pbabe c myc zeo plasmid  (Addgene inc)

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: Figure 7. 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: Plasmid transfection, RNA interference, and ELISA The overexpression of c-Myc, Oct4, Sox2, Klf4, Eno1, vinculin, and Eef2 was conducted by transfecting plasmids (#17758, #19778, #26817, #26815, #27563, #58198; Addgene, Cambridge, MA, USA; and HG13966-G, Sino Biological, Wayne, PA, USA), while blank plasmids (FLAG-HA-pcDNA3.1; Addgene) were used as a control.

Techniques: Mass Spectrometry, Control, 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: Figure 8. 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: Plasmid transfection, RNA interference, and ELISA The overexpression of c-Myc, Oct4, Sox2, Klf4, Eno1, vinculin, and Eef2 was conducted by transfecting plasmids (#17758, #19778, #26817, #26815, #27563, #58198; Addgene, Cambridge, MA, USA; and HG13966-G, Sino Biological, Wayne, PA, USA), while blank plasmids (FLAG-HA-pcDNA3.1; Addgene) were used as a control.

Techniques: Over Expression, Recombinant, Control, 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: Figure 9. 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: Plasmid transfection, RNA interference, and ELISA The overexpression of c-Myc, Oct4, Sox2, Klf4, Eno1, vinculin, and Eef2 was conducted by transfecting plasmids (#17758, #19778, #26817, #26815, #27563, #58198; Addgene, Cambridge, MA, USA; and HG13966-G, Sino Biological, Wayne, PA, USA), while blank plasmids (FLAG-HA-pcDNA3.1; Addgene) were used as a control.

Techniques: Control, Recombinant, Over Expression, Derivative Assay