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Proteomics analysis of differentially expressed proteins by p53 status and cholesterol concentration of culture medium in on-top 3D culture using MDA-MB-231

ABSTRACT: Tumor cells had p53 knocked down by CRISPR/Cas9. In addition, Doxycycline-induced p53WT or p53R280K was incorporated into the p53KO cells by the Tet-on system. Furthermore, we created a high cholesterol condition in that Cholesterol was added to the culture medium while p53R280K was being expressed, and a low cholesterol condition in which Cholesterol was reduced by reducing serum.　The cells were cultured for 7 days in on-top 3D with respect to these four conditions. Whole cell lysates were processed for non-targeted proteomic analysis.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Tomoaki Tanaka

PROVIDER: PXD054373 | JPOST Repository | Sun Sep 14 00:00:00 BST 2025

REPOSITORIES: jPOST

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			Action	DRS
	NA-09_p53OK_Tet-on_p53R280K_Normal_1.raw	Raw
	NA-10_p53OK_Tet-on_p53R280K_Normal_2.raw	Raw
	NA-11_p53OK_Tet-on_p53WT_Normal_1.raw	Raw
	NA-12_p53OK_Tet-on_p53WT_Normal_2.raw	Raw
	NA-15_p53OK_Tet-on_p53R280K_Low-Choleterol_1.raw	Raw

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Project description:Derivation and maintenance of pluripotent stem cells (PSCs) including embryonic stem cells(ESCs) and (iPSCs) usually requires optimization of complex culture media, which hinders the generation of PSCs from various species. Expression of Oct4, Sox2, Klf4 and c-Myc (OSKM) can reprogram somatic cells into iPSCs, even for species possessing no optimal culture condition. Here we explored whether expression of OSKM could induce and maintain pluripotency without specific growth factors and signaling inhibitors. Tet-On-OSKM/Oct4-GFP Mouse ESCs (mESCs) were derived from the embryos obtained after mating of Tet-On-OSKM mouse and Oct4-GFP mouse under the 2iL ( N2B27 plus MEK inhibitor PD0325901, GSK3β inhibitor ChIR99021, and leukemia inhibitory factor) condition. Tet-On-OSKM mouse was Gt(ROSA)26Sor tm1(rtTA*M2)Jae Col1a1 tm3(tetO-Pou5f1,-Sox2,-Klf4,-Myc)Jae/J(The Jackson Laboratory,011004), and Oct4-GFP mouse was B6;CBA-Tg(Pou5f1-EGFP)2Mnn/J (The Jackson Laboratory,004654).The culture medium of Tet-On-OSKM/Oct4-GFP mouse ESCs was switched from 2iL to N2B27 plus Doxycycline medium (OSKM medium), under which OSKM trangene was activated. These above cells switched from 2iL to OSKM medium were called OSKM-ESCs. Meanwhile 2iL-ESCs (continuely cultured in 2iL) served as the positive control group. 2iL-ESCs and OSKM-ESCs collected at selective days (d18, 25 and 38) after medium switch were collected for RNA-Seq. Moreover, Tet-On-OSKM mouse embryonic fibroblast (MEF) cells were reprogrammed under OSKM medium (N2B27 plus Doxycycline) and 2iL medium, respectively. The resulting OSKM-iPSCs were reprogrammed and cultured under OSKM medium.The 2iL-iPSCs were reprogrammed under 2iL plus Doxycycline medium and cultured under 2iL medium (without Doxycycline)once clones were picked up.RNA-Seq of these above samples was conducted to analyze gene expression. We found that via continuous expression of OSKM,OSKM-ESCs and OSKM-iPSCs propagated stably, expressed pluripotency marker genes, and formed three germ layers in teratomas. Importantly, OSKM-iPSCs could produce gene-modified animals through germline transmission.Transcriptional landscapes of OSKM-iPSCs resembled 2iL-ESCs, and were more similar to those of ESCs cultured in serum/LIF.

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Proteomics analysis of differentially expressed proteins by p53 status and cholesterol concentration of culture medium in on-top 3D culture using MDA-MB-231

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