Journal: Translational Oncology

Article Title: A machine learning-based prognostic signature utilizing MSC proteomics for predicting bladder cancer prognosis and treatment response

doi: 10.1016/j.tranon.2025.102349

Figure Lengend Snippet: Clinical information of bladder cancer patients.

Article Snippet: After blocking, added the prepared TIMP2 (MCE, HY-P80356, 1:200) primary antibody to each slice and incubate overnight.

Techniques:

Journal: Translational Oncology

Article Title: A machine learning-based prognostic signature utilizing MSC proteomics for predicting bladder cancer prognosis and treatment response

doi: 10.1016/j.tranon.2025.102349

Figure Lengend Snippet: Correlation analysis between TIMP2 and MSC. (A) Thumbnail of TMA for TIMP2 immunohistochemistry. (B) IHC of tissues with high TIMP2 expression and corresponding regions stained with mFIHC (where the gold-colored cells indicated by arrows represent MSCs). (C) IHC of tissues with lowTIMP2 expression and corresponding regions stained with mFIHC. (D) Correlation analysis between TIMP2 expression and MSC proportion in 23 cases of BLCA tissues.

Article Snippet: After blocking, added the prepared TIMP2 (MCE, HY-P80356, 1:200) primary antibody to each slice and incubate overnight.

Techniques: Immunohistochemistry, Expressing, Staining

Journal: bioRxiv

Article Title: Youth-associated protein TIMP2 alters microglial state and function in the context of aging

doi: 10.1101/2025.05.20.655226

Figure Lengend Snippet: (a) Schematic diagram of isolation, culturing, and assays performed on TIMP2 KO and WT primary microglia. (b) TIMP2 immunoblotting from primary microglia lysates isolated from TIMP2 WT and KO littermates (N = 4 samples per genotype, comprised of 5-6 pups (P5-P8) pooled per sample). (c) Representative images from cultures of TIMP2 KO and WT primary microglia (N = 3 pups (P6-P7) pooled per genotype; repeated in 2 additional experiments) stained with anti-TIMP2 and anti-IBA1 antibodies by ICC (scale bar = 100 μm). (d) Gene expression (transcript per million) values for Timp2 from bulk RNAseq experiments from TIMP2 KO and WT primary microglia (N = 7 samples per genotype, with each sample representing a pool of 5 pups (P6-P7); sex-matched) with subsequent (e) over-representation analysis of GO terms ( P adj < 0.05) using upregulated and downregulated DEGs in TIMP2 KO vs. WT primary microglia. (f) Representative images from WT primary microglia cultures stained by ICC with DAPI, as well as anti-TIMP2 and anti-CD68 antibodies for co-localization analyses (N = 8 mice, 5-6-month-old mice; sex-matched; 2 independent experiments combined; scale bar = 100 μm), with (g) quantification of the fraction of TIMP2 + cells that are TIMP2 + CD68 + .

Article Snippet: Gels were transferred, the blots probed with anti-TIMP2 (1:5000; D18B7, Cell Signaling) or anti-actin (1:10000; A5060; Sigma), and then developed as previously described .

Techniques: Isolation, Western Blot, Staining, Gene Expression

Journal: bioRxiv

Article Title: Youth-associated protein TIMP2 alters microglial state and function in the context of aging

doi: 10.1101/2025.05.20.655226

Figure Lengend Snippet: (a) Representative confocal images of dentate gyrus (DG) of 3-4-month-old TIMP2 KO and WT mice stained with anti-IBA1 and anti-CD68 antibodies (left; scale bar = 100 μm) and magnified inset (right; scale bar = 50 μm) (N = 9-13 mice per group; sex-matched; arrowheads indicate IBA1 + CD68 + cells) with corresponding (b) quantification of thresholded DG area covered by IBA1 + staining and (c) the number of IBA1 + CD68 + cells normalized to DG area. (d) Representative confocal images of dentate gyrus (DG) of 6-7-month-old TIMP2 KO and WT mice stained with anti-IBA1 and anti-CD68 antibodies (left; scale bar = 100 μm) and magnified inset (right; scale bar = 50 μm) (N = 5-8 mice per group; female mice; arrowheads indicate IBA1 + CD68 + cells) with corresponding (e) quantification of thresholded DG area covered by IBA1 + staining and (f) the number of IBA1 + CD68 + cells normalized to DG area. (g) Schematic diagram of the high molecular-weight cut-off (HMWCO,1-MDa) in vivo microdialysis method to dialyze hippocampal ISF proteins and the corresponding (h) timeline of LPS treatment paradigm in mice undergoing microdialysis (N = 4; 6-7-month-old male 5XFAD mice) with (i) brain ISF protein measurements, represented as z-scored NPX values for significant proteins (paired t-test; * P < 0.05, ** P < 0.01, *** P < 0.001). (j) Timeline of HMWCO in vivo microdialysis experiments to evaluate steady state levels of hippocampal ISF proteins in 2-3-month-old TIMP2 WT and KO mice (N = 8-9 mice per group; female mice) with corresponding (k) brain ISF protein measurements, represented as z-scored NPX values for significant proteins (unpaired t-test with Welch’s correction; * P < 0.05, ** P < 0.01). Mean ± SEM. Student’s t test used in panels (b-c) and (e-f); * P < 0.05.

Article Snippet: Gels were transferred, the blots probed with anti-TIMP2 (1:5000; D18B7, Cell Signaling) or anti-actin (1:10000; A5060; Sigma), and then developed as previously described .

Techniques: Staining, High Molecular Weight, In Vivo

Journal: bioRxiv

Article Title: Youth-associated protein TIMP2 alters microglial state and function in the context of aging

doi: 10.1101/2025.05.20.655226

Figure Lengend Snippet: (a) Schematic diagram of tamoxifen-inducible microglial deletion of TIMP2 in mice and subsequent isolation of adult microglia, culturing, and imaging. (b) Representative images from primary microglia cultures of Timp2 fl/fl and Cx3cr1 CreERT2/+ ;Timp2 fl/fl littermates stained by ICC with DAPI and anti-CD68 antibody (arrowheads indicate CD68 + cells; scale bar = 100 μm) with corresponding (c) quantification of the number of CD68 + cells normalized to total cell number (DAPI + ) (N = 8 mice per group; two independent experiments combined; 5-6-month-old mice; sex-matched). (d) Representative images from primary microglia cultures of Timp2 fl/fl and Cx3cr1 CreERT2/+ ;Timp2 fl/fl littermates stained by ICC with DAPI and anti-p16INK4a antibody (arrowheads indicate p16INK4a + cells; scale bar = 100 μm) with corresponding (e) quantification of the number of p16INK4a + cells normalized to total number of cells (DAPI + ) (N = 4 mice per group, 7-month-old mice, sex-matched). (f) Timeline (top) of Cre induction paradigm with tamoxifen in Timp2 fl/fl control and Cx3cr1 CreERT2/+ ;Timp2 fl/fl mice and downstream processing of tissue, with representative confocal images (bottom) of DG of 6-7-month-old Timp2 fl/fl and Cx3cr1 CreERT2/+ ;Timp2 fl/fl mice stained with anti-IBA1 and anti-CD68 antibodies (scale bar = 100 μm) and magnified inset (scale bar = 50 μm) (N = 17-19 mice per group, sex-matched) with corresponding (g) quantification of DG area covered by anti-IBA1 staining and (h) the number of IBA1 + CD68 + cells normalized to area. Mean ± SEM. * P < 0.05; Student’s t test.

Article Snippet: Gels were transferred, the blots probed with anti-TIMP2 (1:5000; D18B7, Cell Signaling) or anti-actin (1:10000; A5060; Sigma), and then developed as previously described .

Techniques: Isolation, Imaging, Staining, Control

Journal: bioRxiv

Article Title: Youth-associated protein TIMP2 alters microglial state and function in the context of aging

doi: 10.1101/2025.05.20.655226

Figure Lengend Snippet: (a) Schematic diagram showing timeline of primary microglia culturing from adult mice and media assay for TIMP2 immunoblotting experiments. (b) TIMP2 immunoblotting (representative) from conditioned serum-free media from primary microglia isolated from 2-month-old WT mice, including Ponceau stain (bottom), with corresponding (c) TIMP2 band intensity quantification (N= 4 mice per timepoint; 4 independent experiments combined, with a full timecourse for each experiment; sex-matched). (d) TIMP2 immunoblotting (representative) from conditioned serum-free media from primary microglia isolated from 2-month-old WT mice at baseline (0 hours with no treatment) or 12 hours following treatment with either PBS, zymosan (10 μg/mL), myelin (10 μg/mL), or LPS (100 ng/mL), with corresponding (e) TIMP2 band intensity quantification (N = 3-4 mice per treatment; 4 independent experiments combined, with all treatments representing an experiment; sex-matched). (f) Schematic diagram of treatment of adult primary microglia with pHrodo-labeled myelin and subsequent Incucyte imaging to assess uptake. ( g ) Timecourse from an experiment with total integrated density (red object mean intensity [RCU, red, x average phase object area (cell, μm 2 )) of pHrodo-labeled myelin signal in primary microglia cultured from tamoxifen-treated 5-7-month-old Cx3cr1 CreERT2/+ ;Timp2 fl/fl and Timp2 fl/fl control mice, with corresponding (h) uptake quantification using area under the curve (AUC; N = 6-7 mice per group; 2 independent experiments combined; sex-matched). (i) Schematic of experimental design to assess clearance of pHrodo-labeled myelin in Cx3cr1 CreERT2/+ ;Timp2 fl/fl and Timp2 fl/fl control mice. ( j ) Timecourse from an experiment with total integrated density (RCU [red calibrated units]) x average phase object area (cell, μm 2 )) of pHrodo-labeled myelin signal in primary microglia cultured from 5-7-month-old tamoxifen-treated Cx3cr1 CreERT2/+ ;Timp2 fl/fl and Timp2 fl/fl control mice, with corresponding (k) clearance rate quantification (from slopes of regressions), represented relative to Timp2 fl/fl control (N = 5-7 mice per group; 2 independent experiments combined; sex-matched). (l) Schematic diagram of neuron-specific TIMP2 deletion using Timp2 fl/fl and Syn Cre/+ ; Timp2 fl/fl mice for confocal imaging experiments. (m) Representative confocal images of DG from 2-3-month-old Timp2 fl/fl and Syn Cre/+ ;Timp2 fl/fl littermates stained with anti-IBA1 and anti-CD68 antibodies (left; scale bar = 100 μm) and magnified inset (right; scale bar = 50 μm; white arrowheads indicate IBA1 + CD68 + cells) with corresponding (n) quantification of IBA1 + CD68 + cell number normalized to DG area (N = 8-9 female mice per group). Mean ± SEM. * P < 0.05, ** P < 0.01; One-way ANOVA with Tukey’s post hoc test (c); One-way ANOVA with Dunnett’s post hoc test (e); Student’s t test (h,k,n).

Article Snippet: Gels were transferred, the blots probed with anti-TIMP2 (1:5000; D18B7, Cell Signaling) or anti-actin (1:10000; A5060; Sigma), and then developed as previously described .

Techniques: Western Blot, Isolation, Staining, Labeling, Imaging, Cell Culture, Control

Journal: bioRxiv

Article Title: Youth-associated protein TIMP2 alters microglial state and function in the context of aging

doi: 10.1101/2025.05.20.655226

Figure Lengend Snippet: (a) Schematic of experimental timeline of microglial isolation from 20-month-old WT mice, followed by incubation with TIMP2 or vehicle. ( b) Representative images from cultures of WT primary microglia stained with anti-IBA1 and anti-CD68 antibodies by ICC (arrowheads indicate IBA1 + CD68 + cells; scale bar = 100 μm), with corresponding (c) quantification of the number of CD68 + cells normalized to total cell number (N = 8 mice per treatment group, 2 independent experiments combined; sex-matched). (d) Schematic diagram showing paradigm of systemic treatment with recombinant TIMP2 or vehicle in 20-month-old WT mice, followed by downstream analyses. (e) Representative confocal images of DG from 20-month-old WT mice treated systemically with TIMP2 or vehicle, stained with anti-IBA1 and anti-CD68 antibodies (scale bar = 100 μm; white arrowheads indicate IBA1 + CD68 + cells), with corresponding (f) quantification of number of IBA1 + CD68 + cells normalized to DG area (N = 9 male mice per group). (g) Uniform Manifold Approximation and Projection (UMAP) plot of hippocampal microglial nuclei (2,831) isolated from 20-month-old WT mice treated systemically with vehicle or TIMP2 (N = 4 male mice per group combined as 2 samples per group). (h) Proportion of nuclei across microglial subclusters for each treatment group with corresponding (i) Significant canonical pathways from IPA for marker genes significantly upregulated in subcluster 2, (j) subcluster 5, and (k) subcluster 6. (l) IPA upstream regulator network based on subcluster 6 marker genes, with predicted relationships for target genes. (m) Violin plots of normalized gene expression values from selected top DEGs; * P < 0.0001, from DESeq2 analysis. (n) Significant canonical pathways from IPA on microglial DEGs ( P -valueadj.< 0.05) from TIMP2-treated vs. vehicle-treated aged mice. (o) Dot plot from Gene Set Enrichment Analysis (GSEA) performed on ranked gene lists derived from differential expression analysis of microglia following TIMP2 treatment. Significantly enriched gene sets derived from published transcriptomic states are shown with corresponding Normalized Enrichment Score (NES), with point size representing number of overlapping genes and color indicating P -valueadj. (p) Representative confocal images (left) of DG from 20-month-old WT mice treated systemically with TIMP2 or vehicle, stained with anti-IBA1, anti-CD68, and anti-Vglut1 antibodies (scale bar = 100 μm), with Imaris-based reconstruction (right) of insetted microglial volume (red), containing engulfed VGLUT1 + puncta (purple with yellow outlines indicated by white arrowheads) within microglial lysosomes (green) (scale bar = 50 μm). (q) Quantification of the volume of engulfed VGLUT1 + signal within CD68 + volume, normalized to microglia volume. Values represent N = 5 mice per group). Mean ± SEM. * P < 0.05, ** P < 0.01; Student’s t test.

Article Snippet: Gels were transferred, the blots probed with anti-TIMP2 (1:5000; D18B7, Cell Signaling) or anti-actin (1:10000; A5060; Sigma), and then developed as previously described .

Techniques: Isolation, Incubation, Staining, Recombinant, Marker, Gene Expression, Derivative Assay, Quantitative Proteomics

Journal: Open Life Sciences

Article Title: Association between TGF-β1 and β-catenin expression in the vaginal wall of patients with pelvic organ prolapse

doi: 10.1515/biol-2022-1058

Figure Lengend Snippet: RT - qPCR primer sequences

Article Snippet: Wuhan Sanying), TIMP2 (Batch No.: A1558, Abcolnal), collagen type I alpha 1 chain (COL1A1; Batch No.: BA0325, Boster), reverse transcription kit (Batch No.: MF166-Plus-01, Juemei), fluorescence quantification kit (Batch No.: MF787-01, Juemei), enhanced RIPA lysate (Batch No.: AR0102-100, Boster), BCA protein quantification kit (Batch No.: AR0146, Boster), protease inhibitor (Batch No.: AR1178, Boster), 5X protein sampling buffer (Batch No.: AR1112-10, Boster), and electrochemiluminescence (ECL) luminescent liquid (Batch No.: MA0186, Meilun).

Techniques: Quantitative RT-PCR, Sequencing

Journal: Open Life Sciences

Article Title: Association between TGF-β1 and β-catenin expression in the vaginal wall of patients with pelvic organ prolapse

doi: 10.1515/biol-2022-1058

Figure Lengend Snippet: (a)–(e) Expression of TGF-β1, β-catenin, MMP2, TIMP2, and COL1A1 in the anterior vaginal wall tissues of patients in the control group. (f)–(j) Expression of TGF-β1, β-catenin, MMP2, TIMP2, and COL1A1 in the anterior vaginal wall tissues of patients in the POP group, respectively. (a)–(j) magnification 50×.

Article Snippet: Wuhan Sanying), TIMP2 (Batch No.: A1558, Abcolnal), collagen type I alpha 1 chain (COL1A1; Batch No.: BA0325, Boster), reverse transcription kit (Batch No.: MF166-Plus-01, Juemei), fluorescence quantification kit (Batch No.: MF787-01, Juemei), enhanced RIPA lysate (Batch No.: AR0102-100, Boster), BCA protein quantification kit (Batch No.: AR0146, Boster), protease inhibitor (Batch No.: AR1178, Boster), 5X protein sampling buffer (Batch No.: AR1112-10, Boster), and electrochemiluminescence (ECL) luminescent liquid (Batch No.: MA0186, Meilun).

Techniques: Expressing, Control

Journal: Open Life Sciences

Article Title: Association between TGF-β1 and β-catenin expression in the vaginal wall of patients with pelvic organ prolapse

doi: 10.1515/biol-2022-1058

Figure Lengend Snippet: (a)–(e) Statistical results of immunohistochemical staining of TGF-β1, β-catenin, MMP2, TIMP2, and COL1A1, respectively.

Article Snippet: Wuhan Sanying), TIMP2 (Batch No.: A1558, Abcolnal), collagen type I alpha 1 chain (COL1A1; Batch No.: BA0325, Boster), reverse transcription kit (Batch No.: MF166-Plus-01, Juemei), fluorescence quantification kit (Batch No.: MF787-01, Juemei), enhanced RIPA lysate (Batch No.: AR0102-100, Boster), BCA protein quantification kit (Batch No.: AR0146, Boster), protease inhibitor (Batch No.: AR1178, Boster), 5X protein sampling buffer (Batch No.: AR1112-10, Boster), and electrochemiluminescence (ECL) luminescent liquid (Batch No.: MA0186, Meilun).

Techniques: Immunohistochemical staining, Staining

Journal: Open Life Sciences

Article Title: Association between TGF-β1 and β-catenin expression in the vaginal wall of patients with pelvic organ prolapse

doi: 10.1515/biol-2022-1058

Figure Lengend Snippet: (a)–(e) Statistical results of TGF-β1, β-catenin, MMP2, TIMP2, and COL1A1 mRNA expression levels in the anterior vaginal wall tissues of control and POP groups.

Article Snippet: Wuhan Sanying), TIMP2 (Batch No.: A1558, Abcolnal), collagen type I alpha 1 chain (COL1A1; Batch No.: BA0325, Boster), reverse transcription kit (Batch No.: MF166-Plus-01, Juemei), fluorescence quantification kit (Batch No.: MF787-01, Juemei), enhanced RIPA lysate (Batch No.: AR0102-100, Boster), BCA protein quantification kit (Batch No.: AR0146, Boster), protease inhibitor (Batch No.: AR1178, Boster), 5X protein sampling buffer (Batch No.: AR1112-10, Boster), and electrochemiluminescence (ECL) luminescent liquid (Batch No.: MA0186, Meilun).

Techniques: Expressing, Control

Journal: Chinese Medical Journal

Article Title: Suppression of METTL3 expression attenuated matrix stiffness-induced vaginal fibroblast-to-myofibroblast differentiation and abnormal modulation of the extracellular matrix in pelvic organ prolapse

doi: 10.1097/CM9.0000000000003409

Figure Lengend Snippet: (A–E) Representative gene expression of vaginal fibroblasts cultured on PA gels with variable stiffness (0.2 kPa vs . 12 kPa). WB analysis of protein expression and RT–qPCR analysis of the mRNA expression of α-SMA (A), COL1A1 (B), TIMP1 (C), TIMP2 (D), COL1A1/COL3A1 (E). n = 5; (F) EdU assays comparing vaginal fibroblast proliferation between 0.2 kPa and 12 kPa. (G) Representative images (IF) showing the effect of matrix stiffness on α-SMA, a marker of fibroblast-to-myofibroblast differentiation. GAPDH was used as an internal control. Scale bar = 100 μm. * P <0.05, † P <0.01, ‡ P <0.001. α-SMA: α-smooth muscle actin; COL1A1: Collagen Type 1 Alpha 1; COL3A1: Collagen Type 3 Alpha 1; DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: Glyceraldehyde-phosphate dehydrogenase; IF: Immunofluorescence; PA: Polyacrylamide; RT–qPCR: Real-time polymerase chain reaction; TIMP1: Tissue inhibitor of matrix metalloproteinase 1; TIMP2: Tissue inhibitor of matrix metalloproteinase 2; WB: Western blot.

Article Snippet: The membranes were blocked for 30 min and incubated with the following antibodies overnight at 4°C: anti-METTL3 (1:1000; rabbit polyclonal antibody, ABclonal; Wuhan, China); anti-α-smooth muscle actin (α-SMA) (1:1000; rabbit monoclonal antibody, ABclonal); anti-collagen Type I Alpha 1 (COL1A1) (1:800; rabbit polyclonal antibody, ABclonal); anti-collagen Type 3 Alpha 1 (COL3A1) (1:800; rabbit monoclonal antibody, ABclonal); anti-tissue inhibitor of matrix metalloproteinase 1 (TIMP1) (1:1000; rabbit polyclonal antibody, ABclonal); anti-tissue inhibitor of matrix metalloproteinase 2 (TIMP2) (1:1000; Proteintech, rabbit polyclonal antibody, Wuhan, China); anti-glyceraldehyde-phosphate dehydrogenase (GAPDH) (1:1000; rabbit monoclonal antibody, ABclonal).

Techniques: Gene Expression, Cell Culture, Expressing, Quantitative RT-PCR, Marker, Control, Immunofluorescence, Real-time Polymerase Chain Reaction, Western Blot