Journal: Toxicology

Article Title: Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

doi: 10.1016/j.tox.2012.12.010

Figure Lengend Snippet: Fig. 4. Ptaquiloside treatment increases Mt2 expression in splenic NK cells that was inhibited by selenium co-treatment. Splenic NK cells were isolated from mice treated with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg) by daily gavage for 14 days. The differential gene expression detected using microarray analysis (5 mice/group) was

Article Snippet: Next, splenic cell suspensions were prepared from the other six untreated mice, and the non-adherent cells were incubated or not with the TrueORFTM vector containing M. musculus Mt2 cDNA (OriGene) as described above.

Techniques: Expressing, Isolation, Gene Expression, Microarray

Journal: Toxicology

Article Title: Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

doi: 10.1016/j.tox.2012.12.010

Figure Lengend Snippet: Fig. 7. Overexpression of Mt2 and its role in splenic NK cells. (A) Note the reduction in the free zinc level in the Mt2-transfected NK cells (*p = 0.0343, Student’s t-test). (B) The NK cytotoxicity is also reduced in the cells overexpressing Mt2 (*p = 0.0260, Mann–Whitney test). The data represent the mean and the error, n = 6.

Article Snippet: Next, splenic cell suspensions were prepared from the other six untreated mice, and the non-adherent cells were incubated or not with the TrueORFTM vector containing M. musculus Mt2 cDNA (OriGene) as described above.

Techniques: Over Expression, Transfection, MANN-WHITNEY

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: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Identification of the KLK14 hydrolysis sites within the CleavEx proMMP protein. CleavEx proMMP fusion proteins were separated using SDS-PAGE and electrotransferred for N-terminal sequencing. Identified sequences are represented in the bold font and the underscore denotes where the location of the expected activation cleavage P1-P1′ in the proMMP-derived sequence.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 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-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Sequencing, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: N-terminal identification of KLK14-mediated processing of recombinant proMMPs. The KLK14 hydrolysis product sequences were analyzed by N-terminal sequencing using Edman degradation. The bold font denotes the amino acid sequences identified. The underscored residues represent changes to the native protein sequence, as reported by the manufacturer (R&D Systems, Abingdon, United Kingdom). KLK14 recognized the sequence 3-aa upstream of the native MMP17 activation site, likely because the native site was modified by the manufacturer. All residues are numbered according to the Uniprot reported sequence of the full-length proteins. Bands are labeled according to the notation explained at Figure 2 .

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 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-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Recombinant, Sequencing, Activation Assay, Modification, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Gelatin zymography of proMMPs by KLK14-mediated processing. Activation of proMMPs by KLK14 results in a fully functional mature enzyme. Each proMMP was incubated with the indicated concentrations of KLK14 for 1 h 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, Abingdon, United Kingdom)). KLK = kallikrein-related peptidase; MMP = matrix metalloproteinase.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 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-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

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

Journal: International Journal of Molecular Sciences

Article Title: Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)—A CleavEx Based Analysis

doi: 10.3390/ijms21124383

Figure Lengend Snippet: Primers used for generating the proMMP CleavEx fusion proteins using three consecutive PCRs.

Article Snippet: A total of 0.5 μg native proMMP2 (catalog no. 902-MP-010, R&D Systems, Abingdon, United Kingdom), 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-Aldrich, St. Louis, MO, USA) for 1 h at 37 °C in PBS.

Techniques: Sequencing

Journal: Frontiers in physiology

Article Title: Maternal Melatonin Therapy Rescues Prenatal Dexamethasone and Postnatal High-Fat Diet Induced Programmed Hypertension in Male Rat Offspring.

doi: 10.3389/fphys.2015.00377

Figure Lengend Snippet: FIGURE 5 | Representative western blots showing (A) melatonin receptor-1 (MT1) (37 kDa), and −2 (MT2) (40 kDa), and RORα (59 kDa) bands in control, prenatal dexamethasone exposure (DEX), postnatal high-fat diet (HF), prenatal DEX plus postnatal (DEX+HF), and melatonin treatment (DEX+HF+M) offspring rats at 16 weeks of age. Relative abundance of renal cortical (B) MT1, (C) MT2, and (D) RORα as quantified. (E) Effect of DEX, HF, and melatonin on renal melatonin level. N = 6/group. *P < 0.05 vs. control; #P < 0.05 vs. DEX; ‡P < 0.05 vs. DEX+HF.

Article Snippet: We used the following antibodies: a goat anti-rat MT1 antibody (1:1000, overnight incubation; Santa Cruz Biotechnology, Santa Cruz, CA, USA); a rabbit anti-rat MT2 antibody (1:1000, overnight incubation; Biorbyt, AllBio Science Inc., Taichung, Taiwan); and a rabbit anti-rat RORα antibody (1:2000, overnight incubation; Proteintech Group, Inc., Chicago, IL, USA).

Techniques: Western Blot, Control

Journal: Molecular Cancer

Article Title: Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

doi: 10.1186/1476-4598-5-69

Figure Lengend Snippet: HEK293 cells transfected with MT1-MMP cDNA invade 3D collagen gels in response to LPA . (A) Tumor cell lysates were prepared for Western blot analysis. Lysates were probed for MT1-MMP to assess protein expression in the four tumor cell lines. Lysates were probed for Actin as a loading control. (B) HEK293 cells were transfected with the pAdTrack-CMV plasmid as a control, or plasmids encoding MT1-MMP, MT2-MMP, or MT3-MMP cDNA 24 hours prior to placement in invasion assays. Cells were allowed to invade 2.0 mg/ml collagen gels in the presence or absence of 1 μM LPA. Data are expressed as mean numbers of invading cells per HPF (20×) (± S.D.) from a minimum of 20 fields. (C) Lysates from HEK293 cells transfected with cDNAs encoding the designated genes were prepared for Western blot analysis and probed for GFP, MT1-MMP, MT2-MMP, MT3-MMP, or Actin as a loading control. TRK = pAdTrack-CMV, MT1 = MT1-MMP, MT2 = MT2-MMP, MT3 = MT3-MMP.

Article Snippet: Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously [ ]. si GENOME SMART pool ® reagents were products of Dharmacon (Lafayette, CO).

Techniques: Transfection, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Toxicology

Article Title: Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

doi: 10.1016/j.tox.2012.12.010

Figure Lengend Snippet: Fig. 4. Ptaquiloside treatment increases Mt2 expression in splenic NK cells that was inhibited by selenium co-treatment. Splenic NK cells were isolated from mice treated with ptaquiloside (5.3 mg/kg) and/or selenium (1.3 mg/kg) by daily gavage for 14 days. The differential gene expression detected using microarray analysis (5 mice/group) was

Article Snippet: Briefly, 5 × 105 cells suspended in OptiMEM I (Invitrogen) were incubated with or without 0.2 g of TrueORFTM vector containing a Mus musculus Mt2 cDNA (OriGene) mixture with 0.5 l Lipofectamine (Invitrogen) per well at 37 ◦C in a humidified atmosphere at 5% CO2, following the manufacturer’s instructions.

Techniques: Expressing, Isolation, Gene Expression, Microarray

Journal: Toxicology

Article Title: Ptaquiloside reduces NK cell activities by enhancing metallothionein expression, which is prevented by selenium.

doi: 10.1016/j.tox.2012.12.010

Figure Lengend Snippet: Fig. 7. Overexpression of Mt2 and its role in splenic NK cells. (A) Note the reduction in the free zinc level in the Mt2-transfected NK cells (*p = 0.0343, Student’s t-test). (B) The NK cytotoxicity is also reduced in the cells overexpressing Mt2 (*p = 0.0260, Mann–Whitney test). The data represent the mean and the error, n = 6.

Article Snippet: Briefly, 5 × 105 cells suspended in OptiMEM I (Invitrogen) were incubated with or without 0.2 g of TrueORFTM vector containing a Mus musculus Mt2 cDNA (OriGene) mixture with 0.5 l Lipofectamine (Invitrogen) per well at 37 ◦C in a humidified atmosphere at 5% CO2, following the manufacturer’s instructions.

Techniques: Over Expression, Transfection, MANN-WHITNEY

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, Control, 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: 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: Knockdown, 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, Knockdown, 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, Knockdown, 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: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 1. Melatonin receptor (MT1 and MT2) overexpression prevented ventral spinal cord 4.1 (VSC4.1) motoneuron death. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h).(A) ApopTag assayshowingrepresentative imagesfromeach treatmentgroup.Arrowsindicateapoptoticcells. (B)Bargraphsindicating the percentage of apoptotic cells (ApopTag assay) and viability (trypan blue dye exclusion test) counted from each group. (C) Representative pictures show levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (D) Bar graphs indicating the changes in expression of MT1, MT2, and GPR50 over Con [Correction added on 8 Oct 2012, after first online publication: Fig. 1 Panel A has been corrected].

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 2. Overexpression of MT1 and MT2 increases calbindin D28K and parvalbumin for suppressing Ca2+ rise and calpain:calpastatin ratio in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G- protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overexpressed cells + 25 lm LGA (24 h exposure), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Determination of intracellular free Ca2+ levels at 24 h. (B) Western blot analysis to show levels of calbindin D28K, parvalbumin, calpain, calpastatin, and b-actin. (C) Bar graphs indicating the changes in expression of calbindin D28K and parvalbumin over Con. (D) Densi- tometric analysis showing the calpain:calpastatin ratio.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 3. Overexpression of MT1 and MT2 increase estrogen recep- tor (ERb:ERa) ratio and suppression inflammatory factors in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glu- tamate (LGA) (24 h), 100 nm melatonin (24 h pretreat- ment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50- overexpressed cells + 25 lm LGA (24 h). (A) Western blot anal- ysis to show levels of ERb, ERa, NF-jB, COX-2, and b-actin. (B) Densitometric analysis showing the ERb: ERa ratio. (C) Bar graphs indicating the changes in the expression of NF-jB and COX-2 over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 4 Overexpression of MT1 and MT2 increases survival and angiogenesic factors in ventral spinal cord 4.1 (VSC4.1) cells. Plas- mid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2– overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overex- pressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Western blot analysis to show levels of p-Akt, Bcl-2, p-Bad, Flk-1, Flt-1, VEGF, and b-actin. (B) Bar graphs indicating the changes in expression of p-Akt, Bcl-2, and p-Bad over Con. (C) Bar graphs indicating changes in the expression of Flk-1, Flt-1, and VEGF over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 5. Overexpression of MT1 and MT2 suppresses apoptotic pathways in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h). (A) Western blot analysis to show levels of Bax, Bcl-2, active caspase-9, active caspase-3, and b-actin. (B) Densitometric analysis showing the Bax:Bcl-2 ratio. (C) Bar graphs indicating the changes in expression of active caspase-9 and active caspase-3 over Con. (D) Colorimetric determination of caspase-9 and caspase-3 activities.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 6. Silencing MT1 and MT2 enhanced glutamate toxicity in ventral spinal cord 4.1 (VSC4.1) cells. We indicated the use of RNA interference (RNAi) to cause a decrease in expression. Treatment groups: control cells (Con), 25 lm L-glutamate (LGA) (24 h exposure), MT1-silenced cells + 25 lm LGA (24 h exposure); MT2-silenced cells + 25 lm LGA (24 h), MT1 + MT2-silenced cells + 25 lm LGA (24 h exposure); G-protein receptor 50 (GPR50) silenced cells + 25 lm LGA (24 h), 25 lm LGA (24 h), 25 lm LGA + 100 nm melatonin (24 h exposure), MT1-silenced cells + 25 lm LGA+ 100 nm melatonin (24h exposure), MT2 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), MT1 + MT2-silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), GPR50 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure). (A) Representative pictures showing levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (B) Bar graphs indicating the percentage of viability (trypan blue dye exclusion test) counted from each group. (C) Colorimetric determination of caspase-3 activity.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Expressing, Control, Western Blot, Activity Assay

Journal: bioRxiv

Article Title: X chromosome factor Kdm6a enhances memory independent of its demethylase function in the aging XY male brain

doi: 10.1101/2022.07.18.500498

Figure Lengend Snippet: (A) Hippocampal Kdm6a mRNA expression in aged XX and XY mice (age 20-35 months; n=10-13 mice per group, shown relative to XX mice). (B) Construct maps showing the mutations rendering dead the demethylase activity of Kdm6a (Kdm6a DeM-Dead) along with the tetrapeptide repeat (TPR) domain. (C) Experimental strategy of lentivirus-mediated overexpression of Kdm6a DeM-Dead in XY mouse primary cortical neurons. (D) Kdm6a mRNA levels in primary XY neurons transfected with lentivirus expressing control or Kdm6a DeM-dead, shown relative to control (n=3 wells per experimental group from 10 XY pups). (E) Experimental strategy of lentiviral injection followed by testing in behavioral tasks. (F) Kdm6a mRNA expression following lentiviral transfection of Kdm6a DeM-Dead measured in the dentate gyrus of the hippocampus, relative to XY controls (n=3 mice per group) *P < 0.05 (two-tailed t tests in A, D; one-tailed in F). Data are presented as means ± SEM.

Article Snippet: The Kdm6a Enzyme-Dead (Kdm6a DeM-dead) plasmid was purified and validated by Addgene (#40619) in which alanine (A) substitutions were made at histidine (H) 1146 [ ] and glutamic acid (E) 1148 [ ] of a sequence encoding protein, Kdm6a (NCBI Reference Sequence: NM_009483.2; 4275 bp).

Techniques: Expressing, Construct, Activity Assay, Over Expression, Transfection, Control, Injection, Two Tailed Test, One-tailed Test

Journal: bioRxiv

Article Title: X chromosome factor Kdm6a enhances memory independent of its demethylase function in the aging XY male brain

doi: 10.1101/2022.07.18.500498

Figure Lengend Snippet: (A) Diagram of the experimental strategy for cognitive testing in the elevated plus-maze, open field testing, and the Morris water maze in aged XY mice (age 17-20 months, n=13-14 mice per experimental group). (B) Percentage of time spent in the open arms of the elevated plus-maze during 10 min exploration period. (C) Total number of movements during exploration of the open field for 5 min. (D) Spatial learning curves (platform hidden) of aged mice, control or Kdm6a DeM-dead, in the Morris water maze. Overexpressing Kdm6a DeM-dead mRNA enhanced learning. Two-way ANOVA: treatment *P < 0.05. (E) Probe trial results 1 hour, 24 hours, and 48 hours after completion of hidden platform learning, indicating spatial memory, showed that Kdm6a DeM-dead overexpressing mice had attenuated spatial deficits measured by increased frequency of entries into the target zone, compared to control mice. Two-way ANOVA: treatment *P < 0.05; **P < 0.01 (Bonferroni-Holm). Data are presented as means + SEM.

Article Snippet: The Kdm6a Enzyme-Dead (Kdm6a DeM-dead) plasmid was purified and validated by Addgene (#40619) in which alanine (A) substitutions were made at histidine (H) 1146 [ ] and glutamic acid (E) 1148 [ ] of a sequence encoding protein, Kdm6a (NCBI Reference Sequence: NM_009483.2; 4275 bp).

Techniques: Control