full length human mt2a expression vector  (OriGene)

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