Down-regulated NEDD4L facilitates tumor progression through activating Notch signaling in lung adenocarcinoma

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Lin et al. examine the correlation between NEDD4L level and clinical outcome in LUAD patients using the data from The #Cancer #Genome Atlas (TCGA) database Read the full article https://t.co/wy7wh5QFez #LungCancer #CellBiology
Lin et al. examine the correlation between NEDD4L level and clinical outcome in LUAD patients using the data from The #Cancer #Genome Atlas (TCGA) database Read the full article https://t.co/LfqMBWYCsd #LungCancer #CellBiology
Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials and Methods

LUAD tissues and cell lines

qRT-PCR

Western blot

Co-immunoprecipitation (Co-IP)

Cycloheximide (Chx) chase assay

Overexpression and RNA interference (RNAi) of NEDD4L

Cell proliferation assay

Colony formation assay

Xenograft mouse model

Statistical analysis

Results

Down-regulated NEDD4L was associated with poor prognosis in LUAD

NEDD4L negatively regulated LUAD cell proliferation in vitro and in vivo

NEDD4L promoted ubiquitination and proteasomal degradation of Notch2

Down-regulated NEDD4L contributed LUAD cell proliferation by activating Notch signaling

Discussion

Supplemental Information

Figure S1 raw data.

DOI: 10.7717/peerj.13402/supp-1

Figure S2 raw data.

DOI: 10.7717/peerj.13402/supp-2

Overexpression and knockdown of NEDD4L.

Recombinant plasmids (pcDNA-NEDD4L) were transfected into A549 cells, and then the mRNA (A) and protein (B) levels of NEDD4L were assessed using qRT-PCR and western blot analysis. siRNAs against NEDD4L (siNEDD4Ls) were transfected into H1299 cells, and then the mRNA (C) and protein (D) levels of NEDD4L were assessed using qRT-PCR and western blot analysis.

DOI: 10.7717/peerj.13402/supp-3

The association of NEDD4L with Notch pathway.

(A) qRT-PCR was carried out to assess NOTCH1-4 mRNA level in A549 cells after NEDD4L overexpression. Immunofluorescence analysis was carried out to assess the cellular localization of NEDD4L (B) and NOTCH2 (C) in A549 cells. (D) The mRNA levels of NOTCH2, NICH, Hes1, and Hey1 was assessed using qRT-PCR analysis in A549 cells after NEDD4L overexpression. **p < 0.01.

DOI: 10.7717/peerj.13402/supp-4

Figure 1 raw data.

DOI: 10.7717/peerj.13402/supp-6

Figure 2 raw data.

DOI: 10.7717/peerj.13402/supp-7

Figure 3 raw data.

DOI: 10.7717/peerj.13402/supp-8

Figure 4 raw data.

DOI: 10.7717/peerj.13402/supp-9

Figure 5 raw data.

DOI: 10.7717/peerj.13402/supp-10

Author checklist.

DOI: 10.7717/peerj.13402/supp-11

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Liping Lin conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Xuan Wu conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Yuanxue Jiang performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Caijiu Deng performed the experiments, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Xi Luo performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Jianjun Han performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Jiazhu Hu performed the experiments, prepared figures and/or tables, and approved the final draft.

Xiaolong Cao conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Animal Ethics

The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers):

The animal experiment protocol was approved by the Ethics Committee of Panyu Central Hospital (PYRC-2021-086, PYRC-2021-087).

Data Availability

The following information was supplied regarding data availability:

The raw measurements are available in the Supplemental Files.

Funding

This work was supported by Science and Technology Project of Guangzhou (No. 202002030085). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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