antibody against timp3  (R&D Systems)

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

Journal: Platelets

Article Title: Transcriptomic and functional characterization of megakaryocytic-derived platelet-like particles: impaired aggregation and prominent anti-tumor effects.

doi: 10.1080/09537104.2024.2449344

Figure Lengend Snippet: Figure 7. Signaling axes promoting or inhibiting oncogenicity. (A) Schematic representation of potential signaling axes mediating platelet-PCa pro- oncogenic interactions and PLP-PCa anti-oncogenic interactions. (B) RNA-seq expression values in TPMs of components in the platelet-PCa cell signaling axes. (C) RNA-seq expression values of components in the PLP-PCa cell signaling axes. Data in (B) and (C) presented as the mean ± SEM of n = 44 for platelets and n = 8 independent determinations for PLPs (determinations for MEG-01- and K-562-derived PLPs combined). RNA-Seq TPM values for MDA and RC77 were from a single determination. Statistical analysis was performed using a t-test (*p < .05). (D) Far left panel depicts relative 48-hour matrigel invasion by MDA-PCa-2b (MDA) and RC77T/E (RC77) cells incubated with vehicle (veh) + 5 ug/ml IgG isotype control, PLP + IgG, or PLP + 5 ug/ml neutralizing antibody against TIMP3 (β-TIMP3). Middle panel depicts relative 24-hour caspase activity in MDA and RC77 cells incubated with vehicle (veh) + 5 ug/ml IgG isotype control, PLP + IgG, or PLP + 5 ug/ml neutralizing antibody α-vegfb. Far right panel depicts relative 24-hour caspase activity in MDA cells incubated with conditioned medium (CM) derived from 24-hour culturing of MEG-01 PLPs (CM1), PCa cells + PLPs (CM2), and PCa cells + PLPs + platelets (CM3). Closed and open bars indicate incubation with 5 ug/ml IgG or α-TIMP3, respectively. Cell:PLP ratio was 1:500 and Cell:PLP:platelet ratio was 1:500:500 for all experiments. Data presented as the mean ± SEM of n = 4 independent determinations and analyzed by ANOVA and Dunnett’s post-hoc test. *significantly different (p < .05) compared to corresponding control (veh-containing group). #Significantly different (p < .05) compared to corresponding IgG group. Abbreviations: RC77, RC77T/E; MDA, MDA-PCa -2b; ITGA2B, integrin alpha 2b; ITGB3, integrin beta 3; FN1, fibronectin 1; IL32, interleukin 32; EGF, epidermal growth factor; TGFA, transforming growth factor alpha; EGFR, epidermal growth factor receptor; HER2, human epidermal growth factor receptor 2; ERBB3, human epidermal growth factor receptor 3; EPHA, EPH receptor A; EFNA, ephrin A; TIMP3, tissue inhibitor of metalloproteinases 3; MMP15, matrix metalloproteinase 15; VEGFB, vascular endothelial growth factor B; FGFR1, fibroblast growth factor receptor 1; IGFBP4, insulin-like growth factor-binding protein 4; LPR6, low-density lipoprotein receptor-related protein 6.

Article Snippet: Cells alone included an isotype IgG control (5 μg/ mL), and cells with PLPs were incubated with isotype IgG control (5 μg/ml) or a neutralizing antibody against TIMP3 (5 μg/ml; R&D Systems, Cat #MAB973-SP) or VEGFB (5 μg/ml; R&D Systems, Cat #MAB3372).

Techniques: RNA Sequencing, Expressing, Derivative Assay, Incubation, Control, Activity Assay, Binding Assay

Journal: RSC advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3.

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: Fig. 3 TIMP3 was a direct target of miR-132 in HaCaT cells. (A) The miR-132 binding sites predicted in the 30-UTR of TIMP3 mRNA and the mutant in seed sites. (B) The relative luciferase activity was detected in HaCaT cells cotransfected with TIMP3-WT or TIMP3-MUT constructs and miR-NC mimics or miR-132 mimics. (C) HaCaT cells were transfected with miR-NC mimics or miR-132 mimics, and the enrichment of TIMP3 mRNA was measured with anti-Ago2 or anti-IgG by qRT-PCR assay. (D) HaCaT cells were transfected with miR-NC mimics, miR-132 mimics, anti-miR-NC or anti-miR-132, followed by the detection of TIMP3 level by Western blot. **P < 0.01 or ***P < 0.001 vs. respective control.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specic primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or b-actin (sc517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Construct, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: RSC advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3.

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: Fig. 4 TGF-b1 repressed the expression of TIMP3. HaCaT (A) and NHEK (B) cells were treated with different concentration (0, 2, 5 and 10 ng ml1) of TGF- b1 for 24 h, and then TIMP3 expression was detected by Western blot. HaCaT (C) and NHEK (D) cells were treated with 5 ng ml1 of TGF-b1 at different time period (0, 6, 12 and 24 h), followed by the measurement of TIMP3 expression by Western blot. *P < 0.05 or **P < 0.01 or ***P < 0.001 vs. control.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specic primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or b-actin (sc517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Expressing, Concentration Assay, Western Blot, Control

Journal: RSC advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3.

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: Fig. 5 TIMP3 inhibited the proliferation and migration of HaCaT cells under TGF-b1 treatment. HaCaT cells were treated with TGF-b1 (5 ng ml1) or transfected with pcDNA or pcDNA-TIMP3 prior to TGF-b1 treatment. (A) and (B) qRT-PCR for miR-132 expression in treated cells. (C) and (D) MTT assay for the proliferation ability in treated cells. (E) and (F) Transwell assay for the migration capacity in treated cells. *P < 0.05 or **P < 0.01 vs. control or TGF-b1 + pcDNA.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specic primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or b-actin (sc517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Migration, Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Transwell Assay, Control

Journal: RSC advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3.

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: Fig. 6 MiR-132-mediated pro-proliferation and pro-migration effects were antagonized by TIMP3 in TGF-b1-treated HaCaT cells. HaCaT cells were transfected with miR-132 mimics + pcDNA, miR-132 mimics + pcDNA-TIMP3, anti-miR-132 + si-NC or anti-miR-132 + si-TIMP3 prior to TGF-b1 (5 ng ml1) treatment, followed by the detection of cell proliferation ability by MTT assay (A) and (B), cell migration capacity by transwell assay (C) and (D). *P < 0.05 or **P < 0.01 vs. control or TGF-b1 + miR-NC or TGF-b1 + miR-132 + pcDNA.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specic primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or b-actin (sc517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Migration, Transfection, MTT Assay, Transwell Assay, Control

Journal: RSC Advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: TIMP3 was a direct target of miR-132 in HaCaT cells. (A) The miR-132 binding sites predicted in the 3′-UTR of TIMP3 mRNA and the mutant in seed sites. (B) The relative luciferase activity was detected in HaCaT cells cotransfected with TIMP3-WT or TIMP3-MUT constructs and miR-NC mimics or miR-132 mimics. (C) HaCaT cells were transfected with miR-NC mimics or miR-132 mimics, and the enrichment of TIMP3 mRNA was measured with anti-Ago2 or anti-IgG by qRT-PCR assay. (D) HaCaT cells were transfected with miR-NC mimics, miR-132 mimics, anti-miR-NC or anti-miR-132, followed by the detection of TIMP3 level by Western blot. ** P < 0.01 or *** P < 0.001 vs. respective control.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specific primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or β-actin (sc-517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 °C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Binding Assay, Mutagenesis, Luciferase, Activity Assay, Construct, Transfection, Quantitative RT-PCR, Western Blot, Control

Journal: RSC Advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: TGF-β1 repressed the expression of TIMP3. HaCaT (A) and NHEK (B) cells were treated with different concentration (0, 2, 5 and 10 ng ml −1 ) of TGF-β1 for 24 h, and then TIMP3 expression was detected by Western blot. HaCaT (C) and NHEK (D) cells were treated with 5 ng ml −1 of TGF-β1 at different time period (0, 6, 12 and 24 h), followed by the measurement of TIMP3 expression by Western blot. * P < 0.05 or ** P < 0.01 or *** P < 0.001 vs. control.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specific primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or β-actin (sc-517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 °C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Expressing, Concentration Assay, Western Blot, Control

Journal: RSC Advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: TIMP3 inhibited the proliferation and migration of HaCaT cells under TGF-β1 treatment. HaCaT cells were treated with TGF-β1 (5 ng ml −1 ) or transfected with pcDNA or pcDNA-TIMP3 prior to TGF-β1 treatment. (A) and (B) qRT-PCR for miR-132 expression in treated cells. (C) and (D) MTT assay for the proliferation ability in treated cells. (E) and (F) Transwell assay for the migration capacity in treated cells. * P < 0.05 or ** P < 0.01 vs. control or TGF-β1 + pcDNA.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specific primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or β-actin (sc-517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 °C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Migration, Transfection, Quantitative RT-PCR, Expressing, MTT Assay, Transwell Assay, Control

Journal: RSC Advances

Article Title: Retracted Article: MiR-132 enhances proliferation and migration of HaCaT cells by targeting TIMP3

doi: 10.1039/c8ra10552a

Figure Lengend Snippet: MiR-132-mediated pro-proliferation and pro-migration effects were antagonized by TIMP3 in TGF-β1-treated HaCaT cells. HaCaT cells were transfected with miR-132 mimics + pcDNA, miR-132 mimics + pcDNA-TIMP3, anti-miR-132 + si-NC or anti-miR-132 + si-TIMP3 prior to TGF-β1 (5 ng ml −1 ) treatment, followed by the detection of cell proliferation ability by MTT assay (A) and (B), cell migration capacity by transwell assay (C) and (D). * P < 0.05 or ** P < 0.01 vs. control or TGF-β1 + miR-NC or TGF-β1 + miR-132 + pcDNA.

Article Snippet: Blocked by a buffer containing 5% non-fat milk in TBS with 0.1% Tween-20, the membrances were probed overnight with specific primary antibodies against TIMP3 (sc-373839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1 : 500) or β-actin (sc-517582, Santa Cruz Biotechnology; dilution 1 : 1000) at 4 °C, followed by the incubation with HRP-conjugated secondary antibodies (sc-516102, Santa Cruz Biotechnology; dilution 1 : 5000).

Techniques: Migration, Transfection, MTT Assay, Transwell Assay, Control