Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Cell-to-cell communications within the TIME for human Group3 and Group4 MB. (A) The UMAP plot of cell type clusters show the identification of 15 distinct cell clusters from the single-cell RNA-seq data of Group3 MB samples. OPC: Oligodendrocyte Precursor Cell. APC: Astrocyte Precursor Cell. M2_1: M2 macrophages cluster 1. M2_2: M2 macrophages cluster 2. NSC: Neural stem cell. Complement-M, complement macrophage. inflam dendritic cell: inflammatory dendritic cell. (B) The violin plot of the marker gene expression for each cell cluster. (C) CellCrossTalker predicted ligand-receptor-mediated tumor-immune cell communications using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis indicates the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (D) CellCrossTalker predicted ligand-receptor-mediated communications between M2 macrophages and tumor cells, M2 macrophages and T cells, M2 macrophages and B cells, as well as M2 macrophages and myeloid cells, using scRNA-seq data from Group3 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (E) The UMAP plot of cell type clusters show the identification of 13 distinct cell clusters. Single-cell RNA-seq data from Group4 MB samples were obtained from Hendrikse et al. (F) The heatmap illustrates the expression of marker genes used to annotate each cell cluster. (G) CellCrossTalker predicted ligand-receptor-mediated cell-cell communications using scRNA-seq data from Group4 MB samples. The vertical axis represents ligands and their corresponding receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor. (H) The heatmap shows the interaction strength of MIF-CD74 between various pairs of cell types, as predicted by CellCrossTalker using scRNA-seq data from Group4 MB samples. (I) CellCrossTalker predicted ligand-receptor-mediated communication co-receptors between tumor cells and immune compartment, using scRNA-seq data from Group3 and Group4 MB samples. The vertical axis represents ligands and their corresponding receptors and co-receptors, while the horizontal axis shows the sender cell type associated with the ligand and the receiver cell type associated with the receptor.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Single Cell, RNA Sequencing, Marker, Gene Expression, Expressing

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Restricted expression of CD74 and MIF in human normal tissues and MB subgroups. (A) Graph depicting the expression of CD74 (ENSG00000019582.14) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (B) CD74 protein expression in normal human cerebellum. Scale bar, 100 um. (C) Graph depicting the expression of MIF (ENSG00000240972.1) in normal human tissue RNA-sequencing data obtained from the GTEx consortium. The dataset comprises 7859 samples across 31 distinct normal tissues, with sample sizes ranging from 5 to 1152 samples per tissue. Expression levels are presented as relative expression levels in transcripts per million (TPM). (D) MIF protein expression in normal human cerebellum. Scale bar, 100 um. (E-J) Protein levels across human MB subgroups and subtypes (based on DNA methylome classification) from Ayrault cohort for the three selected proteins: CD74, CD68 and MIF. Boxplots show median (line), upper and lower quartiles (boxes), and lines extending to highest and lowest observations (whiskers). (K) CD74 immunohistochemistry staining analysis of paired human pediatric diagnostic (left) and relapse (right) MB samples. Black arrows depict CD74 positivity. Scale bar represents 100 µM. (L) Immunohistochemistry membrane staining depicting CD74 expression across a subgrouped human diagnostic MB tissue microarray. Scale bar represents 200 µM.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Expressing, RNA Sequencing, Immunohistochemistry, Staining, Diagnostic Assay, Membrane, Microarray

Journal: Neuro-Oncology

Article Title: MIF-CD74 signaling drives immune modulation in medulloblastoma

doi: 10.1093/neuonc/noag020

Figure Lengend Snippet: Inhibition of CD74 via the lateral ventricle demonstrates immune-modulation and decreased tumor burden in primary and relapsed MB. (A) IHC of treatment-naïve primary (top row) and radiation-treated (bottom row) GTML allografts displaying H&E, CD74, and MIF expression within the tumor. Results representative of 3 independent replicates. Much of the tumor outside the cerebellum was removed for downstream RNA sequencing analyses, Scale bar, 100 µm. (B) Experimental schematic for evaluating the CD74-MIF blocking peptide C36L1. GTML primary or relapse cells expressing firefly luciferase were allografted into the cerebellum of FVBNRJ mice. C36L1 was administered either intraperitoneally or locoregionally through the lateral ventricle at time of engraftment and 1-week after, C36L1 vehicle was used as a control. Bioluminescence imaging was conducted to monitor tumor engraftment, progression, and/or regression up to 14 days post-therapy. At the endpoint, the tumor and CNS were harvested and assessed for immune infiltration and tumor burden. (C) Growth rate of mice bearing primary GTML allografts ( n = 3-4 per group) treated with vehicle control (black), C36L1 peptide through the lateral ventricle (red), or C36L1 peptide intraperitoneally (blue) was determined by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (D) UMAP projection of flow cytometry analysis of tumor/cerebellum of mice treated vehicle control or the C36L1 peptide via the lateral ventricle. The left panel display cells colored by experimental group (Control - black, C36L1 delivered via lateral ventricle - red, C36L1 delivered intraperitoneally - blue), highlighting the distribution and intensity target expression across different cell populations within the tumor and cerebellum of treated and control mice. The visualization provides insights into the immune cell infiltration and its association with the treatment groups. (E) UMAP projection of flow cytometry results, colored by the expression of CD74. (F) UMAP projection of flow cytometry results, colored by the expression of MHCII. (G) UMAP projection of flow cytometry results, colored by the expression of CD11b. (H) IHC of control (top row) and peptide-treated (bottom row) GTML primary tumor allografts displaying H&E, CD3, F4/80 and CD74 within the tumor. Results representative of 3 independent replicates, Scale bar, 100 um. (I) Growth rate of mice bearing recurrent GTML allografts ( n = 5 per group) treated with vehicle control (black) or C36L1 peptide through the lateral ventricle (blue) was calculated by calculating the slope of tumor growth between day 7 and 14 (endpoint). Line represents mean growth rate with individual points representing individual mice. Points below the dashed line indicate tumor regression. Statistical significance was calculated by two-way ANOVA with Tukey’s post-test. (J) Bar chart to illustrate the proportion of tumor-associated immune cells identified as microglia. A higher proportion of microglia is observed in the TME of CD36L1 peptide treatment versus scrambled control. (K) Bar chart to illustrate the level of CD74 expression in the TME of mice treated with CD36L1 versus scrambled control. (L) Bar charts displaying the proportion of CD38+ cells in the microglia population. Statistical analysis was performed using a two-way ANOVA with Tukey’s post-test to compare the groups. Error bars represent the SD. Significant differences between groups are indicated by * P < .05, ** P < .01, and *** P < .001.

Article Snippet: In brief, tissue sections were incubated in Tris-EDTA buffer (cell conditioning 1; CC1) at 95 ̊C for 1-h to retrieve antigenicity, followed by incubation with CD74 antibody (Origene CF507339 ) at 1:500 for 1-h.

Techniques: Inhibition, Expressing, RNA Sequencing, Blocking Assay, Luciferase, Control, Imaging, Flow Cytometry

Journal: bioRxiv

Article Title: T CELLS PROMOTE THE GROWTH OF SMALL-CELL LUNG CARCINOMA VIA AN IL-6/CD74 AXIS

doi: 10.64898/2025.12.19.694894

Figure Lengend Snippet: A. Experimental approach. To identify candidate genes mediating the pro-tumor effects of T cells in SCLC, bulk RNA-seq was performed on KP1 cells cultured alone or with TILs ( in vitro ), and on KP11 tumors grown in NSG mice with or without TILs ( in vivo ). B. Venn diagram of the number of genes significantly up- and down-regulated in bulk RNA-seq data, as in (A). C. Correlation of CD3ε (T cells) and CD74 in RNA-seq data from 81 human SCLC tumors. D. Cell death analysis of KP1 SCLC cells treated with IL-6 blocking antibody with or without TILs cells for 48 hours (n=4). Statistical significance was assessed using a t-test; error bars represent SD. E. Quantification for Cd7 4 mRNA levels relative to Gapdh in KP1 cells co-culture with TILs, with or without IL-6-blocking antibodies (n=3). Statistical significance was assessed using a t-test; error bars represent s.e.m. F. Representative immunofluorescence staining of CD74 (red) in SCLC tumors from control (Ctrl) and T cell-depleted (Depl.) RPR2 mutant mice. DAPI stains DNA in blue. Scale bar, 50 µm. G. Quantification of CD74 staining as in (F), normalized to DAPI staining (n=4 mice). Statistical significance was assessed using a t-test; error bars represent s.e.m. H. Correlation of CD3 + T cells and CD74 + cells from n=110 SCLC samples. Low T cell infiltration (green) and high T cell infiltration (orange) tumors are highlighted. I. Immunofluorescence staining of low T cell infiltration (green, right) and high T cell infiltration (orange, left) in human SCLC tumor sections, as in (H). CD3 (green) and CD74 (red). DAPI stains DNA in blue. Scale bar, 50 µm. J. Cell viability assay (AlamarBlue) of KP1 SCLC cells treated with ISO-1 (100 µg/mL and 250 µg/mL) or DMSO control (Ctrl) for 4 days (n=3). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. K. Cell death analysis of KP1 SCLC cells treated with ISO-1 (100 µg/mL) or DMSO control (Ctrl), with or without TILs, for 2 days (n=5-7). Statistical significance was assessed using a t-test; error bars represent SD. L. Representatives immunoblot and quantification of CD74 protein levels relative to HSP90 in control (shCtrl) and CD74 knock-down (sh Cd74 #1 and #2) KP1 cells (n=3). Statistical significance was assessed using a t-test; error bars represent SD. M. Cell viability assay (AlamarBlue) of control (shCtrl) and CD74 knock-down KP1 (sh Cd74 #1 and #2) cells for 4 days (n=3). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. N. Tumor growth curves of KP1 SCLC allografts in control (shCtrl) and T cell-depleted mice (Depl.), with or without Cd74 knock-down (n=8-9 mice per group, 2 tumors per mouse, 1 experiment). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. O. Representative images of KP1 SCLC allografts (terminal time point) as in (N). P. Fold change in tumor size as in (N). A depletion ratio score was calculated for each time point by dividing tumor volume by the mean of control tumors. Ratio >1 indicates an anti-tumor effect of T cells, ratio = 1 indicates no effect, and ratio <1 indicates a pro-tumor effect of T cells. In controls the ratio decreased (<1), whereas in CD74 knock-down tumors it remained ∼1. Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. Q. Tumor growth curves of control KP11 SCLC allografts and allografts co-injected with TILs, with or without Cd74 knock-down (n=7-8 mice per condition, 1-2 tumors per mouse, 1 experiment). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. R. Representative images of KP11 SCLC allografts (terminal time point), as in (Q). S. Fold change in tumor size as in (Q) calculated at each time point by dividing tumor volume by the mean of control tumors. Ratio >1 indicates a pro-tumor effect of T cells, ratio = 1 indicates no effect, and ratio <1 indicates an anti-tumor effect of T cells. A pro-tumor effect was observed in control tumors (ratio >1), whereas no effect was observed in CD74 knock-down tumors (ratio ∼1). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m.

Article Snippet: The following antibodies were used: HSP90 (4877S, Cell Signaling), CD74 (AF7478, R&D Systems), STAT3 (4904, Cell Signaling), phospho-STAT3 (9145, Cell Signaling), Akt (4691, Cell Signaling), Phospho-Akt (4058, Cell Signaling).

Techniques: RNA Sequencing, Cell Culture, In Vitro, In Vivo, Blocking Assay, Co-Culture Assay, Immunofluorescence, Staining, Control, Mutagenesis, Viability Assay, Western Blot, Knockdown, Injection

Journal: bioRxiv

Article Title: T CELLS PROMOTE THE GROWTH OF SMALL-CELL LUNG CARCINOMA VIA AN IL-6/CD74 AXIS

doi: 10.64898/2025.12.19.694894

Figure Lengend Snippet: A-D. Correlation of CD3ε (T cells) with expression of HLA-G (A), CCD80 (B), PIGR (C), and PBHMT (D) in RNA-seq data from 81 human SCLC tumors. E. Apoptosis analysis of KP11 SCLC cells treated with IL-6 blocking antibody in the presence or absence of TILs for 48 hours (n=4-6). Statistical significance was assessed by a t-test; error bars represent SD. F. Quantification for Cd7 4 mRNA levels relative to Gapdh in KP11 cells co-culture with TILs, with or without IL-6-blocking antibodies (n=3). Statistical significance was assessed using a t-test; error bars represent s.e.m. G. Quantification of CD74 staining normalized to DAPI (n=8 mice, one tumor per mouse). Statistical significance was assessed using a t-test; error bars represent s.e.m. H. Representative immunofluorescence staining of CD74 (red) in KP1 SCLC allografts from control (Ctrl) and T cell-depleted (Depl.) mice, as in (G). DAPI stains DNA in blue. Scale bar, 50 µm. I. Representative immunofluorescence staining of CD3 (green) and CD74 (red) in KP1 SCLC allografts from control tumors (Ctrl) and tumors mixed with TILs cells (+TILs). DAPI stains DNA in blue. Scale bar, 50 µm. J. Quantification of CD3 staining as in (I), normalized to DAPI (n=7 mice, n=1-2 tumors per mouse). Statistical significance was assessed using a t-test; error bars represent s.e.m. K. Quantification of CD74 as in (I), normalized to DAPI (n=7 mice, n=1-2 tumors per mouse). Statistical significance was assessed using a t-test; error bars represent s.e.m. L. Representative immunofluorescence staining for CD3 (green) and CD74 (red) in KP11 SCLC allografts from control tumors (Ctrl) and tumors mixed with TILs cells (+TILs). DAPI stains DNA in blue. Scale bar, 50 µm. M. Quantification of CD3 staining as in (L), normalized to DAPI (n=7-9 mice, one tumor per mouse). Statistical significance was assessed using a t-test; error bars represent s.e.m. N. Quantification of CD74 staining as in (L), normalized to DAPI (n=7-9 mice, one tumor per mouse). Statistical significance was assessed using a t-test; error bars represent s.e.m. O. Representative immunoblot and quantification of phosphorylated AKT (pAKT) relative to total AKT normalized to HSP90 in KP11 SCLC cells cultured alone or co-cultured with TILs or splenic T cells (T Spleen ). Statistical significance was assessed by a t-test; error bars represent SD (n=3). P. Cd74 and Mif expression in KP1 and TILs co-culture assay, as described in . Q. Cd74 and Mif expression in KP11 allograft tumors with or without TILs, as described in . R. Cell viability assay (AlamarBlue) of KP11 SCLC cells treated with ISO-1 (100 µg/mL and 250 µg/mL) or DMSO control (Ctrl) for 4 days (n=3). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. S. Cell death analysis of KP11 SCLC cells treated with ISO-1 (100 µg/mL) or DMSO control (Ctrl), with or without TILs cells for 48 hours (n=4-6). Statistical significance was assessed using a t-test; error bars represent SD.

Article Snippet: The following antibodies were used: HSP90 (4877S, Cell Signaling), CD74 (AF7478, R&D Systems), STAT3 (4904, Cell Signaling), phospho-STAT3 (9145, Cell Signaling), Akt (4691, Cell Signaling), Phospho-Akt (4058, Cell Signaling).

Techniques: Expressing, RNA Sequencing, Blocking Assay, Co-Culture Assay, Staining, Immunofluorescence, Control, Western Blot, Cell Culture, Co-culture Assay, Viability Assay

Journal: bioRxiv

Article Title: T CELLS PROMOTE THE GROWTH OF SMALL-CELL LUNG CARCINOMA VIA AN IL-6/CD74 AXIS

doi: 10.64898/2025.12.19.694894

Figure Lengend Snippet: A. Representative immunoassay and quantification of CD74 protein levels relative to HSP90 in control (shCtrl) and CD74 knock-down (sh Cd74 #1 and #2) KP11 cells (n=3). Statistical significance was assessed using a t-test; error bars represent SD. B. Cell viability assay (AlamarBlue) of control (shCtrl) and Cd74 knock-down (sh Cd74 #1 and #2) KP11 cells for 4 days (n=3). Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. C. Tumor weight of KP1 SCLC allografts at terminal time point as in (n=8-9 mice, 2 tumors per mouse, 1 experiment). Statistical significance was assessed using a t-test; error bars represent s.e.m. D. Tumor growth curves of KP11 SCLC allografts (n=7-8 mice, 2 tumors per mouse, 1 experiment) in control (shCtrl) and T cell-depleted mice (Depl.), with or without Cd74 knock-down. Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. E. Representative images of KP11 SCLC allografts at terminal time point as in (D). F. Fold change in tumor size as in (D). Depletion ratio score calculated by dividing tumor volume at each time point by the mean volume of control tumors. Ratio >1 indicates an anti-tumor effect of T cells, ratio = 1 indicates no effect, and ratio <1 indicates a pro-tumor effect. In controls, ratio decreases (<1), whereas in CD74 knock-down tumors, ratio remains ∼1. Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. G. Tumor weight of KP11 SCLC allografts at terminal time point as in (D). Statistical significance was assessed using a t-test; error bars represent s.e.m. H. Tumor weight of KP11 SCLC allografts at terminal time point as in . Statistical significance was assessed using a t-test; error bars represent s.e.m. I. Tumor growth curves of control KP1 SCLC allografts and allografts co-injected with TILs cells (n=7-9 mice, 1-2 tumors per mouse, 1 experiment), with or without Cd74 knock-down. Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. J. Representative images of KP1 SCLC allografts at terminal time point, as in (I). K. Fold change in tumor size as in (I). Tumor volume at each time point was divided by the mean volume of control tumors. As expected, the ratio increases for control tumors (TILs promote tumor growth), whereas it remains closer to 1 for Cd74 knock-down tumors. Statistical analysis was performed using two-way ANOVA; error bars represent s.e.m. L. Tumor weight of KP1 SCLC allografts at terminal time point, as in (I). Statistical significance was assessed using a t-test; error bars represent s.e.m.

Article Snippet: The following antibodies were used: HSP90 (4877S, Cell Signaling), CD74 (AF7478, R&D Systems), STAT3 (4904, Cell Signaling), phospho-STAT3 (9145, Cell Signaling), Akt (4691, Cell Signaling), Phospho-Akt (4058, Cell Signaling).

Techniques: Control, Knockdown, Viability Assay, Injection

Journal: bioRxiv

Article Title: T CELLS PROMOTE THE GROWTH OF SMALL-CELL LUNG CARCINOMA VIA AN IL-6/CD74 AXIS

doi: 10.64898/2025.12.19.694894

Figure Lengend Snippet: A. Cartoon of the approach used to test the combination therapy. B. Tumor weight of KP1 SCLC allografts at the terminal time point as in . Statistical significance was assessed using a t-test; error bars represent s.e.m. C. Tumor weight of KP11 SCLC allografts at the terminal time point as in . Statistical significance was assessed using a t-test; error bars represent s.e.m. D. Table showing the percentage of each immune cell cluster in each treatment condition as in . E. Uniform Manifold Approximation and Projection (UMAP) plots of scRNA-seq data from SCLC cells isolated from KP11 allografts (n = 4 per treatment), comparing untreated, PD-1 blockade, IL-6 blockade, and combined treatments. Cell clusters are color-coded by population, illustrating changes in intratumoral SCLC heterogeneity. F. Box plot of NF-κB pathway scores in SCLC cells across the four experimental conditions. Statistical analysis by t-test; error bars represent SD. G. Forest plots showing progression-free survival (PFS) hazard ratios with 95% confidence intervals (CI) for atezolizumab plus carboplatin/etoposide (atezo+CE) versus placebo plus CE (placebo+CE) in the T signature > median cohort, stratified by gene expression. Low gene expression (≤ median) cohort shown in blue (Gene low ), high gene expression (> median) cohort shown in red (Gene high ). “Cohort” refers to the T signature > median without further subsetting. H. As in (G) for the T signature ≤ median cohort, stratified by gene expression. I. Model: (Left) In most cases of SCLC, T cells fail to control SCLC tumor growth, in part because of low cytotoxic activity and also because IL-6 produced by T cells can activate pro-survival signals in SCLC cells, including via induction of CD74. (Right) Combining immune checkpoint inhibition (ICI) to activate the cytotoxic activity of T cells and IL-6 blockade to suppress pro-survival signals may help inhibit SCLC growth.

Article Snippet: The following antibodies were used: HSP90 (4877S, Cell Signaling), CD74 (AF7478, R&D Systems), STAT3 (4904, Cell Signaling), phospho-STAT3 (9145, Cell Signaling), Akt (4691, Cell Signaling), Phospho-Akt (4058, Cell Signaling).

Techniques: Isolation, Gene Expression, Control, Activity Assay, Produced, Inhibition

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: The total cholesterol (mg/dl) of ( A ) female ( B ) male WT and β2GPI KO mice fed with NC or HF diet, respectively. The triglyceride concentration (mg/dl) of female ( C ) and male ( D ) WT and β2GPI KO mice when fed with NC or HF diet, respectively. n = 5, * p < 0.05 by two tailed Student’s t-test. WT = wild type, mg/dl = milligrams/decilitre = WT, = β2GPI KO, NC= Normal Chow, HF= high fat, KO = Knockout

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Concentration Assay, Two Tailed Test, Knock-Out

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: Lipoprotein separation of plasma samples from WT and β2GPI KO female and male mice fed a HF or NC diet using FPLC. ( A-D ) ( A ) Female WT and β2GPI KO mice fed a NC and ( B ) HF diet ( n = 5). ( C ) Male WT and KO mice fed NC ( n = 5) and ( D ) HF diet ( n = 4). Cholesterol levels are expressed in µg/ml. The numbers on the X axis denote FPLC fractions. VLDL eluted in fractions 13 to 21, LDL in fractions 22 to 39 and HDL in fractions 40 to 51. WT = wild type, KO = Knockout, NC = normal chow, HF= high fat = β2GPI KO = WT

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Clinical Proteomics, Knock-Out

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: Western blot analysis of ApoE and ApoAI from individual HDL FPLC fractions comparing WT and β2GPI KO mice fed NC or HF diet. ( A ) Immunoreactivity of ApoE and ApoAI in FPLC HDL fractions from individual mice, 5 WT and 5 β2GPI KO ( A ) female mice fed NC ( B ) female mice fed a HF diet. ( C ) male mice fed NC. ( D ) male mice fed a HF diet. The ratio of Apo E to Apo AI in the HDL fractions are shown in the bar graphs below the Western blots ( A ), ( B ), ( C ), and ( D ) respectively. WT = Wild type, KO = β2GPI Knockout, NC = normal chow, HF = high fat, = WT, = β2GPI KO, ST = standard (normal plasma), Apo E = Apolipoprotein E, Apo AI = Apolipoprotein AI, MW = molecular weight, kDa = kilodaltons

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Western Blot, Knock-Out, Clinical Proteomics, Molecular Weight

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: The plasma ApoE concentration of (A) female and (B) male WT and β2GPI KO mice when fed either a NC or HF diet. n = 5, * p < 0.05 by two-tailed Student’s t-test. NC = normal chow, HF = high fat diet, Apo E = Apolipoprotein E, ng/ml = nanograms per milliliter = WT, = β2GPI KO

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: ( A ) β2GPI immunoreactivity in the pooled plasma lipoprotein fractions (VLDL, LDL, HDL) in female WT mice fed NC or HF diet. β2GPI reactive bands were only detected in the HDL fractions. ( B ) Quantifications of the scanned β2GPI immunoreactive bands in the HDL fractions of WT mice. Density of β2GPI in the HDL FPLC fractions from female WT mice fed a NC or HF diet. n = 5, * p < 0.05. ( C ) Immunoprecipitation of ApoE with an anti-β2GPI monoclonal antibody using plasma from female WT and β2GPI KO mice. Immunoprecipitates from WT or β2GPI KO mouse plasma was subjected to Western blot using an antibody to ApoE or β2GPI. Immunoreactive bands for β2GPI and ApoE were detected in the immunoprecipitates from WT (lane 1) but not from β2GPI KO mice (lane 2). Plasma controls consisted of WT mouse plasma demonstrating immunoreactive bands to β2GPI and ApoE (lane 3), β2GPI KO plasma did not have an immunoreactive band to β2GPI but had an immunoreactive band to ApoE (lane 4). VLDL = Very low density lipoproteins, LDL = low density lipoproteins, HDL = High density lipoproteins, KO = Knockout, HF = high fat diet, NC = normal chow, WT = Wild type

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Clinical Proteomics, Immunoprecipitation, Western Blot, Knock-Out

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: Lipoprotein separation of human plasma samples using FPLC. ( A ) females ( B ) males. Plasma used was from 3 patients deficient in β2GPI (1 female and 2 males) and 7 age and sex matched controls (3 females and 4 males), Cholesterol levels are expressed in µg/ml. The numbers on the horizontal axis denote FPLC fractions. VLDL eluted in fractions 5 to 11, LDL in fractions 12 to 30 and HDL in fractions 31 to 46. VLDL = Very Low-density lipoprotein, LDL = low density lipoprotein, HDL = high density lipoprotein controls, β2GPI deficient patients, β2GPI = βeta-2-glycoprotein-I

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Clinical Proteomics

Journal: Biology of Sex Differences

Article Title: βeta-2 glycoprotein I is a novel regulator of Apolipoprotein E containing HDL particles in females

doi: 10.1186/s13293-025-00766-9

Figure Lengend Snippet: Effect of β2GPI deficiency on levels of ApoE in human HDL plasma samples. ( A) Western blot analysis of β2GPI, Apo E and Apo AI in pooled VLDL, LDL and HDL FPLC fractions from β2GPI deficient or control patient plasma samples. C = normal controls D = β2GPI deficient ( B ) Immunoprecipitates from one β2GPI deficient patient sample (lanes 1,3) and one normal control (lanes 2, 4) were subjected to Western blot using anti-β2GPI monoclonal antibody (lanes 1,2), or with an isotype control IgG monoclonal antibody (lanes 3, 4) and plasma from a female β2GPI deficient patient (lane 5) or normal control (lane 6). Immunoreactivity to β2GPI was not detected in any of the β2GPI deficient patient samples. In the β2GPI deficient samples ApoE immunoreactivity was only detected in the plasma sample (lane 5) but not in the immunoprecipitation samples (lane 1). Immunoreactivity for β2GPI and ApoE was detected using specific anti-human β2GPI or ApoE antibodies. ( C ) Binding of biotinylated ApoE to β2GPI and domain deletion mutants DI-IV and DII-V. % binding is expressed as a percentage of the binding of ApoE to full length β2GPI (DI-DV) which is set as 100. β2GPI = β2 glycoprotein I, ApoE = Apolipoprotein E, ApoAI = Apolipoprotein A I, MW = molecular weight, kDa = Kilodaltons

Article Snippet: Pre-cleared plasma was obtained following centrifugation for 30 s at 14,000 g. Individual plasma samples were then incubated for 14–16 h at 4°C with either sheep anti-mouse β2GPI antibody (R&D systems) (10 μg/ml) or rabbit anti-human β2GPI antibody, [ ] at 5 μg/ml final antibody concentration, [ ] followed by the addition of 20 μl of protein G-Sepharose beads.

Techniques: Clinical Proteomics, Western Blot, Control, Immunoprecipitation, Binding Assay, Molecular Weight