Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: Clinicopathological data of the patients.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Expressing

Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: Primer sequences.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Sequencing

Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: High expression of TREM2 in RCC tissues. Reverse transcription-quantitative PCR was used to detect the expression of TREM2 in (A) 10 cases of RCC and matched adjacent normal tissues and (B) HK-2 and ACHN cells. **P<0.01, ****P<0.0001. TREM2, triggering receptor expressed on myeloid cells-2.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: TREM2 promotes the proliferation of RCC cells. ACHN cells were infected with the corresponding lentiviruses (sh-NC, sh-TREM2, vector and TREM2). After 48 h, cells were collected and the results of the knock-down and overexpression of TREM2 was assessed using (A) reverse transcription-quantitative PCR and (B) western blotting. (C) Cell Counting Kit-8 and (D) EdU assays were used to detect the effect of the knockdown or overexpression of TREM2 on the viability and proliferation of RCC cells. **P<0.01. TREM2, triggering receptor expressed on myeloid cells-2; RCC, renal cell carcinoma; sh, short hairpin; NC, negative control; EdU, 5-ethynyl-2′-deoxyuridine.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Infection, Plasmid Preparation, Knockdown, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Cell Counting, Negative Control

Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: TREM2 promotes migration of RCC cells. (A) Wound healing assay and (B) Transwell assays were used to assess the effect of the knockdown or overexpression of TREM2 on the migration and invasion of RCC cells. (A) Magnification, ×100. (B) Scale bar, 50 µm. **P<0.01. TREM2, triggering receptor expressed on myeloid cells-2; RCC, renal cell carcinoma; sh, short hairpin; NC, negative control.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Migration, Wound Healing Assay, Knockdown, Over Expression, Negative Control

Journal: Oncology Letters

Article Title: TREM2 promotes the proliferation and invasion of renal cell carcinoma cells by inhibiting the P53 signaling pathway

doi: 10.3892/ol.2024.14671

Figure Lengend Snippet: TREM2 promotes the proliferation of renal cell carcinoma cells by inhibiting the P53 signaling pathway. (A) Western blotting was used to assess the effects of the knockdown or overexpression of TREM2 on the protein and phosphorylation levels of P53 and P21, as well as the phosphorylation levels. (B) Semi-quantitative results of P53, p-P53, P21 and p-P21 protein bands. Relative ratio of (C) p-P53/P53 and (D) p-P21/P21. **P<0.01. TREM2, triggering receptor that is expressed on myeloid cells-2; sh, short hairpin; NC, negative control.

Article Snippet: The antibodies used included β-actin (1:5,000; cat. no. 13E5; CST Biological Reagents Co., Ltd.), TREM2 (1:1,000; cat. no. 91068; CST Biological Reagents Co., Ltd.), P53 (1:2,000; cat. no. 2527; CST Biological Reagents Co., Ltd.), p-P53 (1:1,000; cat. no. 82530; CST), P21 (1:2,000; cat. no. 2947; CST Biological Reagents Co., Ltd.) and p-P21 (1:500; cat. no. ab47300; Abcam).

Techniques: Western Blot, Knockdown, Over Expression, Negative Control

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lipid associated macrophage marker genes are increased in obese tumor-adjacent mammary adipose tissue (mAT Tum-adj ) and positively correlate with tumor mass. B) Schematic for magnetically sorting CD11b + cells from E0771 tumors using Miltenyi Octomacs. C) Trem2 , Cd36 , and Cd9 relative gene expression by RT-PCR in tumor CD11b + fraction. C) Anatomical schematic of subcutaneous mAT Tum-adj and contralateral mammary adipose tissue (mAT Contra ). D - F) Trem2 , Plin2 , Cd36 relative gene expression by RT-PCR from whole tissue lysates of mAT Contra and mAT Tum-adj . G-H) Correlation analysis of tumor mass vs. Trem2 and Plin2 gene expression in mAT Contra and mAT Tum-adj with linear regression fit. Figures D-F, solid bar = mAT Contra , hashed bar = mAT Tum-adj . One-way ANOVA tests were used to compare tumor LAM marker gene expression among groups. Two-way ANOVA with Tukey’s multiple comparison tests were used to compare groups for all mAT gene expression. Simple linear regression analysis was applied to correlation plots and the p value and coefficient of determination (r 2 ) is displayed on each graph. All data plotted as mean ± SEM for 8-12 mice per group. *denotes diet effect, †denotes tissue effect (mAT Contra vs. mAT Tum-adj ), **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, †††p<0.001, ††††p<0.0001. & C were created with Biorender.com.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Trem2 ablation attenuates tumor growth, decreases lipid associated macrophages, and increases adipocyte hypertrophy in a chow-fed postmenopausal breast cancer model. A) Schematic of study design for chow-fed postmenopausal breast cancer model in Trem2 +/+ and Trem2 -/- mice. B) sTREM2 (ng/mL) in plasma from female Trem2 +/+ and Trem2 -/- mice measured by ELISA. C) Blood glucose (mg/dL) measured during 14-week time point intra-peritoneal glucose tolerance test (ipGTT) and corresponding integrated area under the curve (iAUC). D) Tumor volume (mm 3 ) over time measured every 1-2 days. E) Tumor mass (g) at study endpoint. F) Relative gene expression by RT-PCR of LAM and macrophage markers from oAT whole tissue lysates. G) Adipocyte sizing frequency in tumor-adjacent mammary adipose tissue (mAT Tum-adj ). Diameter: small = 25-49μm, medium = 50-69μm, large = 70-99μm, very large = 100-150μm, extremely large = >150μm. H-I) Representative image of Trem2 +/+ and Trem2 -/- mAT Tum-adj stained with hematoxylin and eosin. Scale bar represents 100μm. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare blood glucose over time in the ipGTT. Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes. Unpaired two-tailed t-tests were used to compare groups for sTREM2 ELISA, iAUC, tumor mass, LAM gene expression, and each binning diameter size in adipocyte sizing analysis. All data plotted as mean ± SEM for 10-13 mice per group. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. was created with Biorender.com.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lean, but not obese or weight loss, Trem2 -/- mice exhibit decreased tumor growth. A) Body weight (g) measured weekly with dashed lines to indicate time of diet switch and E0771 PD-L1 cell injection. For diet groups and genotypes, light green = lean Trem2 +/+ , dark green = lean Trem2 -/- , light blue = obese Trem2 +/+ , dark blue = obese Trem2 -/- , light pink = weight loss Trem2 +/+ , and maroon = weight loss Trem2 -/- . B) sTREM2 (ng/mL) in plasma from Trem2 +/+ and Trem2 -/- mice measured by ELISA.C) Tumor mass (g) at study endpoint. D-F) Tumor volume (mm 3 ) over time for lean, obese, and weight loss Trem2 +/+ and Trem2 -/- mice, respectively. G) Diagram of tumor and tumor-adjacent mammary adipose tissue CD45 + cell sorting and processing for single cell RNA Sequencing. Mixed effects analysis with uncorrected Fisher’s LSD test was used compare tumor volumes over time between genotypes in the lean group (one missing value in lean Trem2 +/+ group at day 28). Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes in obese and weight loss groups. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare tumor mass between genotypes in lean, obese, and weight loss groups. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ), †denotes diet effect, *p<0.05, **p<0.01, ****p<0.0001 for genotype differences (Trem2 +/+ vs. Trem2 -/- ) and ††††p<0.0001 for diet differences. All data plotted as mean ± SEM for n=4-23 per group. Figure G was created with Biorender.com.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, FACS, RNA Sequencing Assay, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Myeloid populations in tumors and tumor-adjacent mammary adipose tissues (mAT Tum-adj ) analyzed by single-cell RNA sequencing. A-B) Uniform Manifold Approximation and Projection (UMAP) of tumor (A) and mAT Tum-adj (B) myeloid cell subclusters from merged conditions labeled broadly by cell type category and colored by high-resolution cell type identities. C-D) Myeloid cell subcluster proportions expressed as frequency (%) split by diet-genotype groups for tumor (C) and mAT Tum-adj (D). E-F) Trem2 expression in lean, obese, and weight loss Trem2 +/+ mice in tumor (E) and mAT Tum-adj (F). G) Dot plot of the top 5 differentially expressed genes (DEGs) between lean Trem2 +/+ and lean Trem2 -/- tumor Macro_C1qc subclusters. Genes include H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 . Log fold change is shown for all 6 diet-genotype groups. H) Dot plot of H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 log fold change in the mAT Tum-adj LAM subcluster shown in all 6 diet-genotype groups.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: RNA Sequencing Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Visualization and binning of Tumor T cell clones reveals expansion of highly clonal CD4 + Th1 cells in lean Trem2 -/- tumors. A) Stacked bar graph of tumor T cell subclusters indicating cells per gram of tumor across all 6 diet-genotype groups. B) Tumor T cell subcluster proportions expressed as frequency (%) across all 6 diet-genotype groups. C) Uniform Manifold Approximation and Projection (UMAP) of tumor T cell subclusters from merged conditions colored by high-resolution cell type identities. D-I) Tumor T cell clones with a clonal population of two or more visualized by mapping clonal cell counts, indicated by color, onto the tumor T cell UMAP. The data is split as labeled by diet and genotype. J) Stacked bar graph showing the distribution of tumor T cell subclusters, binned as single cells or clonal low, clonal moderate, or clonal high (low = x ≤ 12 (median); moderate = 13 ≤ x ≤ 79 (3 rd quartile cutoff); high = x >79) shown for all 6 diet-genotype groups.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: Clone Assay, Labeling

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Obese mice do not respond to αPD-1 therapy and do not exhibit an increase of T cell activation markers in tumor-adjacent mammary adipose tissue (mAT Tum-adj ) or tumor. A) Schematic of study design for lean, obese, and weight loss breast cancer model treated with IgG and αPD-1. During the last two weeks of tumor monitoring, mice were injected with either αPD-1 or IgG isotype control. B) Tumor mass (g) at study endpoint. C-E) Tumor volume (mm 3 ) over time for lean, obese, and weight loss IgG and αPD-1 treated mice with dashed line to indicate start of antibody treatment. F) Schematic of isolating the stromal vascular fraction from the mAT Tum-adj and subsequently isolating a pooled sample of CD4 + and CD8 + T cells using the magnetic Miltenyi Octomacs microbead sorting system. G) T cell activation markers, Pdcd1 , Tigit , Tox , and Lag3 relative gene expression by RT-PCR from magnetically sorted CD4 + and CD8 + T cells from the mAT Tum-adj . H) Cd8a , Pdcd1 , Lag3 , and Trem2 gene expression by RT-PCR from whole tumor lysates. Due to the response to αPD-1 treatment in the lean mice, only two tumors from the lean αPD-1 group could be analyzed by RT-PCR. For diet groups, solid colors: green = lean IgG, blue = obese IgG, pink = weight loss IgG; open circles: dark green = lean αPD-1, dark blue = obese αPD-1, maroon = weight loss αPD-1. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare groups for tumor mass. Mixed effects analysis was used for statistical analysis of tumor volumes over time. Two-way ANOVA with Sidak’s multiple comparison test was used to compare groups in the T cell activation marker gene expression analysis. All data plotted as mean ± SEM for 2-6 mice per group. *denotes antibody effect (IgG vs. αPD-1), †denotes diet effect, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, ††p<0.01. , F, & H were created with Biorender.com.

Article Snippet: A 96-well ELISA plate was coated overnight with TREM2 capture antibody (R&D Systems; cat. #MAB17291; RRID: AB_2208679).

Techniques: Activation Assay, Injection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Marker

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lipid associated macrophage marker genes are increased in obese tumor-adjacent mammary adipose tissue (mAT Tum-adj ) and positively correlate with tumor mass. B) Schematic for magnetically sorting CD11b + cells from E0771 tumors using Miltenyi Octomacs. C) Trem2 , Cd36 , and Cd9 relative gene expression by RT-PCR in tumor CD11b + fraction. C) Anatomical schematic of subcutaneous mAT Tum-adj and contralateral mammary adipose tissue (mAT Contra ). D - F) Trem2 , Plin2 , Cd36 relative gene expression by RT-PCR from whole tissue lysates of mAT Contra and mAT Tum-adj . G-H) Correlation analysis of tumor mass vs. Trem2 and Plin2 gene expression in mAT Contra and mAT Tum-adj with linear regression fit. Figures D-F, solid bar = mAT Contra , hashed bar = mAT Tum-adj . One-way ANOVA tests were used to compare tumor LAM marker gene expression among groups. Two-way ANOVA with Tukey’s multiple comparison tests were used to compare groups for all mAT gene expression. Simple linear regression analysis was applied to correlation plots and the p value and coefficient of determination (r 2 ) is displayed on each graph. All data plotted as mean ± SEM for 8-12 mice per group. *denotes diet effect, †denotes tissue effect (mAT Contra vs. mAT Tum-adj ), **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, †††p<0.001, ††††p<0.0001. & C were created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Trem2 ablation attenuates tumor growth, decreases lipid associated macrophages, and increases adipocyte hypertrophy in a chow-fed postmenopausal breast cancer model. A) Schematic of study design for chow-fed postmenopausal breast cancer model in Trem2 +/+ and Trem2 -/- mice. B) sTREM2 (ng/mL) in plasma from female Trem2 +/+ and Trem2 -/- mice measured by ELISA. C) Blood glucose (mg/dL) measured during 14-week time point intra-peritoneal glucose tolerance test (ipGTT) and corresponding integrated area under the curve (iAUC). D) Tumor volume (mm 3 ) over time measured every 1-2 days. E) Tumor mass (g) at study endpoint. F) Relative gene expression by RT-PCR of LAM and macrophage markers from oAT whole tissue lysates. G) Adipocyte sizing frequency in tumor-adjacent mammary adipose tissue (mAT Tum-adj ). Diameter: small = 25-49μm, medium = 50-69μm, large = 70-99μm, very large = 100-150μm, extremely large = >150μm. H-I) Representative image of Trem2 +/+ and Trem2 -/- mAT Tum-adj stained with hematoxylin and eosin. Scale bar represents 100μm. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare blood glucose over time in the ipGTT. Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes. Unpaired two-tailed t-tests were used to compare groups for sTREM2 ELISA, iAUC, tumor mass, LAM gene expression, and each binning diameter size in adipocyte sizing analysis. All data plotted as mean ± SEM for 10-13 mice per group. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. was created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Staining, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Lean, but not obese or weight loss, Trem2 -/- mice exhibit decreased tumor growth. A) Body weight (g) measured weekly with dashed lines to indicate time of diet switch and E0771 PD-L1 cell injection. For diet groups and genotypes, light green = lean Trem2 +/+ , dark green = lean Trem2 -/- , light blue = obese Trem2 +/+ , dark blue = obese Trem2 -/- , light pink = weight loss Trem2 +/+ , and maroon = weight loss Trem2 -/- . B) sTREM2 (ng/mL) in plasma from Trem2 +/+ and Trem2 -/- mice measured by ELISA.C) Tumor mass (g) at study endpoint. D-F) Tumor volume (mm 3 ) over time for lean, obese, and weight loss Trem2 +/+ and Trem2 -/- mice, respectively. G) Diagram of tumor and tumor-adjacent mammary adipose tissue CD45 + cell sorting and processing for single cell RNA Sequencing. Mixed effects analysis with uncorrected Fisher’s LSD test was used compare tumor volumes over time between genotypes in the lean group (one missing value in lean Trem2 +/+ group at day 28). Two-way ANOVA with Sidak’s multiple comparison test was used to compare tumor volumes over time between genotypes in obese and weight loss groups. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare tumor mass between genotypes in lean, obese, and weight loss groups. *denotes genotype effect (Trem2 +/+ vs. Trem2 -/- ), †denotes diet effect, *p<0.05, **p<0.01, ****p<0.0001 for genotype differences (Trem2 +/+ vs. Trem2 -/- ) and ††††p<0.0001 for diet differences. All data plotted as mean ± SEM for n=4-23 per group. Figure G was created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, FACS, RNA Sequencing Assay, Comparison

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Myeloid populations in tumors and tumor-adjacent mammary adipose tissues (mAT Tum-adj ) analyzed by single-cell RNA sequencing. A-B) Uniform Manifold Approximation and Projection (UMAP) of tumor (A) and mAT Tum-adj (B) myeloid cell subclusters from merged conditions labeled broadly by cell type category and colored by high-resolution cell type identities. C-D) Myeloid cell subcluster proportions expressed as frequency (%) split by diet-genotype groups for tumor (C) and mAT Tum-adj (D). E-F) Trem2 expression in lean, obese, and weight loss Trem2 +/+ mice in tumor (E) and mAT Tum-adj (F). G) Dot plot of the top 5 differentially expressed genes (DEGs) between lean Trem2 +/+ and lean Trem2 -/- tumor Macro_C1qc subclusters. Genes include H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 . Log fold change is shown for all 6 diet-genotype groups. H) Dot plot of H2-Eb1 , H2-Ab1 , H2-Aa , Cd74 , and Trem2 log fold change in the mAT Tum-adj LAM subcluster shown in all 6 diet-genotype groups.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: RNA Sequencing Assay, Labeling, Expressing

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Visualization and binning of Tumor T cell clones reveals expansion of highly clonal CD4 + Th1 cells in lean Trem2 -/- tumors. A) Stacked bar graph of tumor T cell subclusters indicating cells per gram of tumor across all 6 diet-genotype groups. B) Tumor T cell subcluster proportions expressed as frequency (%) across all 6 diet-genotype groups. C) Uniform Manifold Approximation and Projection (UMAP) of tumor T cell subclusters from merged conditions colored by high-resolution cell type identities. D-I) Tumor T cell clones with a clonal population of two or more visualized by mapping clonal cell counts, indicated by color, onto the tumor T cell UMAP. The data is split as labeled by diet and genotype. J) Stacked bar graph showing the distribution of tumor T cell subclusters, binned as single cells or clonal low, clonal moderate, or clonal high (low = x ≤ 12 (median); moderate = 13 ≤ x ≤ 79 (3 rd quartile cutoff); high = x >79) shown for all 6 diet-genotype groups.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Clone Assay, Labeling

Journal: bioRxiv

Article Title: Trem2 deficiency attenuates breast cancer tumor growth in lean, but not obese or weight loss, mice and is associated with alterations of clonal T cell populations

doi: 10.1101/2024.09.25.614811

Figure Lengend Snippet: Obese mice do not respond to αPD-1 therapy and do not exhibit an increase of T cell activation markers in tumor-adjacent mammary adipose tissue (mAT Tum-adj ) or tumor. A) Schematic of study design for lean, obese, and weight loss breast cancer model treated with IgG and αPD-1. During the last two weeks of tumor monitoring, mice were injected with either αPD-1 or IgG isotype control. B) Tumor mass (g) at study endpoint. C-E) Tumor volume (mm 3 ) over time for lean, obese, and weight loss IgG and αPD-1 treated mice with dashed line to indicate start of antibody treatment. F) Schematic of isolating the stromal vascular fraction from the mAT Tum-adj and subsequently isolating a pooled sample of CD4 + and CD8 + T cells using the magnetic Miltenyi Octomacs microbead sorting system. G) T cell activation markers, Pdcd1 , Tigit , Tox , and Lag3 relative gene expression by RT-PCR from magnetically sorted CD4 + and CD8 + T cells from the mAT Tum-adj . H) Cd8a , Pdcd1 , Lag3 , and Trem2 gene expression by RT-PCR from whole tumor lysates. Due to the response to αPD-1 treatment in the lean mice, only two tumors from the lean αPD-1 group could be analyzed by RT-PCR. For diet groups, solid colors: green = lean IgG, blue = obese IgG, pink = weight loss IgG; open circles: dark green = lean αPD-1, dark blue = obese αPD-1, maroon = weight loss αPD-1. Two-way ANOVA with uncorrected Fisher’s LSD test was used to compare groups for tumor mass. Mixed effects analysis was used for statistical analysis of tumor volumes over time. Two-way ANOVA with Sidak’s multiple comparison test was used to compare groups in the T cell activation marker gene expression analysis. All data plotted as mean ± SEM for 2-6 mice per group. *denotes antibody effect (IgG vs. αPD-1), †denotes diet effect, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; †p<0.05, ††p<0.01. , F, & H were created with Biorender.com.

Article Snippet: Biotinylated TREM2 antibody (R&D systems; cat. #BAF1729; RRID: AB_356109) in conjunction with streptavidin HRP (R&D Systems; cat. # DY998) were used to detect sTREM2 at 450 nm on a plate reader.

Techniques: Activation Assay, Injection, Control, Expressing, Reverse Transcription Polymerase Chain Reaction, Comparison, Marker

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for alveolar macrophages and not shared with interstitial macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (b) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for interstitial macrophages and not shared with alveolar macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (c) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for classical monocytes and not shared with interstitial macrophages and alveolar macrophages. Data reanalyzed from previously published scRNA-seq data . (d) Venn diagram showing the overlap between hyperoxia-induced genes (FC > 2; FDR < 0.05) in 3 different myeloid cell subsets; alveolar macrophages (n = 281), interstitial macrophages (n = 275), classical monocytes (n = 215) (left panel). Venn diagram showing the overlap between hyperoxia-induced genes in the three myeloid cell subsets (n = 18) and whole lung tissues (n = 253 in ) (right panel). (e) SNPs in the Trem2 gene between B6 and DBA mice. (f) Body weight in hyperoxia-exposed WT (B6) (same samples as ) and T2KO mice. Data are mean ± SEM (n = 6 for each group).

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques:

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Single-cell RNA-seq analysis of whole lung tissues from B6 mice exposed to 95% oxygen from P0 to P5 . (b) Trem2 mRNA expression in the lungs of B6 and DBA mice. Bar graphs show mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p -adj < 0.01 and *** p -adj < 0.001. (c) Immunoblots for TREM2 protein in whole lung tissues collected on P14 from B6 and DBA mice. Bar graphs show mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01. Uncropped images of the blots are shown in . (d) Soluble TREM2 levels in the plasma of B6 and DBA mice on P14 after neonatal hyperoxia exposure. Bar graphs show mean ± SEM (n = 3-8 per group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. (e) H&E staining was performed to assess the alveolar complexity at P14 in WT (B6) mice (left panels) and T2KO mice (right panels) with scale bars denoting 100 μm. The bar graph represents the results of the quantification of alveolar simplification using mean linear intercept (same WT (B6) samples as ). Data are mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. (f) Bronchoalveolar lavage (BAL) was performed at P14 in WT (B6) and T2KO hyperoxia-exposed and normoxic control mice (n = 14 for WT (B6) and n = 11-13 for T2KO mice). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. n.s.: not significant. (g) Matrix ultrastructural analysis of lung tissues of neonatal hyperoxia-exposed B6 (WT), DBA and T2KO mice on P14. Manifold of hyperoxia-induced pathological architecture with higher pseudotime representing increasingly disrupted architecture and alveolar simplification. Tissue images show representative tiles along the pseudotime trajectory. (h) Architecture-defining parameters. Identification of top 5 individual matrix features associated with low pseudotime (normal-like interstitium) and high pseudotime (more aberrant interstitium), based on Pearson correlations. Parameters are displayed as the absolute value of the Pearson coefficient (i.e. magnitude of correlation, as shown by height of arrows on y-axis), with all p < 0.001. (i) Visualization of hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. Hyperoxia-exposed lungs from DBA and T2KO mice with less lung matrix remodeling localize near the root point of the pseudotime trajectory. (j) Bar graphs of matrix pseudotime for hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. The difference in pseudotime normalized to normoxic control lungs is shown. Data are mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. n.s.: not significant. Each dot represents one mouse (b, d, e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: RNA Sequencing Assay, Expressing, Comparison, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) BMDMs were obtained from B6 (WT), DBA and T2KO mice and exposed to hyperoxia (95%) for 24 h. (b) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed BMDMs obtained from B6 (WT), DBA and T2KO mice and the normoxic controls. Uncropped images of the blots are shown in . (c) RNA-seq of hyperoxia-exposed BMDMs from B6 (WT), DBA and T2KO mice and the normoxic controls. (d) Venn diagram showing the overlap between hyperoxia-induced genes in B6 (WT) BMDMs (n = 173) and hyperoxia-suppressed genes in T2KO (n = 570) and DBA (n = 905) BMDMs. Gene Ontology analysis of 34 genes induced by hyperoxia in B6 (WT) BMDMs and suppressed in T2KO and DBA BMDMs. (e) Bar plots for expression of representative genes belonging to the p53 signaling pathway. Data are mean ± SEM. DSeq2 was used for comparisons. ** p -adj < 0.01 and *** p -adj < 0.001. (f) Caspase 3 activity measured in cytosolic fractions from BMDMs from B6 (WT), DBA and T2KO mice. Data are mean ± SEM. (n = 4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. Each dot represents one mouse (e, f). See also .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot, RNA Sequencing Assay, Expressing, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed (1, 2, 4 or 24 h) BMDMs obtained from B6 (WT) mice and normoxic controls (0 h). Uncropped images of the blots are shown in .

Article Snippet: Plates were washed five times and incubated with mouse TREM2 biotinylated antibody (R&D Systems, BAF1729) for 2 h at RT with constant shaking.

Techniques: Western Blot

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for alveolar macrophages and not shared with interstitial macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (b) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for interstitial macrophages and not shared with alveolar macrophages and classical monocytes. Data reanalyzed from previously published scRNA-seq data . (c) Gene Ontology enrichment analysis of hyperoxia-induced genes specific for classical monocytes and not shared with interstitial macrophages and alveolar macrophages. Data reanalyzed from previously published scRNA-seq data . (d) Venn diagram showing the overlap between hyperoxia-induced genes (FC > 2; FDR < 0.05) in 3 different myeloid cell subsets; alveolar macrophages (n = 281), interstitial macrophages (n = 275), classical monocytes (n = 215) (left panel). Venn diagram showing the overlap between hyperoxia-induced genes in the three myeloid cell subsets (n = 18) and whole lung tissues (n = 253 in ) (right panel). (e) SNPs in the Trem2 gene between B6 and DBA mice. (f) Body weight in hyperoxia-exposed WT (B6) (same samples as ) and T2KO mice. Data are mean ± SEM (n = 6 for each group).

Article Snippet: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques:

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Single-cell RNA-seq analysis of whole lung tissues from B6 mice exposed to 95% oxygen from P0 to P5 . (b) Trem2 mRNA expression in the lungs of B6 and DBA mice. Bar graphs show mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p -adj < 0.01 and *** p -adj < 0.001. (c) Immunoblots for TREM2 protein in whole lung tissues collected on P14 from B6 and DBA mice. Bar graphs show mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01. Uncropped images of the blots are shown in . (d) Soluble TREM2 levels in the plasma of B6 and DBA mice on P14 after neonatal hyperoxia exposure. Bar graphs show mean ± SEM (n = 3-8 per group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. (e) H&E staining was performed to assess the alveolar complexity at P14 in WT (B6) mice (left panels) and T2KO mice (right panels) with scale bars denoting 100 μm. The bar graph represents the results of the quantification of alveolar simplification using mean linear intercept (same WT (B6) samples as ). Data are mean ± SEM (n = 3-4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. (f) Bronchoalveolar lavage (BAL) was performed at P14 in WT (B6) and T2KO hyperoxia-exposed and normoxic control mice (n = 14 for WT (B6) and n = 11-13 for T2KO mice). ANOVA was performed followed by Tukey’s post hoc comparison. ** p < 0.01 and *** p < 0.001. n.s.: not significant. (g) Matrix ultrastructural analysis of lung tissues of neonatal hyperoxia-exposed B6 (WT), DBA and T2KO mice on P14. Manifold of hyperoxia-induced pathological architecture with higher pseudotime representing increasingly disrupted architecture and alveolar simplification. Tissue images show representative tiles along the pseudotime trajectory. (h) Architecture-defining parameters. Identification of top 5 individual matrix features associated with low pseudotime (normal-like interstitium) and high pseudotime (more aberrant interstitium), based on Pearson correlations. Parameters are displayed as the absolute value of the Pearson coefficient (i.e. magnitude of correlation, as shown by height of arrows on y-axis), with all p < 0.001. (i) Visualization of hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. Hyperoxia-exposed lungs from DBA and T2KO mice with less lung matrix remodeling localize near the root point of the pseudotime trajectory. (j) Bar graphs of matrix pseudotime for hyperoxia-exposed and normoxic control lung tissues of B6 (WT), DBA and T2KO mice. The difference in pseudotime normalized to normoxic control lungs is shown. Data are mean ± SEM (n = 3 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. n.s.: not significant. Each dot represents one mouse (b, d, e, f). See also .

Article Snippet: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: RNA Sequencing Assay, Expressing, Comparison, Western Blot, Staining, Control

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) BMDMs were obtained from B6 (WT), DBA and T2KO mice and exposed to hyperoxia (95%) for 24 h. (b) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed BMDMs obtained from B6 (WT), DBA and T2KO mice and the normoxic controls. Uncropped images of the blots are shown in . (c) RNA-seq of hyperoxia-exposed BMDMs from B6 (WT), DBA and T2KO mice and the normoxic controls. (d) Venn diagram showing the overlap between hyperoxia-induced genes in B6 (WT) BMDMs (n = 173) and hyperoxia-suppressed genes in T2KO (n = 570) and DBA (n = 905) BMDMs. Gene Ontology analysis of 34 genes induced by hyperoxia in B6 (WT) BMDMs and suppressed in T2KO and DBA BMDMs. (e) Bar plots for expression of representative genes belonging to the p53 signaling pathway. Data are mean ± SEM. DSeq2 was used for comparisons. ** p -adj < 0.01 and *** p -adj < 0.001. (f) Caspase 3 activity measured in cytosolic fractions from BMDMs from B6 (WT), DBA and T2KO mice. Data are mean ± SEM. (n = 4 for each group). ANOVA was performed followed by Tukey’s post hoc comparison. * p < 0.05. Each dot represents one mouse (e, f). See also .

Article Snippet: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: Western Blot, RNA Sequencing Assay, Expressing, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Genetic variation in the activity of a TREM2-p53 signaling axis determines oxygen-induced lung injury

doi: 10.1101/2024.09.13.612775

Figure Lengend Snippet: (a) Immunoblots of TREM2 protein in whole cell lysates of hyperoxia-exposed (1, 2, 4 or 24 h) BMDMs obtained from B6 (WT) mice and normoxic controls (0 h). Uncropped images of the blots are shown in .

Article Snippet: 96-well plate was coated overnight at 4 °C with TREM2 coating antibody (MAB17291-100, R&D Systems) in ELISA Coating Buffer (421701, Biolegend).

Techniques: Western Blot

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Clone Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Clone Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: A 647-conjugated anti-TREM2 antibody (R&D, FAB17291A) or an isotype control antibody (R&D, IC103A) was used to stain surface TREM2 by incubation for 30 min on ice.

Techniques: Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A-B. Proteomic hiSPECS analysis comparing BV2 iRhom2 -/- (A) or ADAM17 -/- (B) microglia to BV2 NTC microglia. Volcano plots depict the log2 abundance change in the secretome and the negative log10 p-value for each protein (two-sample t-test, N = 12). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. For ease of visualization, the secretome data of the two iRhom2 (iR2 -/- : KO #1 and #2) and ADAM17 (A17 -/- : KO #1 and #2) clones were pooled and compared to the pooled secretome of the two NTC clones (NTC #1 and NTC #2) non-targeting control clones. C. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using the web tool Quantitative Analysis of Regulated Intramembrane Proteolysis (QARIP) ( Ivankov et al , 2013 ). D. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in BV2 cells. The supernatant prepared for the hiSPECS analysis in (A and B) was subjected to ELISA. sTREM2 levels were quantified in conditioned media of the indicated BV2 KO cell lines (N ≥5). NTC (N = 10) contains the combined data of NTC #1 (N = 5) and NTC #2 (N = 5) clones. NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. E. Immunoblot analysis of TREM2 in BV2 KO cell lines. Highly glycosylated, mature species of TREM2 were enriched in the ADAM17 and iRhom2 KO lines. Calnexin served as loading control. F. Surface abundance of TREM2 in BV2 KO cells. Cells were labeled with an antibody against TREM2 or isotype control to assess the geometric mean intensity of indicated BV2 KO cell lines using flow cytometry (N ≥ 3). NTC (N = 6) contains the combined data of NTC #1 (N = 3) and NTC #2 (N = 3) clones. BV2 NTC was used as baseline, and its average normalized to 100. One-way ANOVA with Tukey’s correction for multiple comparisons. Data information: Data (D, F) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

Techniques: Clone Assay, Control, Enzyme-linked Immunosorbent Assay, Western Blot, Labeling, Flow Cytometry

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Proteomic hiSPECS analysis comparing the secretome of primary microglia isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) pups. The volcano plot displays protein abundance changes between WT and iR2 KO supernatants. The log2 fold change is plotted against the p-value (-log10). The permutation-based false discovery rate estimation is represented by the black hyperbolic dashed line (FDR, p = 0.05, s0 = 0.1). Proteins with a p-value below 0.05 are highlighted with red (FDR significant) or black circles. Vertical dotted lines indicate changes of larger or smaller than two-fold in the log scale. B. The peptide distribution according to the specific protein domains of the indicated iRhom2/ADAM17 substrates was visualized using QARIP (see ). C. Validation of iRhom2/ADAM17-mediated TREM2 ectodomain shedding in primary iRhom2 -/- microglia. The supernatant prepared for the hiSPECS analysis in (A) was subjected to ELISA. sTREM2 levels were quantified in supernatants of primary microglia (N ≥ 7). Two-sided independent Student’s t-test was performed. D. Immunoblot analysis of TREM2 levels in primary microglia lysates derived from wild-type or iRhom2 -/- pups. Three biological replicates of either genotype are shown. Highly glycosylated, mature species of TREM2 were enriched in the lysates of iRhom2-deficient microglia. Actin served as loading control. Data information: Data (C) are represented as means ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

Techniques: Isolation, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Control

Journal: bioRxiv

Article Title: The late onset Alzheimer’s disease risk factor iRhom2/RHBDF2 is a modifier of microglial TREM2 proteolysis

doi: 10.1101/2024.09.13.612888

Figure Lengend Snippet: A. Immunoblot analysis of TREM2 signaling in cell lysates isolated from wild-type (WT) or iRhom2 -/- (iR2 KO) BMDMs. Three biological replicates of either genotype are shown. The membranes were probed with antibodies against iRhom2, ADAM17, TREM2, or antibodies against SYK or phospho-SYK-Y519/520. Calnexin and Actin served as loading control. B. Quantification of phospho-SYK-Y519/520 levels in immunoblots as shown in (A). iRhom2 -/- BMDMs showed stronger SYK phosphorylation than wild-type BMDMs (N = 6). The phospho-SYK/total-SYK ratio was normalized to wild-type. Two-sided independent Student’s t-test was performed. C. iRhom2/ADAM17-mediated TREM2 ectodomain shedding in iRhom2 -/- BMDMs. The sTREM2 levels were quantified in supernatants from cultures in (A) by ELISA (N = 6). Two-sided independent Student’s t-test. Data information: Data (B, C) are represented as means ± SD from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Plates were washed and incubated with rat anti-TREM2 detection antibody (Sigma Aldrich, clone 5F4, stock: 1 mg/ml) at 1:1000 dilution for 1 hour.

Techniques: Western Blot, Isolation, Control, Enzyme-linked Immunosorbent Assay