got1 expression vector  (Sino Biological)

Journal: Journal of Cancer

Article Title: Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells survival under glucose deprivation

doi: 10.7150/jca.64195

Figure Lengend Snippet: Glutaminolysis related enzymes served different roles in response to different glucose conditions. a-b , The GOT1 protein levels ( a ) and GDH1 protein levels ( b ) in SK-Hep-1 and PLC/PRF/5 cells transfected with shRNAs were detected by Western blot. β-actin was used as an internal control. ( c ) The effect of GOT1 knockdown on cell viability in high glucose (25mM) medium was detected by CCK-8 assay. * P < 0.05 , vs. shCont. d, The effect of GOT1 knockdown on cells colonies formation viability in high glucose (25mM) medium was detected by colony formation assay. Newly formed colonies in each well were counted and expressed as a percentage relative to the control group. * P < 0.05 , vs. shCont. e , The effect of GDH1 deficiency on cell viability in low glucose (1.0mM) medium was detected by CCK-8 assay. * P < 0.05, vs. shCont. f , The efficacy of silencing GOT1 in colony-formation viability of HCC cells in low glucose (1.0mM) medium was detected by colony formation assay. Newly formed colonies in each well were counted and expressed as a percentage relative to the control group. * P < 0.05 , vs. shCont. Results were expressed as the average of three independent experiments (n=3 per group).

Article Snippet: GOT1 expression vector (#HG14196-CM) were obtained from Sino Biological Inc. (Beijing, China).

Techniques: Transfection, Western Blot, CCK-8 Assay, Colony Assay

Journal: Journal of Cancer

Article Title: Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells survival under glucose deprivation

doi: 10.7150/jca.64195

Figure Lengend Snippet: GDH1 was upregulated in glucose-poor liver cancer tumor, accompanying with decreased GOT1 . a , Relative glucose levels in 30 paired clinical liver cancer tissues. The central line represents the mean and the error bars indicate the standard deviation. ** P < 0.01 , vs. adjacent nontumoral tissue group. b , Images of Western blot of GDH1 and GOT1 protein levels in glucose-poor liver cancer specimens.

Article Snippet: GOT1 expression vector (#HG14196-CM) were obtained from Sino Biological Inc. (Beijing, China).

Techniques: Standard Deviation, Western Blot

Journal: Journal of Cancer

Article Title: Glutamate dehydrogenase 1 mediated glutaminolysis sustains HCC cells survival under glucose deprivation

doi: 10.7150/jca.64195

Figure Lengend Snippet: A schematic diagram of GDH1 and GOT1 mediated pathways in different glucose conditions in HCC.

Article Snippet: GOT1 expression vector (#HG14196-CM) were obtained from Sino Biological Inc. (Beijing, China).

Techniques:

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: Primers used in QPCR analysis of gene expression.

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Variant Assay

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: Impact of 1,25(OH)2D on Oxidative Stress in MCF10A-ras Epithelial Cells. MCF10A-ras cells were treated with vehicle or 1,25(OH)2D (10 nM). (A&B) The ratios of reduced to oxidized NADPH and GSH were measured after indicated treatment duration and are expressed relative to vehicle treated results of each time point. (C) Intracellular ROS was measured at time indicated and is expressed relative to vehicle treated cells. (D) Cell viability was quantified after a 24 h challenge with indicated H2O2 concentrations following treatment of cells with vehicle or 1,25(OH)2D for 96 h. (E) The percent decrease in cell viability in 1,25(OH)2D-treated MCF10A-ras cells is represented relative to vehicle treated cells at each indicated H2O2 concentration. Values are expressed as mean ± S.E.M. Asterisk indicates significant difference from vehicle (P < 0.05). Bars with different letters are significantly different (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: Impact of 1,25(OH)2D on Oxidative Stress in MCF10A-ErbB2 Epithelial Cells. MCF10A-ErbB2 cells were treated with vehicle or 1,25(OH)2D (10 nM) for 96 h and the ratios of reduced to oxidized NADPH and GSH were measured. The results are expressed relative to vehicle treated cells and as mean ± S.E.M. Asterisk (*) indicates significant difference from vehicle (P < 0.05) (2-tailed (A) and 1-tailed (B), respectively).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques:

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: 1,25(OH)2D-Mediated Regulation of the Pentose Phosphate Pathway. MCF10A-ras cells were treated with vehicle or 1,25(OH)2D (10 nM). (A) G6PD mRNA was measured after 48 h of treatment and is expressed relative to vehicle treated cells. (B) Proportion of PPP to glycolytic glucose flux was measured after 96 h of treatment. (C) Intracellular ribose-5-phosphate concentration was measured after 96 h of treatment and is expressed as ng/mg protein. (D) MCF10A-ras cells were treated with vehicle or 1,25(OH)2D for 96 h with a concurrent DHEA treatment for the last 24 h. ROS values are expressed relative to vehicle treated cells with no DHEA (0 μM). All values are presented as mean ± S.E.M. Asterisk (*) indicates significant difference from vehicle (P < 0.05). Bars with different letters are significantly different (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Concentration Assay

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: Effect of 1,25(OH)2D on GOT1 Expression. MCF10A-ras cells were treated with vehicle or 1,25(OH)2D (10 nM). (A) ME1 expression was measured after 96 h of treatment using qRT-PCR and normalized to 18S. (B) GOT1 mRNA expression was measured after indicated hours of treatment using qRT-PCR and normalized to 18S. (C) GOT1 protein expression was measured after indicated hours of treatment and normalized to actin. Values are presented relative to vehicle treated cells at each time point and representative images are shown for each time point. (D) GOT1 mRNA expression was measured after cell transfection with a pCMV6-Neo or GOT1 expressing plasmid and 96 h vehicle or 1,25(OH)2D treatment. (E) NEO and GOT1 transfected MCF10A-ras cells were treated with vehicle or 1,25(OH)2D for 96 h with a concurrent H2O2 challenge (100 μM) for the last 24 h. Values are expressed as mean ± S.E.M. Asterisk (*) indicates significant difference from vehicle treatment (P < 0.05). Bars with different letters are significantly different (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: 1,25(OH)2D-Mediated Effect on Redox Balance Through its Regulation of Mitochondrial Pyruvate Metabolism, (A) MCF10A-ras cells were pretreated with vehicle or 1,25(OH)2D (10 nM) for 72 h followed by treatment for an additional 24 h concurrently with H2O2 in the presence or absence of OAA (2 mM) and UK-5099 (25 μM). (B, C&D) Intracellular ROS, NADPH/NADP+ and GSH/GSSG ratios were measured after 96 h of treatment with vehicle or 1,25(OH)2D and a concurrent UK-5099 treatment for the last 24 h. Values are expressed as mean ± S.E.M. Asterisk indicates significant difference from vehicle treatment (P < 0.05). Bars with different letters are significantly different (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques:

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: 1,25(OH)2D-Mediated Effect on PC mRNA and Protein Expression. MCF10A-ras cells were treated with vehicle or 1,25(OH)2D (10 nM). (A) PC mRNA expression was measured after indicated hours of treatment using qRT-PCR and normalized to 18S. (B) PC protein expression was measured after indicated hours of treatment and normalized to actin. Values are presented relative to vehicle treated cells at each time point and representative images for each time point are provided. Values are expressed as mean ± S.E.M. (*) Asterisk indicates significant difference from vehicle treatment (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Expressing, Quantitative RT-PCR

Journal: Cancer letters

Article Title: Inhibition of pyruvate carboxylase by 1α,25-dihydroxyvitamin D promotes oxidative stress in early breast cancer progression

doi: 10.1016/j.canlet.2017.09.045

Figure Lengend Snippet: Effect of 1,25(OH)2D on oxidative stress under PC knockdown. Stably transfected MCF10A-ras cells expressing Dox inducible shRNA targeting PC were treated with vehicle or 1,25(OH)2D (10 nM) for 96 h with a concurrent Dox (10 μg/mL) treatment for the last 72 h (A) PC mRNA expression was measured after 96 h of treatment using qRT-PCR and normalized to 18S. (B) PC protein expression was measured after 96 h of treatment. Values were normalized to β-tubulin and are expressed relative to vehicle Dox(−) treated cells. Representative blot images are provided. (C–E) ROS levels, NADPH/NADP+ and GSH/GSSG ratios were measured after 96 h of treatment and are expressed relative to vehicle Dox(−) treated group. (F&G) Total GSSG and GSH levels are normalized to protein content. Values are expressed as mean ± S.E.M. (*) Asterisk indicates statistical significance relative to vehicle treated cells. Bars with different letters are statistically significant (P < 0.05).

Article Snippet: GOT1 overexpression MCF10A- ras cells were transfected with the pCMV6-Neo or pCMV6-GOT1 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_002079","term_id":"197304792","term_text":"NM_002079"}} NM_002079 ) expressing plasmid (Origene Technologies, Rockville, MD) using lipofectamine 2000 and Opti-MEM Reduced Serum Medium (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Techniques: Stable Transfection, Transfection, Expressing, shRNA, Quantitative RT-PCR