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: PLoS ONE

Article Title: Systematic Analysis Reveals Elongation Factor 2 and α-Enolase as Novel Interaction Partners of AKT2

doi: 10.1371/journal.pone.0066045

Figure Lengend Snippet: A: Activity of immuno-captured α-enolase (ENO1) from HEK293T cells after stimulation with insulin and AKT-inhibition with CCT128930. Data represent means ± SD of n = 7 experiments. B: Detection of immuno-captured α-enolase after α-enolase activity assay. After detection of α-enolase blots were incubated with anti-pan AKT antibody. Note that AKT was also detected after isolation of α-enolase by the α-enolase antibody.

Article Snippet: Primary Antibodies (Cell Signaling Technology, abcam): AKT2 Mouse mAB (5239), eEF2-Antibody (2332), GAPDH Rabbit mAB (2118), AKT1 Mouse mAB (2967), anti-ENO1-antibody (ab85086).

Techniques: Activity Assay, Inhibition, Incubation, Isolation

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