Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Sorsby Fundus Dystrophy in an Asian Pedigree: Pathogenic Timp3 P.Y191c Variant Impairs Its Binding with Mmp2/9 and Cellular Localization

doi: 10.2147/OPTH.S556592

Figure Lengend Snippet: Whole exome sequencing. ( A ) Sanger sequencing chromatograms of twelve family members identified the heterozygous c.572A>G (p. Y191C ) TIMP3 variant in the proband (III:5), his father (II:6), uncle (II:3, II:8), aunt (II:11), and cousins (III:3, III:10). ( B ) Pedigree analysis illustrating the inheritance of the c.572A>G (p. Y191C ) variant. ( C ) Multiple sequence alignments of TIMP3 Tyr191 across species revealed that the variant occurred in highly conserved residues. ( D ) Schematic structures of the original and mutant amino acids, highlighting the backbone in red and the side chain in black. ( E ) Comparative 3D structures of the wild-type ( a ) and mutant ( b ) proteins.

Article Snippet: Primary antibodies included TIMP3 (Proteintech, 10858-1-AP, WB: 1:2000, IP: 1:1000, IF: 1:400), MMP9 (Proteintech, 10375-2-AP, WB: 1:600, IP:1:500, IF: 1:100), MMP2 (Proteintech, 66366-1-Ig, WB: 1:1000, IP:1:500, IF: 1:200), and GAPDH (Abcam, ab8245, 1:5000).

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Sorsby Fundus Dystrophy in an Asian Pedigree: Pathogenic Timp3 P.Y191c Variant Impairs Its Binding with Mmp2/9 and Cellular Localization

doi: 10.2147/OPTH.S556592

Figure Lengend Snippet: The TIMP3 variant Y191C mitigated the cell viability and promoted the apoptosis of ARPE-19. ( A ) CCK-8 assay demonstrated a significant reduction in cell viability in the MT group compared to the empty vector control group 24 hours post-LPS treatment ( P= 0.0096), while no significant difference was observed in the WT group ( P= 0.7128). ( B and C ) Flow cytometry analysis indicated a significant increase in apoptosis rates in the MT group compared to the empty vector control group following LPS treatment ( P= 0.0005). In contrast, the WT group exhibited a 10% reduction in apoptosis rates compared to the empty vector control group. FIGURE 4 The TIMP3 variant Y191C mitigated the cell viability and promoted the apoptosis of ARPE-19. ( A ) CCK-8 assay demonstrated a significant reduction in cell viability in the MT group compared to the empty vector control group 24 hours post-LPS treatment ( P= 0.0096), while no significant difference was observed in the WT group ( P= 0.7128). ( B and C ) Flow cytometry analysis indicated a significant increase in apoptosis rates in the MT group compared to the empty vector control group following LPS treatment ( P= 0.0005). In contrast, the WT group exhibited a 10% reduction in apoptosis rates compared to the empty vector control group. n=3/group. “n” denotes biological replicates, defined as independently assayed aliquots derived from the same lentiviral infection and differentiation batch.

Article Snippet: Primary antibodies included TIMP3 (Proteintech, 10858-1-AP, WB: 1:2000, IP: 1:1000, IF: 1:400), MMP9 (Proteintech, 10375-2-AP, WB: 1:600, IP:1:500, IF: 1:100), MMP2 (Proteintech, 66366-1-Ig, WB: 1:1000, IP:1:500, IF: 1:200), and GAPDH (Abcam, ab8245, 1:5000).

Techniques: Variant Assay, CCK-8 Assay, Plasmid Preparation, Control, Flow Cytometry, Derivative Assay, Infection

Journal: Clinical Ophthalmology (Auckland, N.Z.)

Article Title: Sorsby Fundus Dystrophy in an Asian Pedigree: Pathogenic Timp3 P.Y191c Variant Impairs Its Binding with Mmp2/9 and Cellular Localization

doi: 10.2147/OPTH.S556592

Figure Lengend Snippet: The Y191C variant inhibited the interaction between TIMP3 and MMP proteins. ( A and B ) The Y191C mutation increases FLAG-TIMP3 complex formation, selectively enhancing interaction with MMP9 proteolytic fragments (45~55 kDa) while reducing MMP2 binding. ( C – E ) Following LPS administration, MMP2 expression levels in the empty vector control group remained comparable to those in the normal control group. However, in the MT group, MMP2 expression was significantly elevated compared to the empty vector group ( P < 0.0001, 1.84 ± 0.21-fold) and showed a modest increase compared to the WT control group ( P= 0.0889, 1.15 ± 0.13-fold). Additionally, MMP9 expression in the MT group was markedly higher than in the empty vector group after LPS treatment ( P < 0.0172, 2.12 ± 0.41-fold). ( F ) IF staining revealed nuclear localization of MMP2 in ARPE-19 cells of the WT group, whereas in the MT group, MMP2 expression extended into the cytoplasm of ARPE-19 cells. n=3/group. “n” refers to the number of independent biological replicates.

Article Snippet: Primary antibodies included TIMP3 (Proteintech, 10858-1-AP, WB: 1:2000, IP: 1:1000, IF: 1:400), MMP9 (Proteintech, 10375-2-AP, WB: 1:600, IP:1:500, IF: 1:100), MMP2 (Proteintech, 66366-1-Ig, WB: 1:1000, IP:1:500, IF: 1:200), and GAPDH (Abcam, ab8245, 1:5000).

Techniques: Variant Assay, Mutagenesis, Binding Assay, Expressing, Plasmid Preparation, Control, Staining

Journal: Stem Cells Translational Medicine

Article Title: Endostatin-expressing endometrial mesenchymal stem cells inhibit angiogenesis in endometriosis through the miRNA-21-5p/TIMP3/PI3K/Akt/mTOR pathway

doi: 10.1093/stcltm/szae079

Figure Lengend Snippet: TIMP3 is a target gene of miR-21-5p, and the expression of the miR-21-5p/TIMP3/PI3K/Akt/mTOR was affected by EMSCs-Endo in vitro. (A) Bioinformatic analysis of the differential expression of miR-21-5p in endometriosis. (B) The levels of miR-21-5p were significantly elevated in the eutopic and ectopic tissues of patients with endometriosis. (C) The TIMP3 3ʹ-UTR contains miR-21-5p binding sites. (D) Dual-luciferase reporter assays confirmed TIMP3 as a target gene of miR-21-5p. (E) The relative expression of miR-21-5p in cocultured HUVECs from each group. (F) Expression analysis of TIMP3/PI3K/Akt/mTOR in cocultured HUVECs from each group by Western blotting. (G-J) Quantification of protein levels of TIMP3 (G), p-PI3K (H), p-Akt (I), p-mTOR (J). The data are presented as the mean ± SD of n = 3 independent experiments. Statistical analysis was performed via 1-way ANOVA with Bonferroni post hoc correction (B, D, E, and G-J), * P < 0.05, *** P < 0.001.

Article Snippet: Next, each membrane was blocked overnight with 5% nonfat milk and then incubated with antibodies against TIMP3 (Proteintech Group, China), p-Akt (CST, USA), Akt (CST, USA), p-PI3K (Proteintech Group, China), PI3K (Proteintech Group, China), p-mTOR (Proteintech Group, China), mTOR (CST, USA), and GAPDH (Proteintech Group, China) at 4° C overnight.

Techniques: Expressing, In Vitro, Quantitative Proteomics, Binding Assay, Luciferase, Western Blot

Journal: Stem Cells Translational Medicine

Article Title: Endostatin-expressing endometrial mesenchymal stem cells inhibit angiogenesis in endometriosis through the miRNA-21-5p/TIMP3/PI3K/Akt/mTOR pathway

doi: 10.1093/stcltm/szae079

Figure Lengend Snippet: The expression of miR-21-5p/TIMP3/p-PI3K/p-Akt/p-mTOR in mouse endometriotic lesions. (A) Expression analysis of TIMP3/p-PI3K/p-Akt/p-mTOR in the endometriotic lesions of each group by Western blotting. (B) Quantification of protein levels of TIMP3, p-PI3K, p-Akt, and p-mTOR. (C) Relative expression of miR-21-5p in the endometriotic lesions of each group. (D) Expression analysis of TIMP3/p-PI3K/p-Akt/p-mTOR in the endometriotic lesions of each group by means of immunohistochemistry. Scale bar = 25 μm. The data are presented as the mean ± SD. Statistical analysis was performed by means of 1-way ANOVA with the Bonferroni post hoc correction. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Next, each membrane was blocked overnight with 5% nonfat milk and then incubated with antibodies against TIMP3 (Proteintech Group, China), p-Akt (CST, USA), Akt (CST, USA), p-PI3K (Proteintech Group, China), PI3K (Proteintech Group, China), p-mTOR (Proteintech Group, China), mTOR (CST, USA), and GAPDH (Proteintech Group, China) at 4° C overnight.

Techniques: Expressing, Western Blot, Immunohistochemistry