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