Cell culture
We prepared four cell lines (BeNa, Culture Collection, Beijing, China), BEAS-2B (The human bronchial epithelial cells, no.BNCC254518), H460 (The human large cell lung cancer cell line, no. BNCC233991), A549 (The human non-small-cell lung cancer cell lines, no. BNCC290808) and H1299 (The human non-small-cell lung cancer cell lines, no. BNCC334400), they cultured in F-12K medium (Hyclone, MA, USA) with 10% fetal bovine serum (Gibco, CA, USA) and kept under 37°C with 5% CO2. Follow-up experiments were performed when the cells were in the logarithmic phase of growth and 80% confluence.
IP/MS
The immunoprecipitation of RPL11 was performed as described previously [20]. Briefly, 10 µg of rabbit RPL11 antibodies (Abcam, Cambridge, UK) and the same amount of rabbit IgG (no. P2173, beyotime, Shanghai, China) were added in A549 cell lysate of the experimental and control groups, respectively, followed by overnight incubation at 4°C. After elution and purification, the immunoprecipitates were separated, then stained with silver. The immunoprecipitation was digested and then analyzed using an LC-MS/MS (TripleTOF, AB Sciex, Boston, MA, USA) instrument, and the results were evaluated. Credibility ≥ 95% and unique peptides ≥ 1 were the criteria to identify proteins [21].
Plasmids, drugs, antibodies, and siRNAs
His-tagged RPL11 expression plasmids were constructed by inserting the RPL11 cDNA into the pET32a His vector at NcoI and XhoI sites (GE Healthcare, Boston, MA, USA) (Supplementary File 1). The RPL11 cDNA was amplified using the following mRNA primers: 5′-GACGACGACAAGGCCATGGCTGCGCAGGATCAAGGTG-3′ and 5′-GTGGTGGTGGTGGTGCTCGAGTTTATTTGCCAGGAAGGATG-3′. A GST-RPS27a Escherichia coli expression vector was constructed by inserting the RPS27a cDNA into the pEGX.6P.1 vector at BamHI and XhoI sites (GE Healthcare) (Supplementary File 1). The RPS27a cDNA was amplified using the following mRNA primers: 5′-GTTCCAGGGGCCCCTGGGATCCATGCAGATTTTCGTGAAAAC-3′ and 5′-CAGTCACGATGCGGCCGCTCGAGTTACTTGTCTTCTGGTTTG-3′. The Flag-RPS27a and His-RPL11 were also generated using polymerase chain reaction (PCR) and cloned into the pIRES2-ZsGreen1 vector.
Lipo2000 (no. 11668019, Invitrogen, CA, USA) was used for RNAi experiments. β-actin (no. ab8227), RPS27a (no. ab74731), p53 (no. ab74731), p21 (no. ab109199) (Abcam, Cambridge, UK), and MDM2 (no. ab16895) (Genetex, NJ, USA) were used for immunoblotting (IB). MG132 (no. HY-13259) was purchased from Medchemexpress (NJ, USA), Doxorubicin (Dox, no. GC16994) and Actinomycin D (ActD, no. GC16866) were purchased from Glpbio (CA, USA). Three different siRNAs of each gene was synthesized by Genepharma (St. Louis, Shanghai, China); their sequences were shown in Supplementary File 2.
GST-fusion assay
His-tagged RPL11 expression plasmids were transfected in Escherichia coli BL21. The His-tagged RPL11 was purified using an Ni2 +-NTA column (Qiagen, Shanghai, China) after expression in E. coli. The GST-fusion assay was conducted as previously described. Then, 50 µg GST-RPS27a or GST was mixed with glutathione Sepharose 4B beads (Sigma) and incubated with 20 µg purified His-RPL11 proteins. anti-S-Tag and GST antibodies were used to analyze protein interactions by IB [22].
Transient transfection, IB, and co-immunoprecipitation (co-IP) analyses
A549 cells were inoculated in six-well plates and cultured in complete medium. When the cells grew to 70% confluence, the cells were transfected with siRNA. The transfected cells for 48 h were collected and treated with lysis buffer. Equal amounts of clear cell lysate (50 mg) were used for IB and co-IP analyses [23].
Immunofluorescence
The immunofluorescence assay was performed as described previously [20]. Cells were then scanned and analyzed using a confocal laser microscope (LSM, Carl Zeiss AG, Germany).
Cell cycle and apoptosis analysis
After 48 h of transfection, the analysis of cell cycle and apoptosis was performed as
described previously [21, 22]. Data of DNA content were collected using CellQuest and analyzed using a ModFit software program. The apoptotic cells were analyzed with a Flowsight imaging flow cytometer (Amnis/Merck Millipore, Darmstadt, Germany).
In vitro ubiquitination assay
In vitro ubiquitination experiments refer to previous studies using Ni2 +-NTA purification method [24]. The Ni2+-NTA pulldown used for ubiquitination experiment, the bead-bound proteins were analyzed using IB.
Reverse transcription and quantitative PCR analyses
TaKaRa company designed and synthesized primers (Dalian, China), andtwo-step
SYBR kit to complete real-time fluorescent quantitative PCR on the FTC-3000 qPCR system (Stratagene, California, USA). The primers used were 5′- AGAAGAAGTCTTACACCACTCCC-3′ and 5′- TGCCATAAACACCCCAGC-3′ (RPS27a); 5′-TCCACTGCACAGTTCGAGGG-3′ and 5′-AAACCTGGCCTACCCAGCAC-3′ (RPL11); 5′- CGACTGTGATGCGCTAATGG-3′ and 5′-AAATCTGTCAGGCTGGTCTGC-3′ (p21); 5′-CTCACCATCATCACACTGGAA-3′ and 5′-TCATTCAGCTCTCGG AACATC-3′ (p53); 5′-AATCATCGGACTCAGGTACATC-3′ and 5′-CTGCTACTGCTTCTTTCACAAC-3′ (MDM2); 5′-TCAAGAAGGTGGTGAAGCAGG-3′ and 5′-TCAAAGGTGGAGGAGTGGGT − 3′ (GAPDH) [25].
Dissociation of ribosomal subunits and measurement of the subunit ratio
The dissociation of ribosomal subunits was performed as described previously [26]. Samples measured at 254 nm absorbance (Biocomp, CA) and quantitative analysis of ribosome peaks. The area under the curve from the lowest point to the lowest point of the 40S, 60S, and 80S peaks was calculated by summing the digital measurements.
Stably knockdown of RPS27a cells constructed
A stable knockdown of the RPS27a (RPS27a knockdown) cell line was achieved by lentiviral infection and drug screening. In brief, a 7.5 × 104 cells/mL suspension was made from A549 cells, and 2 mL of the suspension per well was seeded in a six-well plate. The virus was added 20 h after seeding the cells. The fresh medium was replaced with 2 µg/mL puromycin every 2–3 days..
Transwell cell invasion assay
Transwell chambers (Costar, USA were used for Transwell invasion assays, as previously described [27]. The IC50 of doxorubicin (Dox) on A549 cells and its effect on the survival of A549 cell clones are shown in Supplementary File 3. The incubation was continued for 24 h. The cells were fixed with 4% paraformaldehyde and stained with crystal violet solution..
Cell viability assay
The cell counting kit 8 (CCK-8, B34304, Bimake, USA) was used to detect cell proliferation. A microplate reader (Tecan M200, Switzerland) was used to measure the absorbance at a wavelength of 450 nm. The formula, Cell viability (%) = [A (compound +)–A (blank)]/[A (compound–)–A (blank)] × 100% was used to calculate the growth percentage. Cell viability means cell proliferation [27].
Colony-formation assay
The colony-formation assay of A549 cells was performed as described previously [28]. The colony formation rate was calculated by dividing the number of colonies/number of cells × 100% by the control.
Docking analysis
Briefly, the protein-protein interaction module of Schrodinger software (Schrodinger 2015 suit) was used for analyzing RPL11 and PRS27a interactions. The three-dimensional crystal structures of the human 80S ribosome (PDBID: 4v6x) were extracted from the PDB database. The small-molecule 3D structures were docked from the x-ray crystal structure of RPL11 and PRS27a, and two proteins were extracted from the 80S ribosome. The ubiquitin and water molecules were removed from the two protein structures to simulate the interaction [29].
Tumor xenografts
The management and handling of animals complied with the administrative regulations of the Laboratory Animal Affairs Administration of the Ministry of Science and Technology of China (1988.11.14). The research on experimental animals was approved by the Ethics Committee of the Institute of Modern Physics, Chinese Academy of Sciences, and the Institutional Animal Care and Use Committee. Four-five-old nude mice (female, weight 16–17 g, SPF level) were obtained from the Laboratory Animal Center of obio biotechnology (Shanghai, China). NC and RPS27a knockdown cells in the logarithmic growth phase were injected (2 × 106) subcutaneously into mice to establish a cell xenograft model. Based on the equation of the flat spheroid [tumor volume = (short diameter)2 × large diameter × π/6], the average tumor volume of each group was calculated and expressed in mm3 [30].
Immunohistochemistry analysis
The LUAD samples and xenograft tumors were analyzed by immunohistochemistry was performed as described previously [30]. The sections were examined with an 80i Nikon optical microscope (Nikon, Tokyo, Japan). Image-Pro Plus software was also used to analyze the optical density of protein expression.
Statistical analysis
Prism 8 software (GraphPad Software, CA, USA) was used to analyze the data. Statistical differences were analyzed based on the Student's t test and on one-way analysis of variance test. p value of < 0.05 was represented statistically significant.