Cell culture and transient transfection

NCI-H1299 and NCI-H1650 cell lines were purchased from Procell Life Science and Technology Co (Wuhan, China), whereas HBE, A549, and HCC827 cell lines were gifted by the Key Laboratory of Environment and Genes Related to Diseases of Xi’an Jiaotong University. NCI-H1299, NCI-H1650, HCC827, and HBE cells were cultured in RPMI-1640 medium (Biological Industries, Beit Haemek, Israel) containing 10% fetal bovine serum (FBS, Biological Industries, Beit Haemek, Israel), A549 cells were cultured in F-12 K medium (Hyclone, Thermo Co., USA) supplemented with 10% FBS. All cells were cultured in a humidified incubator at 37 ℃ with 5% CO2.

For vector and small interference RNAs (siRNA) transfection, jetPRIME reagent (polyplus-transfection SA) was used according to the manufacturer’s instructions. RPL11 overexpression vector were constructed by inserting the flag-conjugated RPL11 sequence into the GV141 vector. siRNA fragments of RPL11 (siRPL11) were synthesized by GenePharma (Shanghai, China). The siRPL11 sequences are si-1: Sense, 5’-GUAUGAUGGGAUCAUCCUU-3’, Antisense, 5’-AAGGAUGAUCCCAUCAUAC-3’; si-2: Sense, 5’-GCTUAGAUACACUGUCAGAU-3’, Antisense, 5’-AUCUGACAGUGUAUCUAGC-3’; si-3: Sense, 5’-GGUGCGGGAGUAUGAGUUA-3’, Antisense, 5’-UAACUCAUACUCCCGCACC-3’. The negative control siRNA (si-nc) sequences are Sense, 5’-UUCUCCGAACGUGUCACGU-3’, Antisense, 5’-ACGUGACACGUUCGGAGAA-3’.

Western blotting analysis

Proteins were isolated using a radio Immunoprecipitation assay buffer supplemented with a cocktail of protease and phosphatase inhibitors (TargetMol, MA, USA). Protein concentration was quantified by a BCA protein assay kit (Proteintech, Wuhan, China). Next, equivalent amounts of protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. For western blotting, the unbound sites of the membrane were blocked, and the membrane was sequentially incubated with primary and secondary antibodies and finally with ECL to detect the protein of interest and photographed using Syngene GBox (Syngene, Cambridge, UK). The primary and secondary antibodies used are list as follows: ribosmal protein L1 (RPL11) (Proteintech, 16277-1-AP), cyclin D1 (CCND1) (Proteintech, 26939-1-AP), heat shock protein family A (Hsp70) member 5 (HSPA5/BiP) (Proteintech, 66574-1-Ig), activating transcription factor 4 (ATF4) (Proteintech, 10835-1-AP), DNA damage inducible transcript 3 (DDIT3/CHOP) (Proteintech, 15204-1-AP), microtubule associated protein 1 light chain 3 beta (MAP1LC3B/LC3) (Proteintech, 14600-1-AP), beclin 1 (BECN1) (BOSTER, BM5181), sequestosome 1 (SQSTM1/p62) (BOSTER, M00300-1), CDK4 (Proteintech, 11026-1-AP), FLAG (Proteintech, 66008-3-1 g), phosphorylated endoplasmic reticulum to nucleus signaling 1 (phospho-ERN1/phospho-IRE1) (Affinity, AF7150), endoplasmic reticulum to nucleus signaling 1 (ERN1/IRE1) (Affinity, DF7709), actin beta (ACTB/β-actin) (Proteintech, 66009-1-Ig).

Reverse transcriptionquantitative (RT-q) polymerase chain reaction (PCR)

Total RNA was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing) were used to reverse transcribe and further examine the gene expression. The qPCR processes were as follows: 95 ˚C for 5 min; 95 ˚C for 10 s, 60 ˚C for 1 min, 72 ˚C for 30 s (40 cycles); and 94 ˚C for 90 s, 60 ˚C for 3 min gradully heating from 60 ˚C to 94 ˚C at a speed of 1 ˚C per minute. The relative gene expression levels were normalized to that of β-actin. The primer sequences were as follows:

RPL11 Forward: 5’AAAGGTGCGGGAGTATGAGTT-3’, Reverse: 5’TCCAGGCCGTAGATACCAATG-3’ ;

β-actin Forward: 5’CCAACCGCGAGAAGATGA-3’, Reverse: 5’CCAGAGGCGTACAGGGATAG-3’;

CCND1 Forward: 5’ATCTACACCGACAACTCCATC-3’, Reverse: 5’TGTTCTCCTCCGCCTCTG-3’;

CDK4 Forward: 5’ATGGCTACCTCTCGATATGAGC-3’, Reverse: 5’CATTGGGGACTCTCACACTCT-3’.

The above experiments followed the manufacturer’s protocol. The relative expression levels were calculated using the 2-∆∆Ct method.

Cell counting kit-8 (CCK-8) assay

Expression vectors or siRNAs pre-transfected cells were seeded into 96-well plates and incubated for 0 h, 24 h, 48 h, and 72 h. Then the CCK-8 reagent (TargetMol, USA) was used according to the manufacturer’s instructions to determine cell viablity in proliferation .

Clone formation assay

Pre-treated cells were seeded into 12-well plates at a density of 800 (A549) or 1000 (NCI-H1299) cells per well. After 8 days of culture, the cells were fixed using 4% paraformaldehyde, washed with phosphate-buffered saline (PBS), stained with 0.5% crystal violet(Sigma Aldrich, Merck KGaA), and imaged using a camera.

Scratch wound healing assay

Pre-transfected cells were seeded into 6-well plates, grown to 80-90% confluency, followed by a scratch made on the monolayer in the center with a 10 µL sterile pipette tip. The cells were washed with PBS and further cultured in 1% FBS-containing medium for 72 h. Magnified images of the cells were captured at 0 h, 24 h, 48h and 72 h after wounding (Nikon, Tokyo, Japan).

Transwell migration assay

Cell migration was examined in a 24-well cell culture transwell insert system. Transfected A549 and NCI-H1299 cells suspended in 200 µL serum-free culture medium were plated in the upper chamber and 600 µL complete culture medium were added in the lower chamber. After 48 h incubation, cells in the lower chamber were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 30 min. The images were captured using an inverted microscope (Nikon, Tokyo, Japan). For quantitative data, the imaged cells were further dissolved in dimethyl sulfoxide and absorbance at 570 nm was read on the SpectraMax 190 microplate reader ( MD, USA).

Cell cycle analysis

Pre-treated cells were trypsin-digested and centrifuged; The cell pellet was collected, washed with PBS, and fixed in ice-cold 70% ethanol overnight at 4 ℃. Next, the cells were further washed with PBS and stained with 50 µg/ml propidium iodide (Biosharp, China) for 30 min. Distribution of the cell-cycle stages was examined by flow cytometry (Syngene, USA).

Acidic vesicular organelle (AVO) staining.

For AVO staining, 48 h after transfection, cells were further incubated in 2 µg/mL acridine orange (Med Chem Express, USA) containing medium for 20 min. The cells were then imaged using fluorescence microscopy (Nikon, Tokyo, Japan).

Autophagy flux measurement

Autophagy flux assessment by the mRFP-GFP-LC3 method was performed to detect autophagy levels. For this, 24 h after transfection of the RPL11 overexpression vector (marked as RPL11) or its control vector, NSCLC cells were further infected with the mRFP-GFP-LC3-expressing adenovirus (Hanbio, China) for 48 h according to the manufacturer’s instruction. After that, the cells were imaged using a laser confocal microscope (Zeiss LSM800, Germany).

Chloroquine (CQ) and tauroursodeoxycholic acid (TUDCA) treatment

CQ (HY-17,589 A) and TUDCA (HY-19,696) were all purchased from MedChemExpress (Shanghai, China) and dissolved in DMSO. For cell treatment, CQ and TUDCA were all added 6 h after transfection at a concentration of 20 µM and 2 mM respectively.

Statistical analysis

Experiments were all performed in three independent replicates. Data was analyzed using GraphPad Prism software version 8.0 (GraphPad Software, San Diego, USA) and presented as the mean ± SD. Unpaired student’s t-test was used to evaluate the differences in the data between two groups. One-way analysis of variance (ANOVA) was used to evaluate the differences in that data of multiple-group. P < 0.05 were considered to be statistically significant.