Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Top Candidate Proteins Involved in Oridonin-treated ESCC.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Variant Assay

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Gene Set Enrichment Analysis and western blotting assessed that oridonin inhibits LASP1 and PDLIM1 on ESCC. ( A ) Molecular function classification of the protein candidates involved in oridonin treatment. The top three are cadherin binding, cadherin binding involved in cell–cell adhesion, and heat shock protein binding. ( B ) The top three cellular component classifications are focal adhesion, cell-substrate junction, and cell cortex. ( C ) The top three biological processes are negative regulation of apoptotic signaling pathway, removal of superoxide radicals, and cellular response to oxygen radicals. ( D ) Protein expression of LASP1 and PDLIM1 under oridonin treatment decreased compared to untreated cell lysates. Hsp70 expression increased in oridonin-treated cell lysates compared to untreated while ENO1 decreased a little in treated TE-8.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Western Blot, Binding Assay, Protein Binding, Expressing

Journal: Molecules

Article Title: Oridonin Induces Apoptosis in Esophageal Squamous Cell Carcinoma by Inhibiting Cytoskeletal Protein LASP1 and PDLIM1

doi: 10.3390/molecules28020805

Figure Lengend Snippet: Interested Genes for Protein Assessment Involved in Oridonin Treatment.

Article Snippet: Membranes were blocked with 5% Beef Serum Albumin (BSA) and then incubated with primary antibodies: mouse Hsp70 antibody (mAb; Abcam, Boston, MA, USA), rabbit ENO1, LASP1, GAPDH antibodies (#3810 (pAb), #8636 (pAb), #2218 (mAb); cell signaling, Danvers, MA, USA), rabbit PDLIM1 antibody (ABclonal, Cambridge, MA, USA), and mouse β -Actin antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA).

Techniques: Binding Assay, Protein Binding

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Mediation of PKM2-dependent glycolytic and non-glycolytic pathways by ENO2 in head and neck cancer development

doi: 10.1186/s13046-022-02574-0

Figure Lengend Snippet: AP-III-a4 inhibits cell proliferation and glucose metabolism in HNSCC cells through suppressing ENO2-mediated downstream signaling. a Effect of AP-III-a4 treatment on ENO1 and ENO2 protein levels determined by western blotting. UM-1 and Cal27 cells were treated with AP-III-a4 at a dose range (0, 1, 2.5, 5µM) for 48 h. b - e Effect of AP-III-a4 treatment on ENO2-mediated downstream targets ( b ), cell proliferation ( c ), and intracellular levels of ATP ( d ) and glucose uptake ( e ). UM-1 and Cal27 cells were treated with or without 5µM AP-III-a4 for 48 h. * p <0.05; ** p <0.01

Article Snippet: ERK1/2, p-ERK1/2, AKT, p-AKT, P70S6K, p-P70S6K, cyclin D1 (CCND1), cyclin B, cyclin E, CDK2, CDK4, CDK6 and ENO1 antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Prediction of the tumor suppressors in CM by mass spectrometry-based whole-genome proteomics. The single and double asterisks indicate p < 0.05 and 0.01, respectively. CN = control, Oct4 = Oct4 plasmids, c-Myc = c-Myc plasmids, Sox2 = Sox2 plasmids, Klf4 = Klf4 plasmids, and CM = conditioned medium. ( A ) Summary list of the potential tumor suppressors by mass spectrometry-based whole-genome proteomics. ( B ) Enolase 1 (Eno1), Hsp90ab1 (HSP), Eef2, and vinculin (VCL) as 4 tumor-suppressor candidates based on MTT-based viability. ( C ) Upregulation of Eno1, Hsp90ab1, Eef2, VCL, p53, and Trail in 4T1.2 cell-derived CM with the overexpression of Oct4, c-Myc, and the treatment with OAC2. The overexpression of Sox2 and Klf4 did not alter their levels. ( D ) Alterations in the levels of Eno1 and Hsp90ab1 in Oct4 and c-Myc CM by ELISA. ( E ) Reduction in MTT-based viability of 4T1.2 cells by the treatment with Eno1 and/or Hsp90ab1 recombinant proteins. ( F ) Tumor selectivity from the MTT-based viability of tumor cells (4T1.2 mammary tumor cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells) and human epithelium cells (KTB34-hTERT and KTB22-hTERT). Tumor selectivity is defined as a ratio of (MTT-based reduction in tumor cells) to (MTT-based reduction in non-tumor cells).

Article Snippet: Antibodies against c-Myc, Oct4, Sox2, Klf4, Lrp5, Runx2, Snail, TGFβ, Eno1, Eef2, vinculin, β-catenin, p-Akt, Akt, p-NFkB p65, NFkB p65, p-ERK, ERK (Cell Signaling), MMP9, NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA), TRAIL (Novus, Centennial, CO, USA), p53 (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), Kdm3a (Thermo Fisher Scientific) and β-actin as a control (Sigma) were employed.

Techniques: Mass Spectrometry, Derivative Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Tumor-promoting effects by the overexpression of Eno1, Eef2, and VCL in 4T1.2 mammary tumor cells, and tumor-suppressing effects by the administration of their recombinant proteins. The double asterisk indicates p < 0.01. CN = control, Eno1 = enolase 1, VCL = vinculin, and Hsp = Hsp90ab1. ( A-C ) Elevation in EdU-based proliferation, transwell invasion, and the upregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail by the overexpression of Eno1, Eef2, and VCL in 4T1.2 tumor cells. ( D ) Decrease in EdU-based proliferation by the administration of Eno1, Eef2, and VCL recombinant proteins. ( E ) Reduction in transwell invasion by the administration of Eno1, Eef2, and VCL recombinant proteins. ( F ) Downregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail in 4T1.2 tumor cells by the administration of Eno1, Eef2, and VCL recombinant proteins. ( G ) Co-immunoprecipitation of CD44 by Eno1 in 4T1.2 cells. ( H-I ) Suppression of the reduction in MTT-based viability by Eno1 in response to the silencing of CD44. ( J ) Downregulation of Lrp5, Runx2, MMP9 and Snail in 4T1.2 cells by the administration of Eno1 recombinant proteins, and the suppression by the silencing of CD44. ( K-L ) Western blotting of wild-type and mutant CD44 proteins, which were pulled down by Halo-tagged Eno1 proteins. PD = pull-down assay, NC = negative control, pl = Eno1 or CD44 proteins from plasmid transfection, MT = mutant CD44 without a cytoplasmic domain, and WT = wild-type CD44.

Article Snippet: Antibodies against c-Myc, Oct4, Sox2, Klf4, Lrp5, Runx2, Snail, TGFβ, Eno1, Eef2, vinculin, β-catenin, p-Akt, Akt, p-NFkB p65, NFkB p65, p-ERK, ERK (Cell Signaling), MMP9, NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA), TRAIL (Novus, Centennial, CO, USA), p53 (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), Kdm3a (Thermo Fisher Scientific) and β-actin as a control (Sigma) were employed.

Techniques: Over Expression, Recombinant, Immunoprecipitation, Western Blot, Mutagenesis, Pull Down Assay, Negative Control, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Downregulation of PDL-1 and Kdm3a in 4T1.2 mammary tumor cells by Hsp90ab1, Eno1, Eef2, and VCL, and the suppression of the development of osteoclasts. CN = control, Hsp = Hsp90ab1, Eno1 = enolase 1, VCL = vinculin, Oct4 = Oct4 plasmids, and CM = conditioned medium. The double asterisk indicates p < 0.01. ( A ) Reduction in the size and weight of mammary tumors by the daily intravenous administration of 1 μg/mL Eno1. EO771 mammary tumor cells were inoculated into the mammary fat pad of C57BL/6 female mice (N = 10). (B) Reduction in p-AKT, NFkB p65, p-ERK, and TNFα in 4T1.2 cells in response to Eno1 recombinant proteins. ( C ) Reduction in PDL1 in 4T1.2 cells by Oct4/cMyc-overexpressing 4T1.2 CM, and Eno1, Hsp, Eef2, and VCL recombinant proteins. ( D ) Reduction in Kdm3a in 4T1.2 cells in response to Eno1, Hsp90ab1, Eef2, and/or VCL recombinant proteins. ( E ) Elevation of Kdm3a in 4T1.2 cells by the overexpression of Eno1, Eef2, and VCL. ( F ) Suppression of RANKL-stimulated osteoclast development by Oct4-overexpressing CM. ( G ) Reduction in the levels of cathepsin K and NFATc1 in RANKL-stimulated osteoclasts by Oct4-overexpressing CM. ( H ) Regulatory mechanism by the tumor-suppressive secretomes, which were derived from Oct4- and c-Myc-overexpressing tumor cells.

Article Snippet: Antibodies against c-Myc, Oct4, Sox2, Klf4, Lrp5, Runx2, Snail, TGFβ, Eno1, Eef2, vinculin, β-catenin, p-Akt, Akt, p-NFkB p65, NFkB p65, p-ERK, ERK (Cell Signaling), MMP9, NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA), TRAIL (Novus, Centennial, CO, USA), p53 (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), Kdm3a (Thermo Fisher Scientific) and β-actin as a control (Sigma) were employed.

Techniques: Recombinant, Over Expression, Derivative Assay

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Prediction of the tumor suppressors in CM by mass spectrometry-based whole-genome proteomics. The single and double asterisks indicate p < 0.05 and 0.01, respectively. CN = control, Oct4 = Oct4 plasmids, c-Myc = c-Myc plasmids, Sox2 = Sox2 plasmids, Klf4 = Klf4 plasmids, and CM = conditioned medium. ( A ) Summary list of the potential tumor suppressors by mass spectrometry-based whole-genome proteomics. ( B ) Enolase 1 (Eno1), Hsp90ab1 (HSP), Eef2, and vinculin (VCL) as 4 tumor-suppressor candidates based on MTT-based viability. ( C ) Upregulation of Eno1, Hsp90ab1, Eef2, VCL, p53, and Trail in 4T1.2 cell-derived CM with the overexpression of Oct4, c-Myc, and the treatment with OAC2. The overexpression of Sox2 and Klf4 did not alter their levels. ( D ) Alterations in the levels of Eno1 and Hsp90ab1 in Oct4 and c-Myc CM by ELISA. ( E ) Reduction in MTT-based viability of 4T1.2 cells by the treatment with Eno1 and/or Hsp90ab1 recombinant proteins. ( F ) Tumor selectivity from the MTT-based viability of tumor cells (4T1.2 mammary tumor cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells) and human epithelium cells (KTB34-hTERT and KTB22-hTERT). Tumor selectivity is defined as a ratio of (MTT-based reduction in tumor cells) to (MTT-based reduction in non-tumor cells).

Article Snippet: Protein samples were pretreated with agarose beads conjugated with protein A and rabbit IgG, followed by the overnight immunoprecipitation with the beads conjugated with anti-Eno1 antibodies (Cell Signaling).

Techniques: Mass Spectrometry, Derivative Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Tumor-promoting effects by the overexpression of Eno1, Eef2, and VCL in 4T1.2 mammary tumor cells, and tumor-suppressing effects by the administration of their recombinant proteins. The double asterisk indicates p < 0.01. CN = control, Eno1 = enolase 1, VCL = vinculin, and Hsp = Hsp90ab1. ( A-C ) Elevation in EdU-based proliferation, transwell invasion, and the upregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail by the overexpression of Eno1, Eef2, and VCL in 4T1.2 tumor cells. ( D ) Decrease in EdU-based proliferation by the administration of Eno1, Eef2, and VCL recombinant proteins. ( E ) Reduction in transwell invasion by the administration of Eno1, Eef2, and VCL recombinant proteins. ( F ) Downregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail in 4T1.2 tumor cells by the administration of Eno1, Eef2, and VCL recombinant proteins. ( G ) Co-immunoprecipitation of CD44 by Eno1 in 4T1.2 cells. ( H-I ) Suppression of the reduction in MTT-based viability by Eno1 in response to the silencing of CD44. ( J ) Downregulation of Lrp5, Runx2, MMP9 and Snail in 4T1.2 cells by the administration of Eno1 recombinant proteins, and the suppression by the silencing of CD44. ( K-L ) Western blotting of wild-type and mutant CD44 proteins, which were pulled down by Halo-tagged Eno1 proteins. PD = pull-down assay, NC = negative control, pl = Eno1 or CD44 proteins from plasmid transfection, MT = mutant CD44 without a cytoplasmic domain, and WT = wild-type CD44.

Article Snippet: Protein samples were pretreated with agarose beads conjugated with protein A and rabbit IgG, followed by the overnight immunoprecipitation with the beads conjugated with anti-Eno1 antibodies (Cell Signaling).

Techniques: Over Expression, Recombinant, Immunoprecipitation, Western Blot, Mutagenesis, Pull Down Assay, Negative Control, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Downregulation of PDL-1 and Kdm3a in 4T1.2 mammary tumor cells by Hsp90ab1, Eno1, Eef2, and VCL, and the suppression of the development of osteoclasts. CN = control, Hsp = Hsp90ab1, Eno1 = enolase 1, VCL = vinculin, Oct4 = Oct4 plasmids, and CM = conditioned medium. The double asterisk indicates p < 0.01. ( A ) Reduction in the size and weight of mammary tumors by the daily intravenous administration of 1 μg/mL Eno1. EO771 mammary tumor cells were inoculated into the mammary fat pad of C57BL/6 female mice (N = 10). (B) Reduction in p-AKT, NFkB p65, p-ERK, and TNFα in 4T1.2 cells in response to Eno1 recombinant proteins. ( C ) Reduction in PDL1 in 4T1.2 cells by Oct4/cMyc-overexpressing 4T1.2 CM, and Eno1, Hsp, Eef2, and VCL recombinant proteins. ( D ) Reduction in Kdm3a in 4T1.2 cells in response to Eno1, Hsp90ab1, Eef2, and/or VCL recombinant proteins. ( E ) Elevation of Kdm3a in 4T1.2 cells by the overexpression of Eno1, Eef2, and VCL. ( F ) Suppression of RANKL-stimulated osteoclast development by Oct4-overexpressing CM. ( G ) Reduction in the levels of cathepsin K and NFATc1 in RANKL-stimulated osteoclasts by Oct4-overexpressing CM. ( H ) Regulatory mechanism by the tumor-suppressive secretomes, which were derived from Oct4- and c-Myc-overexpressing tumor cells.

Article Snippet: Protein samples were pretreated with agarose beads conjugated with protein A and rabbit IgG, followed by the overnight immunoprecipitation with the beads conjugated with anti-Eno1 antibodies (Cell Signaling).

Techniques: Recombinant, Over Expression, Derivative Assay

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Prediction of the tumor suppressors in CM by mass spectrometry-based whole-genome proteomics. The single and double asterisks indicate p < 0.05 and 0.01, respectively. CN = control, Oct4 = Oct4 plasmids, c-Myc = c-Myc plasmids, Sox2 = Sox2 plasmids, Klf4 = Klf4 plasmids, and CM = conditioned medium. ( A ) Summary list of the potential tumor suppressors by mass spectrometry-based whole-genome proteomics. ( B ) Enolase 1 (Eno1), Hsp90ab1 (HSP), Eef2, and vinculin (VCL) as 4 tumor-suppressor candidates based on MTT-based viability. ( C ) Upregulation of Eno1, Hsp90ab1, Eef2, VCL, p53, and Trail in 4T1.2 cell-derived CM with the overexpression of Oct4, c-Myc, and the treatment with OAC2. The overexpression of Sox2 and Klf4 did not alter their levels. ( D ) Alterations in the levels of Eno1 and Hsp90ab1 in Oct4 and c-Myc CM by ELISA. ( E ) Reduction in MTT-based viability of 4T1.2 cells by the treatment with Eno1 and/or Hsp90ab1 recombinant proteins. ( F ) Tumor selectivity from the MTT-based viability of tumor cells (4T1.2 mammary tumor cells, EO771 mammary tumor cells, and MDA-MB-231 breast cancer cells) and human epithelium cells (KTB34-hTERT and KTB22-hTERT). Tumor selectivity is defined as a ratio of (MTT-based reduction in tumor cells) to (MTT-based reduction in non-tumor cells).

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Mass Spectrometry, Derivative Assay, Over Expression, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Tumor-promoting effects by the overexpression of Eno1, Eef2, and VCL in 4T1.2 mammary tumor cells, and tumor-suppressing effects by the administration of their recombinant proteins. The double asterisk indicates p < 0.01. CN = control, Eno1 = enolase 1, VCL = vinculin, and Hsp = Hsp90ab1. ( A-C ) Elevation in EdU-based proliferation, transwell invasion, and the upregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail by the overexpression of Eno1, Eef2, and VCL in 4T1.2 tumor cells. ( D ) Decrease in EdU-based proliferation by the administration of Eno1, Eef2, and VCL recombinant proteins. ( E ) Reduction in transwell invasion by the administration of Eno1, Eef2, and VCL recombinant proteins. ( F ) Downregulation of Lrp5, MMP9, Runx2, TGFβ, and Snail in 4T1.2 tumor cells by the administration of Eno1, Eef2, and VCL recombinant proteins. ( G ) Co-immunoprecipitation of CD44 by Eno1 in 4T1.2 cells. ( H-I ) Suppression of the reduction in MTT-based viability by Eno1 in response to the silencing of CD44. ( J ) Downregulation of Lrp5, Runx2, MMP9 and Snail in 4T1.2 cells by the administration of Eno1 recombinant proteins, and the suppression by the silencing of CD44. ( K-L ) Western blotting of wild-type and mutant CD44 proteins, which were pulled down by Halo-tagged Eno1 proteins. PD = pull-down assay, NC = negative control, pl = Eno1 or CD44 proteins from plasmid transfection, MT = mutant CD44 without a cytoplasmic domain, and WT = wild-type CD44.

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Over Expression, Recombinant, Immunoprecipitation, Western Blot, Mutagenesis, Pull Down Assay, Negative Control, Plasmid Preparation, Transfection

Journal: Theranostics

Article Title: Counterintuitive production of tumor-suppressive secretomes from Oct4- and c-Myc-overexpressing tumor cells and MSCs

doi: 10.7150/thno.70549

Figure Lengend Snippet: Downregulation of PDL-1 and Kdm3a in 4T1.2 mammary tumor cells by Hsp90ab1, Eno1, Eef2, and VCL, and the suppression of the development of osteoclasts. CN = control, Hsp = Hsp90ab1, Eno1 = enolase 1, VCL = vinculin, Oct4 = Oct4 plasmids, and CM = conditioned medium. The double asterisk indicates p < 0.01. ( A ) Reduction in the size and weight of mammary tumors by the daily intravenous administration of 1 μg/mL Eno1. EO771 mammary tumor cells were inoculated into the mammary fat pad of C57BL/6 female mice (N = 10). (B) Reduction in p-AKT, NFkB p65, p-ERK, and TNFα in 4T1.2 cells in response to Eno1 recombinant proteins. ( C ) Reduction in PDL1 in 4T1.2 cells by Oct4/cMyc-overexpressing 4T1.2 CM, and Eno1, Hsp, Eef2, and VCL recombinant proteins. ( D ) Reduction in Kdm3a in 4T1.2 cells in response to Eno1, Hsp90ab1, Eef2, and/or VCL recombinant proteins. ( E ) Elevation of Kdm3a in 4T1.2 cells by the overexpression of Eno1, Eef2, and VCL. ( F ) Suppression of RANKL-stimulated osteoclast development by Oct4-overexpressing CM. ( G ) Reduction in the levels of cathepsin K and NFATc1 in RANKL-stimulated osteoclasts by Oct4-overexpressing CM. ( H ) Regulatory mechanism by the tumor-suppressive secretomes, which were derived from Oct4- and c-Myc-overexpressing tumor cells.

Article Snippet: Western blotting was conducted using antibodies against Eno1 and CD44 (Cell Signaling).

Techniques: Recombinant, Over Expression, Derivative Assay

Journal: Medical Oncology (Northwood, London, England)

Article Title: GSK3 inhibitor suppresses cell growth and metabolic process in FLT3-ITD leukemia cells

doi: 10.1007/s12032-022-01899-2

Figure Lengend Snippet: BIO represses the altered metabolite level involved in glycolysis in MV4-11 cells. A Heat map showing metabonomics profiling of 61 metabolites. B Quantification of relatively level of B pyruvate and C ATP in MV4-11 cells vs. SHI-1, THP-1 and RS4;11 cells. The D pyruvate and E ATP levels following BIO treatment were determined with the pyruvate Assay Kit and ATP Assay Kit. The F mRNA and G protein expression of ENO1 were detected using RT-qPCR and Western blot assay with or without BIO incubation. * P < 0.05, ** P < 0.01

Article Snippet: The membranes were incubated with 3% bovine serum albumin for 2 h at room temperature (RT) and then incubated overnight at 4 °C with the following primary antibodies: anti-ENO1 (cell signaling technology, 3810), anti-β-Catenin (Cell Signaling Technology, 8480), anti-caspase3 (cell signaling technology, 14220S), anti-p21 (cell signaling technology, 2947S), anti-cyclin D2 (cell signaling technology, 3741S) and anti-GAPDH (cell signaling technology, 5174).

Techniques: Pyruvate Assay, ATP Assay, Expressing, Quantitative RT-PCR, Western Blot, Incubation