Inhibition of Connexin 43 reverses ox-LDL-mediated inhibition of autophagy in VSMC by inhibiting the PI3K/Akt/mTOR signaling pathway

View article
Biochemistry, Biophysics and Molecular Biology

Main article text

 

Introduction

Materials and Methods

Culture of thoracic aortic VSMC and study design

Reagents and antibodies

Western blot analysis

Immunofluorescence analysis

Quantitative real-time PCR

Transmission electron microscopy (TEM)

Oil red O staining

Statistical analysis

Results

Ox-LDL inhibited autophagy in VSMC via downregulation of autophagy-related protein

Ox-LDL increased Cx43 protein levels in VSMC

Involvement of Cx43 in VSMC autophagy and FCs formation mediated by Ox-LDL

Inhibition of Cx43 up-regulated the autophagy levels of VSMC after ox-LDL treatment via inhibiting the PI3K/AKT/mTOR signaling pathway

Discussion

Supplemental Information

Culture and identification of thoracic aortic VSMCs and construction of the FC model.

(A) Growth of SMCs. (a) At 4–7 days of culture, the cells climbed out from the sides of the blocks. (b) On the 15th day of culture, the cells covered the bottom of the dish. (c) The cells exhibited typical peak-valley growth patterns; scale bar: 100 μm. (B) The expression of α-SMA and Desmin on P3 and P6 VSMCs. (C and D) Statistical analysis of α-SMA and Desmin protein expression (n = 3, P = 0.2746 and 0.7226). (E) Expression and localization of α-SMA and Desmin in P3 and P6 VSMCs. Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. (F) Identification of the FC model. After ox-LDL intervention, the number of oil red O-positive cells in primary cultured VSMCs was significantly higher than that in the control group. The black arrow indicates the red-stained lipid droplets in the cytoplasm. Scale bar: 100 μm.

DOI: 10.7717/peerj.12969/supp-1

Statistics of results.

Mean, SD and confidence interval.

DOI: 10.7717/peerj.12969/supp-2

Raw data for western blotting.

DOI: 10.7717/peerj.12969/supp-3

Raw numeric data.

DOI: 10.7717/peerj.12969/supp-4

The ARRIVE guidelines 2.0: author checklist.

DOI: 10.7717/peerj.12969/supp-5

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Xuqing Qin conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Wenjun He performed the experiments, prepared figures and/or tables, and approved the final draft.

Rui Yang analyzed the data, prepared figures and/or tables, and approved the final draft.

Luqian Liu performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Yingying Zhang performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Li Li analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Junqiang Si analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.

Xinzhi Li conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, and approved the final draft.

Ketao Ma conceived and designed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the paper, 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 Medical Ethics Committee of Shihezi University approved the study (SCXK Xin (2018-001)).

Data Availability

The following information was supplied regarding data availability:

The raw data is available in the Supplemental File.

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 81860085, 81860286), Corps Science and Technology Cooperation Project of China (No. 2020BC004), Central Research Institute Fund of Chinese Academy of Medical Sciences (2020-PT330-003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

10 Citations 2,178 Views 167 Downloads