# Uncovering the Regulatory Role of Proteins in EBSS-Induced Autophagy Using RNA-Seq Analysis

## TL;DR

This study uses RNA-seq to identify proteins involved in autophagy triggered by EBSS, revealing TXNRD1 as a potential regulator.

## Contribution

The study identifies TXNRD1 as a novel regulatory protein in EBSS-induced autophagy using RNA-seq and experimental validation.

## Key findings

- TXNRD1 knockdown reduced LC3B-II levels, indicating a regulatory role in autophagy.
- RNA-seq analysis identified 470 candidate genes, with 27 selected for further validation.
- Protein–protein interaction networks and pathway analyses linked genes to autophagy.

## Abstract

EBSS is a classical autophagy inducer that provides a special culture environment lacking amino acids and serum, causing cell starvation. However, there are almost no relevant omics data related to EBSS-induced autophagy, and consequently, autophagy-regulatory proteins have not been fully elucidated. In this study, we selected EBSS-induced autophagy as our research object and uncovered proteins that have regulatory roles in this process using RNA-seq analysis. Through the combination of omics calculation and biological experiments, this study has revealed, for the first time, that TXNRD1 has a regulatory function for autophagy activation in EBSS-induced autophagy. Our research provides useful omics information that contributes to the clarification of the autophagy mechanism.

Earle’s balanced salt solution (EBSS) is a classical autophagy inducer that provides a special culture environment lacking amino acids and serum, causing cell starvation. However, the production of relevant omics data surrounding EBSS-induced autophagy is still in the early stage. The objective of this study was to identify new potential functional proteins in the autophagy process through omics analysis. We selected EBSS-induced autophagy as our research object and uncovered autophagy-regulatory proteins using RNA-seq analysis. Western blotting showed that EBSS increased LC3B-II protein levels in NRK cells, reaching the maximum amount at 2 h of culture. Then, we used next-generation sequencing to obtain quantified RNA-seq data from cells incubated with EBSS and the bowtie–tophat–cufflinks flow path to analyze the transcriptome data. Using significant differences in the FPKM values of genes in the treated group compared with those in the control group to indicate differential expression, 470 candidate genes were selected. Subsequently, GO and KEGG analyses of these genes were performed, revealing that most of these signaling pathways were closely associated with autophagy, and to better understand the potential functions and connections of these genes, protein–protein interaction networks were studied. Considering all the conclusions of the analysis, 27 candidate genes were selected for verification, where the knockdown of Txnrd1 decreased LC3B-II protein levels in NRK cells, consistent with the results of confocal experiments. In conclusion, we uncovered autophagy-regulatory proteins using RNA-seq analysis, with our results indicating that TXNRD1 may play a role in regulating EBSS-induced autophagy via an unknown pathway. We hope that our research can provide useful information for further autophagy omics research.