Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 are transiently induced in kidney proximal tubular cells in sepsis-associated acute kidney injury models. BUMPT and HK-2 cells were treated with 100 μg/mL lipopolysaccharide (LPS) for 3, 6, and 12 hours. Male C57BL/6J mice were subjected to an i.p. injection of 10 mg/kg LPS or cecal ligation and puncture (CLP) surgery, and kidney samples were collected at 3, 6, and 9 hours after injury. A—D: Quantitative RT-PCR analysis of Gstm2-ps1 or GSTM3P1 expression. A: Gstm2-ps1 in BUMPT. B: GSTM3P1 in HK2. C: Gstm2-ps1 in LPS-treated mouse kidney. D: Gstm2-ps1 in mouse kidney with CLP. A—D: The statistical difference was determined using one-way analysis of variance with Tukey multiple comparison. E and F: Representative images of Gstm2-ps1 in kidney tissue assessed by in situ hybridization and costained with fluorescein isothiocyanate—labeled Lotus tetragonolobus lectin (LTL). E: Mouse kidneys with or without LPS treatment for 3 hours. F: Mouse kidneys with or without CLP injury for 3 hours. n 5 ( A—D ); n 6 ( E and F ). ** P < 0.01, *** P < 0.001. Scale bar 100 μm ( E and F ). NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Injection, Ligation, Quantitative RT-PCR, Expressing, Comparison, In Situ Hybridization, Labeling

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 exacerbate lipopolysaccharide (LPS)—induced apoptosis in cultured proximal tubular cells. RPTC cells with or without GSTM3P1 stable overexpression, BUMPT cells with or without Gstm2-ps1 stable overexpression, and wild-type (WT) or Gstm2-ps1 knockout (KO) primary proximal tubular cells were treated with 100 μg/mL LPS for 19 hours. A—C: Quantitative RT-PCR analysis confirming the expression of GSTM3P1 or Gstm2-ps1. A: RPTC. B: BUMPT. C: Primary proximal tubular cells. D—F: Cell viability assessed by MTT assay following 19 hours of LPS treatment. D: RPTC. E: BUMPT. F: Primary proximal tubular cells. A—F: The statistical difference was determined by unpaired t -test. G—I: Representative immunoblots of full-length caspase 3 and cleaved caspase 3 (C-caspase-3) with β-actin as the loading control (Ctrl; top panels ), and the densitometry analysis of C-caspase-3 normalized to β-actin ( bottom panels ). G: RPTC. H: BUMPT. I: Primary proximal tubular cells. G—I: The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. n =3 ( A , G , and H ); n = 4 ( B , C , and I ); n = 6 ( D—F ). * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Cell Culture, Over Expression, Knock-Out, Quantitative RT-PCR, Expressing, MTT Assay, Western Blot, Control, Comparison

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 overexpression induces oxidative stress in cultured proximal tubular cells. RPTC cells with or without GSTM3P1 stable overexpression and BUMPT cells with or without Gstm2-ps1 stable overexpression were treated with 100 μg/mL lipopolysaccharide (LPS) for 19 hours. A and B: Oxidative stress levels were assessed by measuring the reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio. A: RPTC. B: BUMPT. The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. C: Representative images of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)—stained RPTC and phase-contrast images. n = 6 ( A—C ). * P < 0.05, ** P < 0.01, and *** P < 0.001. Scale bars = 200 μm ( C ).

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Over Expression, Cell Culture, Comparison, Staining

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: Proximal tubular—specific Gstm2-ps1 knockout protects mice from lipopolysaccharide (LPS)—induced sepsis acute kidney injury. Proximal tubular—specific Gstm2-ps1 knockout (KO) mice and their wild-type (WT) littermates were treated with one i.p. injection of 10 mg/kg LPS and sacrificed after 19 hours. The control (Ctrl) mice were given an equal volume of phosphate-buffered saline. A: Blood urea nitrogen (BUN) levels. B: Serum creatinine levels. C: Quantification of apoptotic cells in kidneys by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. A—C: The statistical difference was determined by two-way analysis of variance with Tukey multiple comparison. D: Representative images of TUNEL staining. E: Representative images of neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical staining. F: Representative images of reactive oxygen species (ROS) levels detected by 2′,7′-dichlorodihydrofluorescein diacetate staining. G: Representative immunoblots of kidney injury molecule-1 (KIM-1) and the densitometry analysis of KIM-1 normalized to the internal loading control glyceraldehyde-3-phosphate dehydrogenase. Data are presented as means ± SD ( A—C and G ). n = 6 per group ( A , B , E , and F ); n = 5 per group ( C ); n = 4 per group ( G ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Scale bars: 50 μm ( D and E ); 100 μm ( F ).

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Knock-Out, Injection, Control, Saline, End Labeling, TUNEL Assay, Staining, Comparison, Immunohistochemical staining, Western Blot

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 down-regulate the expression of their parent gene GSTM3/GSTM2. A—F: RPTC cells ( A and D ) or BUMPT cells ( B and E ) were overexpressed with or without GSTM3P1 or Gstm2-ps1, and C57BL/6J mouse kidneys ( C and F ) were treated with 10 mg/kg lipopolysaccharide (LPS) for 3, 6, and 9 hours. A—C: Representative immunoblots of GSTM3 and GSTM2, with β-actin or cyclophilin B as the internal loading marker. D—F: The densitometric analysis of GSTM3 and GSTM2 protein normalized to the internal loading control. The statistical difference was determined by unpaired t -test ( D and E ) or one-way analysis of variance with Tukey multiple comparison ( F ). G: Representative immunofluorescence images of GSTM2 with fluorescein isothiocyanate— Lotus tetragonolobus lectin (LTL) costaining. Gstm2-ps1 knockout (KO) and wild-type (WT) mice were treated with/without 10 mg/kg LPS for 3 hours. n = 6 for cell samples ( D—F ); n = 4 for kidney samples ( D—F ); n = 6 ( G ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Scale bar = 100 μm ( G ). NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Expressing, Western Blot, Marker, Control, Comparison, Immunofluorescence, Knock-Out

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM2/GSTM3 protect cultured renal proximal tubular cell from lipopolysaccharide (LPS)—induced apoptosis. RPTC cells with or without GSTM3 overexpression, and BUMPT cells with or without GSTM2 overexpression, were treated with 100 μg/mL LPS. A and B: Immunoblot analysis confirming the overexpression efficiency of GSTM3 and GSTM2. C and D: Cell viability assessed using the MTT assay following 17 hours of LPS treatment. A—D: The statistical difference was determined by unpaired t -test. E and F: Representative immunoblots ( top panels ) and densitometric analysis ( bottom panels ) of cleaved caspase-3 (C-caspase-3) protein levels. E: RPTC. F: BUMPT. E and F: The statistical difference was determined by one-way analysis of variance with Tukey multiple comparison. n = 3 ( A and B ); n = 6 ( C and D ); n = 4 ( E and F ). ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Ctrl, control.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Cell Culture, Over Expression, Western Blot, MTT Assay, Comparison, Control

Journal: The American journal of pathology

Article Title: Pseudogene-Derived Long Noncoding RNAs GSTM3P1/Gstm2-ps1 Exacerbate Sepsis-Associated Acute Kidney Injury by Suppressing Their Parent Gene Translation

doi: 10.1016/j.ajpath.2026.01.004

Figure Lengend Snippet: GSTM3P1/Gstm2-ps1 may down-regulate GSTM3/GSTM2 protein level through RNA translation inhibition. A: Quantitative RT-PCR (RT-qPCR) of GSTM3 in RPTC cells with or without GSTM3P1 overexpression. The unpaired t -test was used. B: RT-qPCR of GSTM2 in BUMPT cells with or without Gstm2-ps1 overexpression. The unpaired t -test was used. C: RT-qPCR of GSTM2 in mouse kidneys with or without 10 mg/kg lipopolysaccharide (LPS) treatment . The one-way analysis of variance (ANOVA) with Tukey multiple comparison was used. D and E: BUMPT cell lysates with Gstm2-ps1 overexpression or empty vector transfection (pcDNA) were subjected to RNA pull-down assay with Gstm2-ps1 probes. The input lysates (Input) and pull-down products (pull-down) were examined by immunoblotting of human antigen R (HuR), with cyclophilin B as the internal loading control of input lysates. D: Representative immunoblots of HuR and cyclophilin B. E: The densitometry analysis of HuR immunoblotting in pull-down products normalized by its level in the input. The unpaired t -test was used. F—H: BUMPT cells were overexpressed with HuR or Gstm2-ps1. F: Representative immunoblots to confirm the overexpression of HuR with cyclophilin B as the internal loading control. The upper band is the overexpressed HuR. The lower band is the endogenous HuR. G: Representative immunoblots and densitometry analysis of GSTM2 in cells with or without Gstm2-ps1 and HuR overexpression. β-Actin was used as the internal loading control. The one-way ANOVA with Tukey multiple comparison was used. H: The cell viability assessed by MTT assay. The one-way ANOVA with Tukey multiple comparison was used. n = 5 ( A and B ); n = 4 ( C and E—G ); n = 6 ( H ). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. NS, nonsignificant.

Article Snippet: Human GSTM3 or murine Gstm2 open reading frames were subcloned from commercially available plasmids (RC201013 or MR202469; Origene, Rockville, MD), respectively.

Techniques: Inhibition, Quantitative RT-PCR, Over Expression, Comparison, Plasmid Preparation, Transfection, Pull Down Assay, Western Blot, Control, MTT Assay

Journal: Genes & Nutrition

Article Title: African-enriched SLC39A10 (ZIP10) missense variants differentially affect cellular zinc homeostasis and associate with health-related traits

doi: 10.1186/s12263-026-00801-3

Figure Lengend Snippet: African-enriched missense variants in SLC39A10 (ZIP10). (A) Population allele frequencies of African-enriched SLC39A10 variants based on the 1000 Genomes Project. Pie size reflects allele frequency (blue scale = 1.0; green scale = 0.1). (B) Multiple sequence alignment of ZIP10 orthologs via Clustal Omega. Asterisk (*) denotes full conservation, colon (:) indicates strong similarity, and period (.) indicates weak similarity. (C) Predicted membrane topology model of human ZIP10 generated using MEMSAT-SVM, with the positions of the T87S, H194Q, and H609Y substitutions indicated. The H194Q and H609Y variants are located within histidine-enriched regions of the protein sequence

Article Snippet: The human SLC39A10 open reading frame ( NM_001127257 ), which is C-terminally tagged with Myc-DDK, was obtained from OriGene (catalog #RC226199).

Techniques: Sequencing, Membrane, Generated

Journal: Genes & Nutrition

Article Title: African-enriched SLC39A10 (ZIP10) missense variants differentially affect cellular zinc homeostasis and associate with health-related traits

doi: 10.1186/s12263-026-00801-3

Figure Lengend Snippet: Phenotypic associations of African-enriched functional SLC39A10 variants in an African ancestry cohort. (A) Manhattan plot showing phenome-wide association results for rs114992984 (ZIP10 H194Q ) and rs76182486 (ZIP10 H609Y ) using the African ancestry subset of the VA Million Veteran Program. The dashed horizontal line indicates the nominal significance threshold ( P < 0.05). (B , C) Selected traits exhibiting nominal associations for rs114992984 (B) and rs76182486 (C) , grouped by disease category: hematologic (red), metabolic (green), renal (blue), and dermatologic (purple)

Article Snippet: The human SLC39A10 open reading frame ( NM_001127257 ), which is C-terminally tagged with Myc-DDK, was obtained from OriGene (catalog #RC226199).

Techniques: Functional Assay

Journal: Cancers

Article Title: An Inducible BRCA1 Expression System with In Vivo Applicability Uncovers Activity of the Combination of ATR and PARP Inhibitors to Overcome Therapy Resistance

doi: 10.3390/cancers18020309

Figure Lengend Snippet: ( A ) ( Left panel ): dose–response curves of olaparib in colony formation assays in tetracycline-repressor (TR) expressing MDAMB436 cells with or without RAD51B complementation (MDAMB436-B-TR and MDAMB436-TR, respectively) and with (+BRCA1) or without (+LacZ) BRCA1 complementation. ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. ( B ) ( Left panel ) : dose–response curves of olaparib in colony formation assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ), or different BRCA1 hypomorphs (+RING-less, +∆exon11, +BRCT-less). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. ( C ) ( Left panel ): dose–response curves of olaparib in survival assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ), or different BRCA1 hypomorphs (+C64R, +L1407P, +R1699Q). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib for each cell line. Data for BRCA1 hypomorphs with deletions in the RING (RING-less, M48 START) or BRCT (BRCT-less) domains are included for comparative purposes. ( D ) ( Left panel ): dose–response curves of carboplatin in survival assays in MDAMB436-B-TR cells expressing full-length BRCA1 (+FL), no BRCA1 (+LacZ) or different BRCA1 hypomorphs (+RING-less, +∆exon11, +BRCT-less, +CC-mutant). ( Right panel ): Logarithmic half-maximal inhibitory concentration (LogIC50) of carboplatin for each cell line. ( E ) Western blot of immunoprecipitation experiments in MDAMB436-B-TR cells expressing full-length (+FL) or ∆exon11 BRCA1 and exposed to different doxycycline doses. GAPDH was used as a loading control. I = input; F = FLAG immunoprecipitation. ( F ) Logarithmic half-maximal inhibitory concentration (LogIC50) of olaparib at the different doses of doxycycline used. All data are from at least 3 biological replicates. Statistical analysis performed using One-Way ANOVA with Holm–Sidak multiple comparisons, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. CFU = colony-forming units. Dotted lines in the olaparib IC50 graphs represent the minimal free concentration of olaparib (approximately 300 nM) in the plasma of patients on the established monotherapy dose of 300 mg twice daily. The original western blot figures can be found in .

Article Snippet: Plasmids containing the human BRCA1 (NM_007294) and RAD51B (NM_133509) open reading frames (ORFs) were obtained from Genscript (Ohu18572D) and OriGene (RC206457L3), respectively.

Techniques: Expressing, Concentration Assay, Mutagenesis, Western Blot, Immunoprecipitation, Control, Clinical Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: Tumorous CA9 may be the source of circulating CA9 and confers resistance to the combination immunotherapy of anti-PD-L1 and anti-VEGF antibodies in HCC. ( A–C ) Single-cell RNA sequencing data from the HCCDB V.2.0 (Lifeome) portal showing ( A ) UMAP of major cell types and feature plots of ( B ) CA9 and ( C ) AFP expression. ( D ) Western blot analysis of Car9 and Actb in Hep55.1c cells with NC or Car9 OE. ( E ) mRNA levels of Car9 in syngeneic subcutaneous tumors derived from Hep55.1c cells with either negative control or Car9 overexpression (NC: n=28; OE: n=25). ( F ) Growth curves of syngeneic subcutaneous tumors derived from Hep55.1c cells with either negative control (left) or Car9 overexpression (right) (n=8 per group). ( G ) Tumor volume 14 days after inoculation in NC and OE tumors (NC: n=28; OE: n=25). ( H ) Growth curves of syngeneic subcutaneous tumors derived from NC or Car9-overexpression Hep55.1c cells treated with vehicle or anti-PD-L1/VEGF antibodies (treatment) (n=8 per group). ( I ) Relative treatment response calculated for each mouse as the tumor volume in the anti-PD-L1/VEGF-treated group divided by the mean tumor volume of the corresponding vehicle-treated group within the same genotype (NC or Car9 OE) at day 14. Each dot represents an individual tumor, allowing comparison of relative treatment effects between NC-derived and Car9-OE-derived tumors. Data are shown as mean±SEM. Group comparisons were performed using two-tailed unpaired Student’s t-tests or two-way repeated-measures ANOVA, as appropriate; p<0.05 was considered statistically significant. AFP, alpha-fetoprotein; ANOVA, analysis of variance; CA9, carbonic anhydrase 9; HCC, hepatocellular carcinoma; mRNA, messenger RNA; NC, negative control; NK, natural killer; OE, overexpression; PD-L1, programmed death-ligand 1; UMAP, Uniform Manifold Approximation and Projection; VEGF, vascular endothelial growth factor.

Article Snippet: Stable Car9-overexpressing and vector control lines were generated using lentiviral particles encoding the murine Car9 open reading frame (ORIGENE, Rockville, Maryland, USA).

Techniques: Single Cell, RNA Sequencing, Expressing, Western Blot, Derivative Assay, Negative Control, Over Expression, Comparison, Two Tailed Test

Journal: Journal for Immunotherapy of Cancer

Article Title: Carbonic anhydrase 9 as a circulating biomarker and therapeutic target in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab

doi: 10.1136/jitc-2025-013384

Figure Lengend Snippet: Tumorous CA9 flourishes the immunosuppressive and angiogenic tumor microenvironment in HCC. ( A–C ) Relative mRNA expression of the indicated genes in syngeneic subcutaneous tumors derived from Hep55.1c cells with NC or Car9 OE, measured by quantitative RT-PCR (NC: n=28; OE: n=25). Each dot represents one tumor; bars indicate mean±SEM. ( D ) UMAP of single-cell RNA sequencing data from NC and CA9 OE tumors, colored by annotated cell type (eg, malignant cells, T cells, TAMs, endothelial cells). ( E ) Frequency of CTLs per tumor sample, calculated as CTLs divided by total cells profiled by scRNA-seq (NC: n=3; CA9 OE: n=3). ( F, G ) Differential gene expression and representative cytotoxicity-related genes in CD8 + T cells from CA9 OE versus NC tumors. ( H, I ) Differential gene expression and representative TAM-related genes in TAMs from CA9 OE versus NC tumors. ( J–L ) Gene Set Enrichment Analysis (Gene Ontology) showed the top 20 pathways in TAMs ( J ), dendritic cells ( K ), and endothelial cells ( L ). ( M ) Tumor growth curves of syngeneic subcutaneous tumors derived from Car9-overexpressing Hep55.1c cells treated with vehicle, CA9 inhibitor alone, anti-PD-L1 plus anti-VEGF antibodies (Combi), or Combi plus CA9 inhibitor (Combi+CA9 inhibitor) (n=8–12 per group). Data are shown as mean±SEM. Group comparisons were performed using two-tailed unpaired Student’s t-tests or Wilcoxon rank-sum tests, as appropriate; p<0.05 was considered statistically significant. CAF, cancer-associated fibroblast; CA9, carbonic anhydrase 9; CTLs, cytotoxic T lymphocytes; HCC, hepatocellular carcinoma; mRNA, messenger RNA; NC, negative control; NK, natural killer; OE, overexpression; PD-L1, programmed death-ligand 1; RT-PCR, reverse transcription PCR; scRNA-seq, single-cell RNA sequencing; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophils; TPM, transcripts per million; UMAP, Uniform Manifold Approximation and Projection; VEGF, vascular endothelial growth factor; WT, wild type.

Article Snippet: Stable Car9-overexpressing and vector control lines were generated using lentiviral particles encoding the murine Car9 open reading frame (ORIGENE, Rockville, Maryland, USA).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Single Cell, RNA Sequencing, Gene Expression, Two Tailed Test, Negative Control, Over Expression, Reverse Transcription Polymerase Chain Reaction, Reverse Transcription

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: scRNA-seq reveals irradiation-induced Trem2 expression in skin macrophages. (A) tSNE plots showing cell type annotation based on CellMarker 2.0, and distribution of cell clusters in control and irradiated groups. (B) Proportion of each cell population comparing control and irradiation groups. (C) Annotation of macrophage-related clusters using marker genes. (D) Quantification of macrophage cluster ratios between control and irradiated groups. (E) Dotplot showing the expression of representative genes in macrophage subclusters, highlighting Trem2 -specific enrichment. (F) irGSEA of macrophage clusters, including a gene expression heatmap and GSEA plot. (G) AUCell analysis indicating high activity of the PI3K-Akt pathway in MAC2 macrophage subclusters. (H) Separation of macrophage clusters based on Trem2 expression. (I) Comparison of the ratio of Trem2 -positive and Trem2 -negative clusters between control and irradiated samples. (J) Pseudotime trajectory analysis illustrating the developmental progression of Trem2 -positive and Trem2-negative macrophages.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Expressing, Control, Marker, Gene Expression, Activity Assay, Comparison

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation-induced ROS–Nrf2–ADAM17 axis promotes Trem2 shedding. (A) Quantification of sTREM2 in culture supernatants of RAW264.7 cells under control and irradiated conditions ( n = 3). (B) ADAM10 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (C) ADAM17 transcript levels from RNA-seq and relative mRNA expression measured by qPCR ( n = 3). (D and E) Western blotting and analysis of ADAM10 and ADAM17 ( n = 3). (F) Pearson correlation analysis between Trem2 , ADAM10 , ADAM17 , and Nrf2 based on RNA-seq data. (G) GSEA showing enrichment of the “Positive Regulation of Reactive Oxygen Species Metabolic Process” pathway after irradiation. (H) (a) ROS levels detected by fluorescence staining at 2 and 6 h post-irradiation (green), shown with brightfield (BF) (scale bar, 200 μm); (b) quantification of integrated fluorescence intensity (IntDen) ( n = 3). (I) ROS quantification based on fluorescence intensity at excitation/emission wavelengths of 488/525 nm ( n = 3). (J) (a and b) IF detection and quantification of NRF2 (green) with DAPI staining ( n = 3; scale bar, 200 μm); (c and d) Western blotting and quantification of NRF2 ( n = 3). (K and L) Western blotting and quantification of TREM2, BAX, and BCL2 protein levels in control, DMSO, and GW280264X cells with or without irradiation ( n = 3). (M) ELISA detection of sTREM2 in various treatment groups ( n = 3). (N) Cell viability assay using CCK-8 in control (Ctr), DMSO, and GW280264X (ADAM17/ADAM10 inhibitor) groups with or without irradiation ( n = 3). (O) ELISA detection of proinflammatory cytokines IL-1β, TNF-α, and IL-6 in various treatment groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Control, Irradiation, RNA Sequencing, Expressing, Western Blot, Fluorescence, Staining, Enzyme-linked Immunosorbent Assay, Viability Assay, CCK-8 Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 overexpression improves radiation resistance of RAW264.7 cells by reducing apoptosis. (A and B) Live/dead staining using Calcein-AM (live, green) and EthD-1 (dead, red); quantification of dead cells based on integrated fluorescence intensity ( n = 3; scale bar, 200 μm). (C) Cell viability assessed by CCK-8 assay in Blank, Vector, OE-Trem2, siNC, and si-Trem2 groups with or without irradiation ( n = 3). (D and E) Scratch assay and distance closure rate quantified at 6, 12, and 24 h relative to 0 h ( n = 3; scale bar, 200 μm). (F) Apoptosis rates determined by flow cytometry across different treatment groups ( n = 3). (G to J) Western blotting and analysis of CD206, CD86, CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (K) ELISA analysis of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and the anti-inflammatory cytokine IL-10 in different groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; only statistically significant comparisons are shown in panel.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Over Expression, Staining, Fluorescence, CCK-8 Assay, Plasmid Preparation, Irradiation, Wound Healing Assay, Flow Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency exacerbates radiation-induced apoptosis by suppressing ERK phosphorylation. (A and B) Western blotting and quantification of TREM2 expression in LysM Cr e Trem2 flox/flox (Trem2-Cko) and LysM − Trem2 flox/flox (Trem2-Ctr) BMDM, with or without irradiation ( n = 3). (C) Flow cytometry analysis of macrophage polarization based on M1 markers (CD86 + CD206 − ) and M2 markers (CD86 − CD206 + ) ( n = 3). (D) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis based on RNA-seq data. (E) Apoptosis rate assessed by Annexin V/PI flow cytometry in Trem2-Ctr and Trem2-Cko macrophages after irradiation ( n = 3). (F and G) Western blotting and analysis of phosphorylated ERK1/2 (p-ERK), total ERK1/2 (t-ERK), CASPASE-9 (CAS9), total and cleaved CASPASE-3 (pro-CAS3, cleaved CAS3), BCL2, BAX, and cleaved CASPASE-8 (cleaved CAS8) ( n = 3). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6) and anti-inflammatory cytokine IL-10 in Trem2-Ctr and Trem2-Cko groups ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Phospho-proteomics, Western Blot, Expressing, Irradiation, Flow Cytometry, RNA Sequencing, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation delayed wound healing in radiation-induced skin injury (RISI) and induced TREM2 expression. (A) The RISI mouse model: WT mice received 5-Gy total body x-ray irradiation followed by full-thickness excisional skin wounding (black circle, 12-mm inner diameter). (B) Representative wound area measurements ( n = 6). (C) Quantification of wound closure rates at various time points relative to post-injury day 0 (PID0) ( n = 6). (D and E) H&E and Masson staining of wound tissue sections at PID0, 3, 7, and 14, showing regions close to the wound margin; IF costaining of TREM2 (gold), F4/80 (red), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) to identify TREM2 + macrophages in control and irradiated groups (scale bar was shown on panel). (F) Quantification of collagen deposition based on Masson staining ( n = 6). (G) Total macrophage ratio calculated as the proportion of F4/80 + DAPI + cells among total DAPI + cells by immunohistochemistry ( n = 6). (H) TREM2 + macrophage ratio calculated as the proportion of TREM2 + F4/80 + DAPI + cells among total F4/80 + DAPI + macrophages ( n = 6). (I) qPCR analysis of Trem2 mRNA expression at different time points ( n = 6). For within-group comparisons, expression was normalized to day 0 of each group. For between-group comparisons, expression levels in the irradiation group were normalized to the corresponding control group at each time point ( n = 6). (J and K) Western blotting and analysis of TREM2 protein expression across time points in control and irradiated groups ( n = 6). (L) ELISA measurement of soluble TREM2 (sTREM2) levels in skin tissue at different time points ( n = 6). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Staining, Control, Immunohistochemistry, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Radiation reduced RAW264.7 cell viability and induced Trem2 expression. (A) Volcano plot showing DEGs between irradiation and control RAW264.7 cells based on RNA-seq ( n = 3). (B) GSEA identifying Trem2 -associated pathways enriched or suppressed following irradiation. (C) Heatmap of representative genes involved in macrophage differentiation, proliferation, migration, and apoptosis from GSEA-identified pathways. Significantly up-regulated and down-regulated genes are marked with upward (↑) and downward (↓) arrows, respectively. (D and E) Flow cytometry analysis of apoptosis using Annexin V and propidium iodide (PI) staining. Early and late apoptotic cells were quantified from Q2 and Q3 quadrants ( n = 3). (F and G) Live (green)/dead (red) staining; dead cell proportion calculated based on integrated fluorescence density ( n = 3; scale bar, 200 μm). (H) CCK-8 assay shown relative cell viability at 24 h post-irradiation compared to 0 h ( n = 3 biological replicates with 3 technical repeats each). (I) Trem2 transcript level from RNA-seq data. (J) qPCR analysis of Trem2 mRNA expression across different treatment groups ( n = 3). (K and L) Western blot and analysis of TREM2, BAX, and BCL2 protein expression ( n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.)

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Expressing, Irradiation, Control, RNA Sequencing, Migration, Flow Cytometry, Staining, Fluorescence, CCK-8 Assay, Western Blot

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Trem2 deficiency impairs wound healing in RISI. (A) RISI mouse model: Trem2-Ctr and Trem2-Cko mice were subjected to 5-Gy total body x-ray irradiation followed by full-thickness excisional wounding (black circle, 12-mm inner diameter). (B) Representative images of wound areas in each group ( n = 6). (C) Quantification of wound closure rates at indicated time points relative to PID0 ( n = 6). (D) H&E and Masson staining of wound tissues at PID0, 3, 7, and 14 ( n = 6). (E) IF costaining of iNOS (red), CD206 (green), and DAPI (blue) to identify M1- or M2-type macrophages in Trem2-Ctr and Trem2-Cko groups (scale bars shown in panels). (F) Collagen deposition quantified from Masson staining ( n = 6). (G) Quantification of macrophage subsets, calculated as the proportion of iNOS + CD206 − DAPI + cells (M1) or iNOS − CD206 + DAPI + cells (M2) among total DAPI + cells ( n = 6). (H) ELISA quantification of proinflammatory cytokines (IL-1β, TNF-α, and IL-6), anti-inflammatory cytokine IL-10, and total CASPASE3 in Trem2-Ctr and Trem2-Cko groups ( n = 6).

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: Irradiation, Staining, Enzyme-linked Immunosorbent Assay

Journal: Research

Article Title: TREM2 Deficiency Regulates Macrophage Apoptosis and Repair in Radiation-Induced Skin Injury

doi: 10.34133/research.1018

Figure Lengend Snippet: Schematic of TREM2 regulates macrophage apoptosis and impairs wound healing following RISI.

Article Snippet: Briefly, the mouse Trem2 open reading frame (ORF) cDNA clone, containing a C-GFPSpark tag (MG50149-ACG, Sino Biological, Beijing, China), was transfected into RAW264.7 cells to overexpress TREM2 (OE-Trem2).

Techniques: