Fig 1: (A) In vitro effect of Ral activation on the migration of RalGAPa2 knockdown Colon26 cells.Scale bars: 200 µm. *P < .01, repeated-measures analysis of variance with the Holm correction. (B) In vitro effect of Ral activation on the invasion of RalGAPa2 knockdown Colon26 cells. Scale bars: 200 µm. *P < .01, 1-way analysis of variance with the Holm correction. (C) Flow cytometry findings for the isolated colon epithelial cells that were CD326-positive/CD45-negative. (D) The ratio of the gene expression levels of colon epithelial cells in RalGAPa2 KO mice/WT mice. (E) The mRNA expression levels of MMP-9 and MMP-13 were analyzed by quantitative polymerase chain reaction. The average expression levels in WT-N were defined as 1. *P < .01, **P < .05, repeated-measures analysis of variance with the Holm correction. (F) Gelatin zymography of activated MMP-9 in colon mucosa and CAC. (G) Western blot of MMP-13 in colon mucosa and CAC. (H and I) Immunohistochemical staining for MMP-9 and MMP-13 in the CAC of WT mice (a) and the CAC of RalGAPa2 KO mice (b). Scale bars: 100 µm. The proportion of cells that are positive for MMP-9 and MMP-13 at the invasive fronts of tumors from WT mice and RalGAPa2 KO mice (n = 10 per group). **P < .05, Fisher exact test. Data are either representative or shown as the means ± SEM (error bars) of 3 independent experiments.
Fig 2: (A) The protocol for AOM/DSS/NLRP3 inhibitor administration. (B) Endoscopic findings of CAC occurrence with NLRP3 inhibitor administration in WT mice and RalGAPa2 KO mice. (C) Macroscopic findings of CAC occurrence with NLRP3 inhibitor administration in WT mice and RalGAPa2 KO mice. (D) Histopathologic findings of CAC occurrence with NLRP3 inhibitor administration in WT mice and RalGAPa2 KO mice. (E–H) Groups were categorized as follows: WT mice + AOM/DSS, WT mice + AOM/DSS/NLRP3 inhibitor, RalGAPa2 KO mice + AOM/DSS, and RalGAPa2 KO mice + AOM/DSS/NLRP3 inhibitor. (E–H) These 4 groups were compared for (E) tumor number (*P < .01, 1-way analysis of variance with the Holm correction), (F) tumor maximum diameter (*P < .01, 1-way analysis of variance with the Holm correction), (G) the ratio of mice with submucosal invasive tumors (**P < .05, Fisher exact test with the Holm correction), and (H) the number of submucosal invasive tumors in the cancer specimens (**P < .05, Fisher exact test with the Holm correction). M, mucosal tumors. (I) The expression of NLRP3, ASC, and pro/cleaved caspase-1 in colon mucosa and CAC with or without NLRP3 inhibitor administration were analyzed by Western blot. (J and K) The mRNA expression of IL1ß, IL18, MMP-9, and MMP-13 in colon mucosa and CAC with or without NLRP3 inhibitor administration were analyzed by quantitative polymerase chain reaction. Data are either representative or shown as the means ± SEM (error bars) of 3 independent experiments. The average expression level in the WT-N without NLRP3 inhibitor was defined as 1. *P < .01, **P < .05, repeated-measures analysis of variance with the Holm correction.
Fig 3: Validation of mechanism of Cuprorivaite (CaCuSi4O10) microspheres in OA. (A) Hematoxylin and eosin staining. (B) Safranin-O staining/fast green staining. (C) OARSI score. (D) ELISA detection for TNF-α, IL-6 and MMP13 in the serum. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cartilage tissue. (F) RT-qPCR quantification for extracellular matrix components including collagen II and SOX9 in cartilage tissue. (G) Copper content in cartilage tissue. (H) RT-qPCR quantification for cuproptosis biomarkers including ATP7B and FDX1 in cartilage tissue. (I) Western blot detection for ATP7B and FDX1 in cartilage tissue. (J) Representative images of FDX1 expression in cartilage tissue detected by immunohistochemistry. (K) Oxidative stress detection including MDA, SOD and GSH in cartilage tissue. (L) RT-qPCR quantification for Wnt1, GSK3β and β-catenin in cartilage tissue. ∗∗∗P < 0.001.
Fig 4: Anti-inflammatory role of Cuprorivaite (CaCuSi4O10) microspheres in IL-1β-stimulated chondrocytes. (A) Cell counting kit-8 test for cell proliferation. (B) Fluorescein diacetate for cell viability detection. (C) Cell apoptosis by flow cytometry. (D) ELISA detection for TNF-α, IL-6 and MMP13 in cell supernatant. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cell lysates. (F) RT-qPCR quantification for in TNF-α, IL-6 and MMP13. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Fig 5: Cuprorivaite (CaCuSi4O10) microspheres improved IL-1β-induced injury in chondrocytes via inhibiting Wnt/β-catenin pathway. (A) Cell counting kit-8 test for cell viability. (B) Fluorescein diacetate for cell viability detection. (C) Cell apoptosis by flow cytometry. (D) ELISA detection for TNF-α, IL-6 and MMP13 in cell supernatant. (E) Western blot detection for TNF-α, IL-6 and MMP13 in cell lysates. (F) RT-qPCR quantification for extracellular matrix components including collagen II and SOX9. (G) Intracellular copper content. (H) RT-qPCR quantification for cuproptosis biomarkers including ATP7B and FDX1. (I) Western blot detection for ATP7B and FDX1. (J) Oxidative stress detection including MDA, SOD and GSH. (K) RT-qPCR quantification for Wnt1, GSK3β and β-catenin. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.
Supplier Page from CUSABIO Technology LLC for Mouse matrix metalloproteinase 13,MMP-13 ELISA Kit