( G ) FCRL3 overexpression has no effect on Y. pseudotuberculosis attachment and invasion. HeLa cells were transfected with empty vector or FCRL3 plasmid and assayed for attachment and invasion by flow cytometric gentamicin protection assay at 4 hpi. Three biological replicates in each of three experiments were plotted and an unpaired t-test was performed to determine significance. ( C, E-G ) Experiments were normalized by grand mean.

Article Snippet: After cells reached >2 million, they were stained with 200 ng/1 million cells PE CD46 (TRA-2-10) flow antibody (BioLegend) and the CD46 negative population was separated by fluorescence-activated cell sorting.

Techniques: Flow Cytometry, Infection, Transfection, Plasmid Preparation, Bacteria, Over Expression, Western Blot, Incubation, Staining, Membrane, Microscopy, CRISPR, Knock-Out, Control, Subculturing Assay