Journal: Neural Development

Article Title: Meningeal cells and glia establish a permissive environment for axon regeneration after spinal cord injury in newts

doi: 10.1186/1749-8104-6-1

Figure Lengend Snippet: Table of antibodies

Article Snippet: Collagen XII (newt) , Mouse IgG1 mAb , DSHB, MT2 , 1/50, concentrate.

Techniques:

Journal: Environmental pollution (Barking, Essex : 1987)

Article Title: Biotransformation of graphene oxide within lung fluids could intensify its synergistic biotoxicity effect with cadmium by inhibiting cellular efflux of cadmium.

doi: 10.1016/j.envpol.2022.119421

Figure Lengend Snippet: Fig. 3. The cytotoxicity of Cd and intracellular Cd accumulation with or without GOs pre-treatment. (a) The cellular viability of BEAS-2B cells detected by CCK8 assay after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h (n = 5, * indicate P < 0.05, compared to the untreated group). (b) The heat map of the cell viability inspected by CCK8 assay for BEAS- 2B cells after being treated to Cd (1, 5, 10, 20, 25, 30 and 50 μM) for 24 h with or without pre-treatment of P-GO, A-GO, or G-GO at the dose of 10 μg/mL, respectively (n = 5). (c) Intracellular Cd mass quan tification by ICP-MS. BEAS-2B cells were pre-treated with P-GO, A-GO, G-GO at 10 μg/mL for 24 h, and then exposed to CdCl2 at 10 μM for 24 h (n = 5), *P < 0.05. (d) Western blot analysis of MT1M and MT2A protein expression levels in BEAS-2B cells exposed to either GOs (10 μg/mL) or Cd (10 μM) or a combina tion of both (GOs + Cd) for 24 h.

Article Snippet: Antibodies (Abs) used were as follows: anti-NRF2 Ab (1:2000 dilution, Proteintech, USA), anti-SOD1 Ab (1:5000 dilution, Proteintech, USA), anti-SOD2 Ab (1:3000 dilution, Proteintech, USA), anti-MT1M (1:500 dilution, Proteintech, USA), anti-MT2A Ab (1:2000 dilution, Affinity, USA), anti-Caspase-8 Ab (1:5000 dilution, Proteintech, USA), anti-Bax Ab (1:5000 dilution, Proteintech, USA), anti-Bcl-2 Ab (1:5000 dilution, Proteintech, USA), anti-RIP1 Ab (1:2500 dilution, Proteintech, USA), anti-RIP3 Ab (1:2500 dilution, Proteintech, USA), anti-ABCB1 Ab (1:5000 dilution, Proteintech, USA), anti-ABCC1 Ab (1:2500 dilution, Proteintech, USA), anti-ABCG2 Ab (1:1000 dilution, Proteintech, USA).

Techniques: CCK-8 Assay, Western Blot, Expressing

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP associates with ZO-1 in polarized MDCK cells. (A) Western blot analysis of HA, E-cadherin and Rho-GDI in biotinylated cell lysates from Mock, MT2-MMP (MT2FL) and MT2-MMPWK (MT2WK) stable MDCK transfectants pulled down with streptavidin beads; input, unbound and bound fractions are shown (Inp, Unb, Biot). (B) Western blot analysis of ZO-1 and HA in cell lysates from Mock, MT2-MMP and MT2-MMPWK stable MDCK transfectants pulled down with anti-HA antibody; IgG immunoprecipitates and whole lysates (Input) are also shown as controls. A blot of the input lanes after a longer exposure is also shown. (C) Representative maximal projections from apical and basolateral stacks of confocal sections from polarized MDCK transfectants stained for HA (MT2-MMP, green), ZO-1 (red) and nuclei (Hoechst, blue). (D) Orthogonal x–z views of 3D confocal image stacks from C. (E) Representative peak intensity profiles from x–z views of 3D confocal image stacks from C. Graph to the right shows the quantification of MT2-MMP/ZO-1 Pearson correlation coefficient in polarized MT2-FL and MT2-WK MDCK transfectants. Values are mean±s.e.m. n=40 cells per condition in two independent experiments; *P<0.05.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Western Blot, Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP overexpression induces aberrant apical epithelial cell accumulation in polarized MDCK monolayers. (A) Orthogonal x–z projections of 3D confocal image stacks of MDCK transfectants stained for F-actin (Phalloidin, gray), HA (MT2-MMP, green) and Hoechst (nuclei, blue). Scale bar: 10 μm. (B) Quantification of apical epithelial foci per field (left) and the percentage of foci having more than 8 nuclei (right). 10 fields were counted per condition in n=4 independent experiments. (C) Representative maximal projections are shown from subapical and complete stacks of confocal sections from polarized MDCK transfectants stained for E-cadherin (gray). The dotted yellow line marks apical foci. Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants. Data are represented as mean±s.e.m. and were tested by one-way ANOVA versus mock 1 followed by Dunnett's post-test in B and C. **P<0.01, ***P<0.001, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Over Expression, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Apical epithelial cell accumulation depends on MT2-MMP catalytic activity. (A) Representative maximal projections from confocal sections of polarized MDCK transfectants stained for E-cadherin (gray) in the presence of GM6001 (50 μM) or vehicle (DMSO). Orthogonal x–z views are shown below. (B) Line and bar graphs show E-cadherin peak and average intensity, respectively, around the junctions formed by MDCK transfectants treated as in A. Bar graph at the bottom shows the number of apical events on the polarized MDCK monolayer in the presence or absence of DMSO. In the middle and bottom graphs, the difference between mock DMSO and MT2 FL were significant with P<0.01 and P<0.0001, respectively. (C) Representative maximal projections are shown from confocal sections of polarized MDCK transfectants (mock, MT2 and MT2EA) stained for E-cadherin (gray). Orthogonal x–z views are shown to the right. (D) Line and bar graphs show E-cadherin peak and average mean fluorescence intensity (MFI), respectively, around the junctions formed by MDCK transfectants shown in C. Bar graph on the right shows the number of apical events occurring in polarized MDCK monolayers. Data are represented as mean± s.e.m. and were tested by one-way ANOVA followed by Sidak post-test in B. Dunnett's post-test was used in D. *P<0.05, **P<0.01, ****P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Activity Assay, Staining, Fluorescence

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: E-cadherin is cleaved by MT2-MMP after N445 in the EC5 loop. (A) In silico model of canine E-cadherin (green)/human MT2-MMP (blue) interactions in cis association at the plasma membrane; the catalytic MT2-MMP center and the E-cadherin peptide, GPIPEPRNMDFCQKNPQP, are shown in orange and red, respectively. (B) Scheme of E-cadherin structure with the peptide containing the predicted cleavage sites after N445 and N459 in the EC5 loop. (C) Representative extracted ion chromatograms of 3 independent experiments corresponding to the peptides detected following in in vitro digestion of the GPIPEPRNMDFCQKNPQP peptide in the absence or presence of the human MT2-MMP recombinant catalytic domain (rhMT2). (D) Western blot analysis of lysates recovered from MDCK transfectants cultured with different calcium concentrations. Results are representative of two independent experiments. (E) Representative orthogonal x–z views of confocal images for polarized MDCK transfectants co-immunostained for HA (MT2-MMP, green), E-cadherin (red) and nuclei (Hoechst, blue).

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: In Silico, Clinical Proteomics, Membrane, In Vitro, Recombinant, Western Blot, Cell Culture

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP disrupts apical E-cadherin-mediated signals. (A) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants stained for F-actin (Phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (B) Orthogonal x–z projections of 3D confocal image stacks are shown of polarized MDCK transfectants (mock and MT2-MMP) in the presence of 4-HAP (500 nM) or vehicle (DMSO) for 72 h and stained for F-actin (phalloidin, green), myosin IIB (red), and nuclei (Hoechst, blue). (C) Quantification of apical/total MFI of myosin IIB in polarized MDCK transfectants treated with 4-HAP (500 nM) or vehicle (DMSO); n=5 independent experiments. (D) Quantification of cell circularity in MDCK cells presented in C. 25 cells per field were counted in 2 images per condition in 6 independent experiments. (E) Quantification of the number of apical events on polarized MDCK cells presented in panel B. 10 fields were counted per condition in n=4 independent experiments. Difference between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.0001 and P<0.05, respectively. Data are represented as mean±s.e.m. and were tested by one-way ANOVA followed by Sidak post-test. *P<0.05, **P<0.01, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Staining

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: Mislocalization of pSrc in polarized MT2-MMP-MDCK cells contributes to apical cell accumulation. (A) Percentage of cells in G0/G1, S, and G2/M phases of the cell cycle analyzed by flow cytometry in propidium-iodide-stained MDCK transfectants after 72 h of serum deprivation. Means±s.e.m. are shown for 3 independent experiments. (B) Orthogonal x–z projections of 3D confocal image stacks of polarized MDCK transfectants (mock and MT2-MMP) stained for F-actin (Phalloidin, green), pSrc (red), and nuclei (Hoechst, blue). Representative peak intensity profiles are shown on the right for pSrc (red) and F-actin (green). (C) Bar graphs show the apical (left) and junctional (right) pSrc intensity, relative to total mean fluorescence intensity (MFI) in 6 independent experiments. (D) Number of apical events occurring in polarized MDCK cells treated with PP2 or vehicle (DMSO). 10 fields were counted per condition in 3 independent experiments. Differences between mock DMSO1 and MT2 FL1, and mock DMSO2 and MT2 FL2 were significant with P<0.001 and P<0.01, respectively. (E) Representative confocal images of 3D cysts formed by MDCK transfectants in Matrigel and stained for pSrc (green), F-actin (white), E-cadherin (red), and nuclei (Hoechst, blue). (F) Quantification of the percentage of lumenized cysts. Data are represented as the mean±s.e.m. and were tested by two-way ANOVA followed by Dunnett's post-test in A, two-tailed Welch-test comparison was used in B, and one-way ANOVA followed by Sidak post-test was used in C. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Flow Cytometry, Staining, Fluorescence, Two Tailed Test, Comparison

Journal: Journal of Cell Science

Article Title: E-cadherin cleavage by MT2-MMP regulates apical junctional signaling and epithelial homeostasis in the intestine

doi: 10.1242/jcs.203687

Figure Lengend Snippet: MT2-MMP deficiency alters junctional E-cadherin and leads to decreased 3D colon organoid formation ex vivo and smaller crypts in vivo. (A) Representative confocal images of MT2-MMP-silenced colon organoids stained for MT2-MMP (green; top). Graph shows the normalized MT2-MMP mean fluorescence intensity (MFI) in stained organoids 72 h after siRNA transfection (bottom), n=6 images per condition from 3 independent experiments; Mmp15 mRNA levels decreased ∼20% in silenced organoids. (B) Bright-field microscopy images of MT2-MMP-silenced colon organoids. Bar graph shows the percentage of organoid generation efficiency 48 h after siRNA transfection (right) in 3 independent experiments. (C) Representative confocal images of MT2-MMP-silenced colon organoids stained for nuclei (Hoechst/Ho, blue), F-actin (red), E-cadherin (green) and β-catenin (white); magnified views of E-cadherin and β-catenin staining are shown in insets. (D) Representative confocal images of colonic tissues recovered from wild-type or MT2-MMP-null mice, and stained for Ki67 (red) and nuclei (Hoechst, blue). On the right, graph shows the percentage of Ki67-positive cells per crypt. 9–15 crypts were quantified per condition in 3 images taken from 2 mice per genotype (top). (E) Quantification of the cumulative frequency of crypt length (left) and width (right). Data are represented as mean±s.e.m. and were tested by unpaired Student's t-test in A and B and by two-tailed Welch-test comparison in D and E. *P<0.05, **P<0.01, ***P<0.001; ns, not significant.

Article Snippet: Antibodies Antibodies used were against β-actin (Sigma-Aldrich, A5441), GST (Thermo Fisher Scientific, A5800), HA (Covance, MMS-101P), MT1-MMP (LEM2/63; Gálvez et al., 2001 ), MT2-MMP (R&D Systems, MAB9161; and a rabbit polyclonal antibody generated by our group at CNB, Madrid, Spain, directed against 16 aa of hMT2 DEPWTFSSTDLHGNNL), Tubulin (Sigma, T6074), pSrc (Cell Signaling, 2101), ZO-1 (Thermo Fisher Scientific, 40-2300), E-cadherin (Cell Signaling, 3195 and BD Biosciences, 610181), Hoechst 3342 (Thermo Fisher Scientific), Ki67 (Abcam, ab16667), Phalloidin 647 (Thermo Fisher Scientific, {"type":"entrez-nucleotide","attrs":{"text":"A22287","term_id":"641467"}} A22287 ), Myosin IIB (Santa Cruz Biotechnology, sc-15370), β-catenin (BD Biosciences, 610153), Rho-GDI (Santa Cruz Biotechnology, sc-360), Ezrin (Upstate, 07-130), EEA1 (Santa Cruz Biotechnology, sc-6415), TfR (Invitrogen, H68.4), HGS (Abcam, ab72053), TSG101 (Abcam, ab30871).

Techniques: Ex Vivo, In Vivo, Staining, Fluorescence, Transfection, Microscopy, Two Tailed Test, Comparison

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: Expression of MT2A in human bladder tissues and cells. ( A ) The expression of MT2A in bladder cells was determined through RT-qPCR assays (±SE, n = 3). The numbers of mRNA levels indicated the ratio of MT2A/β-Actin in relation to HBdEC cells. RT-qPCR assays using β-Actin ( B ) or 18S ( C ), respectively, as the internal control, was conducted to quantitatively analyze the MT2A mRNA levels in paired bladder cancer and normal tissues. The comparison of the MT2A expressions in normal bladder and cancer tissues was examined by box plot analysis ( n = 26). ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Expressing, Quantitative RT-PCR

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: MT2A modulates the heme oxygenase-1 and endogenous reactive oxygen species in bladder carcinoma cells. ( A ) Protein levels of MT2A and HO-1 after knockdown of MT2A in HT1376 (left) and T24 (right) cells were examined by immunoblot assays. The quantitative data are presented as the intensity of the protein bands of the target proteins/β-actin relative to the mock-knockdown cells (bottom). ( B ) Relative fold-induction mRNA levels of MT2A and HO-1 in HT_shMT2A and T24_shMT2A cells compared with HT_shCOL and T24_shCOL cells, respectively, determined by using RT-qPCR assays. ( C ) Relative luciferase activity of HO-1 reporter vector after co-transfection with various concentrations of MT2A expression vector in TSGH-8301(black bars) and HT1376 (white bars) cells (±SE, n = 6). The endogenous ROS levels in HT_shCOL, HT_shMT2A ( D ), T24_shCOL, and T24_shMT2A ( E ) cells were measured by flow cytometry. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Western Blot, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation, Cotransfection, Expressing, Flow Cytometry

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: MT2A enhances H 2 O 2 treatment-induced cell apoptosis in bladder carcinoma cells. Cell apoptosis was determined by the association of annexin V-FITC with PI staining. The fluorescence intensity of mock-knockdown (T24_shCOL) and MT2A knockdown (T24_shMT2A) cells after 500 μM of H 2 O 2 treatment for 3 h was determined by flow cytometry ( A ). The quantitative data were presented as the percentage of early apoptosis, late apoptosis and necrosis of cells after treatments as indicated in T24 cells ( B ). Flow cytometry was used to determine the fluorescence intensity of mock-overexpressed TSGH-8301 (TSGH-8301_DNA) and MT2A-overexpresssed TSGH-8301 (TSGH-8301-MT2A) after 500 μM of H 2 O 2 treatment for 3 h ( C ). The quantitative data were presented as the percentage of the early apoptosis, late apoptosis and necrosis of cells after treatments as indicated in TSGH-8301 cells ( D ). Fluorescence intensity of the annexin V-FITC is plotted on the x -axis, and the PI is plotted on the y -axis. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Staining, Fluorescence, Flow Cytometry

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: MT2A downregulated H 2 O 2 -induced ROS in bladder carcinoma HT1376 cells. ( A ) Protein levels of MT2A and HO-1 after overexpression of MT2A in HT1376 cells were examined by immunoblot assays (top). The quantitative data were presented as the intensity of the protein bands of the target proteins/β-actin relative to the mock-overexpressed cells (bottom). ( B ) Relative fold-induction of mRNA levels of MT2A and HO-1 in HT-MT2A cells compared with HT-DNA cells was evaluated by RT-qPCR assays. ROS levels ( C ) and quantitative data ( D ) of HT-DNA and HT-MT2A cells after treatment with or without H 2 O 2 were measured by flow cytometry. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Over Expression, Western Blot, Quantitative RT-PCR, Flow Cytometry

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: Caffeic acid phenethyl ester induces MT2A and HO-1 expressions to downregulate endogenous ROS in bladder carcinoma cells. ( A ) Protein levels of MT2A and HO-1 after CAPE treatments in T24_shCOL and T24_shMT2A cells were determined by immunoblot assays (left). The presented quantitative data were the intensity of the protein bands of the target proteins/β-actin relative to the mock-knockdown cells (right). The relative luciferase activity of HT1376 (black bars) and TSGH-8301 (white bars) cells were transfected with the human MT2A ( B ) or HO-1 ( C ) reporter vectors and treated with various concentrations of CAPE. Presented data were the mean percentage (±SE, n = 6) compared with the vehicle-treated cells. ( D ) Protein levels of MT2A and HO-1 after CAPE treatments in HT_shCOL and HT_shMT2A cells were examined by immunoblot assays (left). The quantitative values presented were the intensity of the protein bands of the target proteins/β-actin relative to the mock-overexpressed cells (right). ( E ) Endogenous ROS levels (left) and quantitative data (right) of T24_shCOL and T24_shMT2A after treatment with or without CAPE were determined by flow cytometry. ( F ) Endogenous ROS levels (left) and quantitative data (right) of HT_shCOL and HOT_shMT2A cells after treatment with or without CAPE were measured by flow cytometry. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Western Blot, Luciferase, Activity Assay, Transfection, Flow Cytometry

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: Modulating effect of MT2A on cellular proliferation and invasion in bladder carcinoma cells. The abilities of cellular proliferation in T24_shCOL, T24_shMT2A ( A ), HT_shCOL, HT_shMT2A ( B ), HT-DNA, HT-MT2A ( C ), TSGH-8310, and TSGH-8301-MT2A cells ( D ) were measured by flow cytometry using a Ki67 flow cytometry kit (±SE, n = 4). The cellular invasion ability was determined by in vitro Matrigel invasion assays. Data are presented as the mean percentage (±SE; n = 3) in relation to the ( E ) T24_shCOL, ( F ) HT_shCOL, or ( G ) TSGH-8301-DNA cells. The scale bar is 50 μm. ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Flow Cytometry, In Vitro

Journal: Antioxidants

Article Title: Metallothionein 2A with Antioxidant and Antitumor Activity Is Upregulated by Caffeic Acid Phenethyl Ester in Human Bladder Carcinoma Cells

doi: 10.3390/antiox11081509

Figure Lengend Snippet: Modulating effect of MT2A on tumor growth of bladder carcinoma HT1376 cells. Four-week-old male athymic nude mice were divided randomly into two groups. ( A ) HT_shCOL and HT_shMT2A cells (6 × 10 6 ) were injected subcutaneously in the dorsal area of the mice ( n = 8). ( B ) Tumors from HT_shCOL and HT_shMT2A cells were recorded after mice were sacrificed. The volumes of tumor ( C ) and body weight ( D ) were measured every 2–3 days during a period of 21 days. The ( E ) tumor weight (±SE; n = 8), mRNA levels (±SE; n = 8) of MT2A ( F ) and HO-1 ( G ), and ( H ) protein levels of MT2A and HO-1 (±SE; n = 8) of the tumors from HT_shCOL and HT_shMT2A cells were recorded after mice were sacrificed. * p < 0.05; ** p < 0.01.

Article Snippet: The full-length human MT2A expression vector (pCMV6-XL4-MT2A) was purchased from OriGene (SC125000; Rockville, MD, USA).

Techniques: Injection

Journal: bioRxiv

Article Title: Kallikrein-related peptidase 14 activates zymogens of membrane type matrix metalloproteinases (MT-MMPs) - a CleavEx library-based analysis

doi: 10.1101/2020.04.23.057109

Figure Lengend Snippet: Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for an hour at 37°C. The reaction was stopped by the addition of KLK14-specific inhibitors and the reaction mixture was analyzed by SDS-PAGE, followed by a zymogram with gelatin as a substrate. The proMMP2 (A) negative control was not activated. ProMMP14 (B) , proMMP15 (C) and proMMP16 (D) were activated whereas proMMP17 (E) did not show hydrolysis of gelatin, yet a shift corresponding to the loss of the profragment was observed (note that an amino acid substitution was introduced in proMMP17 by the manufacturer (R&D Systems)).

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D systems), 0.5 μg proMMP14 (catalog no. 918-MP-010, R&D systems), 1 μg proMMP15 (catalog no. 916-MP-010, R&D systems), 0.5 μg ProMMP16 (catalog no. 1785-MP-010, R&D systems) and 1 μg ProMMP17 (catalog no. 7796-MP-010, R&D systems) were separately incubated in 10 μl with a range of KLK14 concentrations (25-250 nM, with molar ratios from around 1:65 to 1:10 KLK14:MMP) in the presence of 5 μM batimastat (Sigma) for 1 hour at 37°C in PBS.

Techniques: Activation Assay, Functional Assay, Incubation, SDS Page, Negative Control