Journal: Journal of Biological Chemistry

Article Title: Modulation of matrix metabolism by ATP-citrate lyase in articular chondrocytes

doi: 10.1074/jbc.ra118.002261

Figure Lengend Snippet: Figure 5. Inhibition of ACLY by HCA attenuated IL-1β-induced augmentation of acetylation H3K9 and H3K27 both globally and specifically in the iNOS, MMP3 and MMP13 promoters.

Article Snippet: Assays of nitric oxide (NO), MMP3, and MMP13 release, and SOX9 acetylation, distribution, and expression Conditioned media were tested for NO generation by Griess reaction assay to quantify nitrite, and MMP3 and MMP13 release measured using Total MMP3 and MMP13 Quantikine ELISA Kits (R&D Systems).

Techniques: Inhibition

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Western blot analysis and semiquantitative evaluation of DLL4, VEGFA, and CD31 expression in PDX mice tissues by densitometry analysis of protein bands reveals a downregulation of DLL4, VEGFA, and CD31 protein expression in PDX mice treated with GSI. The bands were measured compared with the housekeeping GAPDH protein band, for each tissue. Average value of DLL4, VEGFA, and CD31 expression levels among all mouse treated with LY3039478 or vehicle is reported in the graph. P value showed versus vehicle treatment. Tissues PDX mice n = 10 for vehicle treatment in gray, n = 10 for LY3039478 treatment in black. b Representative images with immunofluorescence staining show DLL4 and CD31 downregulation in representative images of PDX tissues treated with LY30349478. DLL4 (green) and CD31 (red) and overlapping staining (yellow) were immunolocalized in PDX tissues. The yellow arrows highlight the detail of the co-localization of DLL4 and CD31 in PDX tissues (#4, #14, #24) not treated with LY339478. DAPI, 4′,6‐diamidino‐2‐phenylindole. c Immunofluorescence staining with MMP13 in red and nucleus in DAPI shown a significantly reduction of MMP13 in iCCA PDX tissues treated with LY3039478. Magnifications: ×20; inset ×60. d Representative images demonstrate a significant ( P < 0.001) destruction of the network created by the HUVECs following the treatment with LY3039478 (1 µM). The concomitant administration of MMP13 counteracts significantly ( P < 0.01) drug effectiveness.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Western Blot, Expressing, Immunofluorescence, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: a Analysis of 31 primary tumors from iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database (GSE107943). Mean expression data were expressed in RPKM (Reads Per Kilobase Million). *** P < 0.001 calculated with Student’s t test. b NOTCH1 gene and its pro-angiogenic targets are overexpressed in human intrahepatic cholangiocarcinoma (iCCA). Levels of NOTCH1, DLL4, VEGFA, and MMP13 mRNA were significantly more elevated in iCCA ( n = 42) than corresponding nontumorous surrounding livers (SL; n = 42), as detected by quantitative reverse-transcription PCR. Number target (NT) = 2 −ΔCt , wherein ΔCt value of each sample was calculated by subtracting the average Ct value of the gene of interest from the average Ct value of the β-actin gene. Mann–Whitney test: vs SL, P < 0.0001. c Expression of the NOTCH1 gene correlates with mRNA levels of putative target genes (HES1, DLL4, VEGFA, and MMP13) in a collection of human intrahepatic cholangiocarcinoma (CCA) samples ( n = 42). Linear regression analysis was used. d Representative expression patterns of CK19, NOTCH1, HES1, DDL4, and MMP13 in human intrahepatic cholangiocarcinoma (iCCA) as detected by immunohistochemistry. Upper panels: CCA case (CCA1) showing strong, concomitant immunoreactivity for NOTCH1, HES1, DDL4, and MMP13. Lower panels: CCA specimens (CCA2) exhibiting low levels of NOTCH1, HES1, DDL4, and MMP13. As expected, both iCCA display robust immunolabeling for CK19 (a biliary marker). Magnification: ×200; scale bar = 100 μm. H&E hematoxylin and eosin staining.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing, Reverse Transcription, MANN-WHITNEY, Immunohistochemistry, Immunolabeling, Marker, Staining

Journal: Cell Death and Differentiation

Article Title: Crenigacestat, a selective NOTCH1 inhibitor, reduces intrahepatic cholangiocarcinoma progression by blocking VEGFA/DLL4/MMP13 axis

doi: 10.1038/s41418-020-0505-4

Figure Lengend Snippet: Levels of tumor microvessel density (MVD) correlate with mRNA expression of NOTCH1 ( a ), HES1 ( b ), DLL4 ( c ), and MMP13 ( e ), but not with those of VEGFA ( d ), in a collection of human intrahepatic cholangiocarcinoma (iCCA) samples ( n = 42). Linear regression analysis was used. f Representative examples of human iCCA specimens with high and low MVD.

Article Snippet: The recombinant protein MMP13 (R&D System, Minneapolis, MN) was used at 50 ng/ml.

Techniques: Expressing

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Intra-articular injection of primed MSCs suppresses OA progression without evidence of direct regeneration. (A) Representative histological analysis using Safranin-O and immunohistochemistry (IHC) (collagen type II, MMP13, collagen type X, DPP4, and human β2-microglobulin) in sham control and DMM-induced rabbits injected with PBS and naïve or primed MSCs. (B) Scoring of OA using OARSI grading system in sham control and DMM-induced OA rabbits that had undergone intra-articular injection of PBS and naïve or primed MSCs. (C to F) Quantification of collagen type II, MMP13, collagen type X, and DPP4 using scoring system. (G) Representative histological analysis using Safranin-O and DPP4 IHC staining in DMM-induced OA rabbits injected with PBS, primed MSCs, low-dose, mid-dose, or high-dose secretome, or triple high-dose secretome isolated from primed MSCs. (H and I) Scoring of OA using OARSI grading system and quantification of DPP4. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Injection, Immunohistochemistry, Control, Isolation

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Paracrine activity of primed MSC suppresses senescence phenotypes of OA chondrocytes. (A) Left: Representative immunofluorescence staining of p16 and p21 (red) in OA chondrocytes (OA), OA + naïve MSC media, OA + MSC priming media, and direct coculture of OA and naïve or primed MSCs. OA chondrocytes were distinguished with CellTracker Dil staining (green), and nuclei were counterstained with DAPI (blue). Right: Bar graphs showing quantification of p16 + OA chondrocytes (top) and quantification of p21 + OA chondrocytes ( n = 3 per group) (bottom). (B) Schematic illustration of the experiments. (C) mRNA levels of p16 and p21 in OA, OA + naïve CM, and OA + primed CM measured by RT-qPCR. (D) Representative SA-β-Gal staining and quantification for OA, OA + naïve CM, and OA + primed CM ( n = 3 per group). (E) Cell counts of OA, OA + naïve CM, and OA + primed CM for 14 d to evaluate proliferation ( n = 3 per group). (F) mRNA expression of matrix degradation factors (ADAMTS5 and MMP13) in OA, OA + naïve CM, and OA + primed CM ( n = 3 per group). (G) Schematic illustration of indirect coculture system using transwell. (H) mRNA levels of p16 and p21 in OA, OA + naïve MSCs, and OA + primed MSCs measured by RT-qPCR. (I) Representative SA-β-Gal staining and quantification for OA, OA + naïve MSCs, and OA + primed MSCs ( n = 3 per group). (J) Cell counts of OA, OA + naïve MSCs, and OA + primed MSCs for 14 d to evaluate proliferation ( n = 3 per group). (K) mRNA expression of matrix degradation factors (ADAMTS5 and MMP13) in OA, OA + naïve MSCs, and OA + primed MSCs ( n = 3 per group). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Activity Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Expressing

Journal: Biomaterials Research

Article Title: Insulin-Like Growth Factor 2 Secreted from Mesenchymal Stem Cells with High Glutathione Levels Alleviates Osteoarthritis via Paracrine Rejuvenation of Senescent Chondrocytes

doi: 10.34133/bmr.0152

Figure Lengend Snippet: Knockdown of IGF2 expression in primed MSCs using siRNA inhibits therapeutic efficacy in vivo. (A) Representative histological analysis using Safranin-O and IHC (collagen type II, MMP13, collagen type X, and DPP4) in sham control and DMM-induced rabbits injected with PBS, primed MSCs, or primed MSCs with IGF2 knockdown (IGF2KD MSCs). (B) Scoring of OA using OARSI grading system in sham control and DMM-induced OA rabbits that had undergone intra-articular injection of PBS, primed MSCs, or IGF2KD MSCs. (C to F) Quantification of collagen type II, MMP13, collagen type X, and DPP4 using scoring system. ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Sections were then incubated with primary antibodies against collagen type II (Thermo Fisher Scientific, MA1-37493), MMP13 (R&D Systems, MAB511), collagen type X (Abcam, ab49945), DPP4 (Thermo Fisher Scientific, MA2607), and human β2 microglobulin (Abcam, ab759) diluted in 4% bovine serum albumin (BSA; Biosesang, AC1025-100-00) for 1 h at 37 °C.

Techniques: Knockdown, Expressing, Drug discovery, In Vivo, Control, Injection

Journal: Journal of Orthopaedic Translation

Article Title: CD73 alleviates osteoarthritis by maintaining anabolism and suppressing catabolism of chondrocytes extracellular matrix

doi: 10.1016/j.jot.2024.08.014

Figure Lengend Snippet: The function of maintaining anabolism and suppressing catabolism of CD73 in chondrocytes with IL-1β treatment. A. qPCR results of mRNA expression levels of Nt5e, Col2a1, and Mmp13 in mouse chondrocytes received siRNA treatment for knocking down Cd73 expression with or without IL-1β. B. Western blot results of Cd73, Col II and Mmp13 in the mouse chondrocytes. C. Immunofluorescence staining of Col II and Mmp13 in the mouse chondrocytes. D. The quantification of western blot in the mouse chondrocytes. E. The quantification of immunofluorescence staining in the mouse chondrocytes. F. qPCR results of mRNA expression levels of Nt5e, Col2a1, and Mmp13 in mouse chondrocytes received lentivirus for overexpressing Cd73 with or without IL-1β. G. Western blot results of Cd73, Col II and Mmp13 in the mouse chondrocytes. H. Immunofluorescence staining of Col II and Mmp13 in the mouse chondrocytes. I. The quantification of western blot in the mouse chondrocytes. J. The quantification of immunofluorescence staining in the mouse chondrocytes. K & L. The extracellular ATP and adenosine levels of the mouse chondrocytes, respectively. Data (n = 3) were shown as mean ± SD. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Article Snippet: Later, the membranes were blocked with 5 % (w/v) milk (Biofroxx) for 1 h at room temperature and then incubated with primary antibodies (1:1000 dilution) of GAPDH (#10494-1-AP, Proteintech, China), CD39 (#14211-1-AP, Proteintech), CD73 (#12231-1-AP, Proteintech), ADORA1 (#20332-1-AP, Proteintech), ADORA2A (#53509, CST, China), ADORA2B (#21071-1-AP, Proteintech), ADORA3 (#ab203298, Abcam, UK), COL2A1 (#BA0533, Boster, China) and MMP13 (#69926, CST).

Techniques: Expressing, Western Blot, Staining

Journal: Journal of Translational Medicine

Article Title: Glibenclamide attenuates osteoarthritis by suppressing NLRP3 inflammasome activation in synovial macrophages through the MAPK pathway

doi: 10.1186/s12967-026-07923-7

Figure Lengend Snippet: Gb attenuates OA progression. A . Representative images of H&E staining of synovium and cartilage, SO-FG and TB staining of mouse knee joints. Scale bar = 100 μm. n = 6. B . Synovitis score comparing the severity of synovial inflammation in mice. n = 6. C . OARSI scoring system comparing cartilage degradation in mice. n = 6. D . Representative IHC images of COL2A and MMP13 in mouse knee cartilage. Scale bar = 100 μm. n = 6. E . Quantitative analysis of COL2A-positive area in cartilage. n = 6. F . Quantitative analysis of MMP13-positive area in cartilage. n = 6. G . Representative micro-CT 3D reconstruction images of mouse knee joints and osteophytes (indicated by red arrows). n = 6. H . Quantitative analysis of osteophyte numbers. n = 6. I . Representative coronal micro-CT images of mouse knee joints. n = 6. J . Quantitative analysis of subchondral bone volume fraction (BV/TV) and trabecular thickness (Tb.Th). n = 6. Data are presented as mean ± SD. One-way ANOVA was used for multiple group comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: MMP13 , Proteintech , 18165-1-AP.

Techniques: Staining, Micro-CT

Journal: Journal of Translational Medicine

Article Title: Glibenclamide attenuates osteoarthritis by suppressing NLRP3 inflammasome activation in synovial macrophages through the MAPK pathway

doi: 10.1186/s12967-026-07923-7

Figure Lengend Snippet: Gb regulates chondrocyte function via synovial macrophages. A . Schematic diagram of the macrophage–chondrocyte co-culture system. B . Western blot analysis of COL2A, Aggrecan, MMP13, and ADAMTS5 protein levels in chondrocytes under different co-culture conditions, and the quantitative analysis of the protein. n = 3. C . Representative micromass Alcian blue staining of chondrocytes under different co-culture conditions. n = 3. D . Flow cytometry analysis of chondrocyte proliferation under different co-culture conditions, with quantitative analysis. n = 6. E . Representative EdU staining images and quantitative analysis of chondrocyte proliferation under different co-culture conditions. Scale bar = 100 μm. n = 6. F . Flow cytometry analysis of chondrocyte apoptosis under different co-culture conditions, with quantitative analysis. n = 6. G . TUNEL assay for chondrocyte apoptosis under different co-culture conditions, with quantitative analysis. Scale bar = 100 μm. n = 6. Data are presented as mean ± SD. One-way ANOVA was used for multiple group comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: MMP13 , Proteintech , 18165-1-AP.

Techniques: Co-Culture Assay, Western Blot, Staining, Flow Cytometry, TUNEL Assay

Journal: Journal of Translational Medicine

Article Title: Glibenclamide attenuates osteoarthritis by suppressing NLRP3 inflammasome activation in synovial macrophages through the MAPK pathway

doi: 10.1186/s12967-026-07923-7

Figure Lengend Snippet: Gb suppresses NLRP3 inflammasome-mediated macrophage inflammation and protects chondrocytes. A . Heatmap of RNA-seq analysis comparing Gb-treated and control macrophages. B . GSEA indicating significant reduction in cytokine activity and metalloproteinase activity in Gb-treated macrophages. C . GO enrichment analysis showing differential mRNAs are mainly involved in maintaining cell polarization, regulating inflammatory response, and regulating NLRP3 signaling. D . Western blot analysis of NLRP3 knockdown efficiency in macrophages. n = 3. E . IF analysis of NLRP3 knockdown efficiency. Scale bar = 100 μm. n = 6. F . Western blot analysis of Caspase-1 and IL-1β expression in shNLRP3 macrophages treated with Gb. n = 3. G . qRT-PCR analysis of IL-1β, TNF-α, and IL-10 mRNA in shNLRP3 macrophages treated with Gb. n = 3. H . Representative micromass Alcian blue staining of chondrocytes under different co-culture conditions. n = 3. I . Western blot analysis of COL2A, Aggrecan, MMP13, and ADAMTS5 protein levels in chondrocytes under different co-culture conditions. n = 3. J . Flow cytometry analysis of chondrocyte proliferation under different co-culture conditions, with quantitative analysis. n = 6. K . Representative EdU staining images and quantitative analysis of chondrocyte proliferation. Scale bar = 100 μm. n = 6. Data are presented as mean ± SD. One-way ANOVA was used for multiple group comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: MMP13 , Proteintech , 18165-1-AP.

Techniques: RNA Sequencing, Control, Activity Assay, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Staining, Co-Culture Assay, Flow Cytometry

Journal: Journal of Translational Medicine

Article Title: Glibenclamide attenuates osteoarthritis by suppressing NLRP3 inflammasome activation in synovial macrophages through the MAPK pathway

doi: 10.1186/s12967-026-07923-7

Figure Lengend Snippet: In vivo knockdown of synovial macrophage NLRP3 weakens Gb-mediated protection against OA. A . Representative IF images of ZsGreen distribution in the knee joint after intra-articular injection of rAAV-shNLRP3, with quantitative analysis of ZsGreen-positive cells in synovium and cartilage. S indicates synovium, and C indicates cartilage. Scale bar = 1 mm (left), 50 μm (right). n = 3. B . Representative IHC images of NLRP3 in synovium and IF images of F4/80 and NLRP3 co-staining in synovium. Scale bar = 100 μm. n = 6. C . Representative HE, SO-FG, and TB staining of synovium and cartilage. Scale bar = 100 μm. n = 6. D . Synovitis score comparing severity of synovial inflammation. n = 6. E. OARSI score comparing cartilage degradation. n = 6. F . Representative IHC images of COL2A and MMP13 in cartilage. Scale bar = 100 μm. n = 6. G . Quantitative analysis of COL2A and MMP13 positive areas. n = 6. H . Representative micro-CT 3D reconstruction images showing osteophytes (red arrows) and quantitative analysis of osteophyte numbers. n = 6. I. Representative coronal micro-CT images, with quantitative analysis of subchondral BV/TV and Tb.Th. n = 6. Data are presented as mean ± SD. Student’s t-test was used for two-group comparisons, and one-way ANOVA for multiple-group comparisons. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: MMP13 , Proteintech , 18165-1-AP.

Techniques: In Vivo, Knockdown, Injection, Staining, Micro-CT