Plasmids and siRNAs
JUNB, RELB, MAFB, and MAFF cDNAs were generated from a human cDNA library and cloned into the pCMV-HA vector (Clontech, USA) at the EcoRI and KpnI sites, respectively. The primers used for polymerase chain reaction (PCR) amplification are listed in Supplementary Table S1. Synthetic siRNA oligonucleotides specific for regions in the human GCN2, JUNB, RELB, MAFB, MAFF, TOX2, FOXP2 and FOSL1 mRNAs were designed and synthesized by GenePharma (Shanghai, China). The sequences for successful gene knockdown are listed in Supplementary Table S2. CCD841 cells at 50–70% confluency were transfected with the various siRNA duplexes using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions.
Cell culture and treatments
Human colon epithelial CCD841 and bronchial epithelial BEAS-2B cells were originally obtained from the American Type Culture Collection (ATCC, USA). All cells were cultured and maintained in Dulbecco’s modified Eagle’s medium (high glucose, 4.5 g/L) supplemented with 10% fetal bovine serum, 50 µg/mL streptomycin, and 50 U/mL penicillin according to the ATCC instructions. Media containing complete amino acids (control) or deficient in all EAAs (leucine, valine, isoleucine, lysine, tryptophan, threonine, phenylalanine, methionine, and histidine) or a single EAA were prepared in regular fetal bovine serum-free Dulbecco’s modified Eagle’s medium. GCN2iB, GCN2-IN-1 (A-92) and GZD824 (MedChemExpress, USA) were dissolved in DMSO as stock solutions (final concentration was less than 0.5% solvent).
Mice, tissue preparation, and IEC isolation
All mice used in this study were of a C57BL/6J background and maintained under specific pathogen-free conditions. Male C57BL/6J wild-type (WT) mice were purchased from the Shanghai Laboratory Animal Co. Ltd (SLAC; Shanghai, China). Gcn2-floxed mice42 were intercrossed with Villin-cre mice (Shanghai Biomodel Organism Science & Technology Development Co.,Ltd., Shanghai, China) to generate the IEC-specific Gcn2 IKO mice. All mice were housed in laboratory cages at a temperature of 23 ± 3°C and humidity of 35 ± 5%, under a 12/12 h dark/light cycle, with free access to a regular chow diet (SLAC) and water.
The tissue samples from 10 organs (heart, liver, spleen, lung, kidney, pancreas, stomach, thigh muscle, subcutaneous white fat, and hypothalamus) along with jejunal, ileal, and colonic epithelial cells were freshly harvested from five wild-type mice, immediately frozen in liquid nitrogen, and stored at -80°C for ACE2 expression analysis. The jejunal, ileal, and colonic epithelial cells were isolated according to the method described in a previous study43. In brief, the jejunum, ileum, and colon were removed from the sacrificed mice and cut into 0.5 cm thin pieces, and these were then rinsed in cold phosphate-buffered saline to remove debris. Primary IECs were then isolated by incubating the pieces in phosphate-buffered saline containing 2 mmol/L dithiothreitol and 1 mmol/L ethylenediaminetetraacetic acid at 37°C for 30 min with gentle shaking.
RNA isolation and quantitative real-time polymerase chain reaction
Total RNA content was extracted from the tissue and cell samples using the TRIzol reagent (Invitrogen, USA) and treated with RNase-free DNase (MBI Fermentas, Germany). The extracted RNA was then reverse transcribed to cDNA using the RevertAid RT Kit (Invitrogen, USA) according to the manufacturer’s instructions. The quantitative real-time PCR was carried out using SYBR Green Master Mix (Invitrogen, USA) on QuantStudio 6 (Applied Biosystems, Foster City, CA, USA). The reaction was carried out according to the following conditions: predenaturation 5 min at 95°C, 95°C 10s followed by 60°C 30s for 40 cycles, melting curve stage 95°C 15s, 60°C 60s, and 95°C 15s. The sequences of primers used in this study are listed in Supplementary Table S3. All PCR amplifications were performed in triplicate for each RNA sample, and the expression level of each gene was quantified relative to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The results were analyzed using the 2 − ΔΔCt method.
RNA sequencing and data analysis
Total RNA content was extracted from CCD841 cells using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions, and the integrity and quality were checked using a NanoDrop 2000 spectrophotometer and formaldehyde agarose gel electrophoresis. Only RNA samples with a ratio of absorbance at 260 nm to that at 280 nm (A260 nm/A280 nm) greater than 1.8 were used. For RNA sequencing library synthesis, 3 µg of total RNA was first depleted of rRNA using the Ribo-Zero rRNA Magnetic Kit (Plant Seed/Root kit, Epicentre, Madison, USA). Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Sequencing was then performed on an Illumina HiSeq 2500 platform and 150 bp paired-end reads were generated.
Raw data (raw reads) of fastq format were first processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low-quality reads from raw data. The clean reads were aligned to the Ensembl 70 gene annotation of the NCBI38/mm10 genome using Bowtie with default parameters. FeatureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Differential expression analysis of two conditions/groups was performed using the DESeq2 R package44. DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq2 were assigned as differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was preformed to determine the biological functions of the differentially expressed genes using the online bioinformatics database DAVID (https://david.ncifcrf.gov/). Significant KEGG categories with a P value of less than 0.05 were selected. The accession number for the sequencing data reported in this paper is SRA: SUB10520187.
Western blot analysis
Protein samples were extracted from the samples using a protein lysis buffer [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.6), 1.5 mM MgCl2, 0.5 mM dithiothreitol, 10 mM KCl, 10 mM NaF, 1 mM Na3VO4, and 0.5 mM phenylmethylsulfonyl fluoride] supplemented with a protease inhibitor cocktail according to the manufacturer’s protocol. After determination of the protein concentration, the samples were subjected to western blot analysis using primary antibodies raised against ACE2 (Abclone, China), AXL, GCN2 and p-EIF2A (Cell Signaling Technology, USA), and beta-actin (Santa Cruz Biotechnology). The specific proteins were visualized by enhanced chemiluminescence using ECL Plus (Amersham Biosciences, UK). The band intensities were quantitated using Quantity One (Bio-Rad Laboratories) and normalized to that of beta-actin.
Statistical analysis
Statistical analysis of the data was performed using GraphPad Prism version 8.0 (San Diego, CA, USA). All values are presented as the means ± standard error of the means. Statistical comparisons were performed using the unpaired two-tailed Student t-test or one-way analysis of variance. Significant differences between data are indicated in the figures by *P < 0.05, ** P < 0.01.