Chemicals and reagents
Dulbecco's modified Eagle medium (DMEM), RPMI-1640, 0.25% trypsin, Ly294002, and fetal bovine serum (FBS) were obtained from ThermoFisher Scientific (Cleveland, OH, USA). MG132, S3I-201, DMSO, paraformaldehyde (PFA), lysogeny broth (LB), MS222, isopropyl β- d-1-thiogalactopyranoside (IPTG), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Merck, Darmstadt, Germany). Protein G Mag Sepharose, glutathione Sepharose 4B GST-tagged protein purification resin, Ni Sepharose high-performance histidine-tagged protein purification resin, and Amicon ultra-centrifugal filters were obtained from GE (Marlborough, MA, USA). Primers were purchased from Purigo Biotech (Taipei, Taiwan). KAPA HiFi polymerase chain reaction (PCR) kits were purchased from Roche (Wilmington, MA, USA). PolyJet was purchased from SignaGen Laboratories (Rockville, MD, USA). The luciferase assay reagent was purchased from Promega (Madison, WI, USA). Anti-UBE2S, anti-lamin-A, and anti-GAPDH antibodies were obtained from GeneTex (Irvine, TX, USA). The p65 antibody was acquired from Cell Signaling Technology (Danvers, MA, USA). Anti-IκBα, anti-phosphorylated (p)-IκBα, anti-matrix metalloproteinase (MMP)-9, anti-Twist, and anti-E-cadherin (E-Cad) antibodies were obtained from Abcam (Cambridge, UK). Anti-hemoagglutinin (HA), anti-flag, anti-His, and anti-β-glutathione S-transferase (GST) antibodies were purchased from Santa Cruz (Capitola, CA, USA).
Database
The survival rate of lung adenocarcinoma patients correlated with UBE2S mRNA was analyzed by GEPIA2 web site (http://gepia2.cancer-pku.cn) [35]. The protein level of UBE2S in lung cancer patients was obtained from staining with CAB015228 antibody in the Human Atlas Protein Database (https://www.proteinatlas.org) [36].
Cell culture
The H441, A549, PC9, and H460 human lung cancer cell lines were obtained as described previously [52]. PC-9 and H460 cells were incubated with RPMI-1640 medium containing 10% FBS (ThermoFisher Scientific) in a 5% CO2 atmosphere at 37 °C.
Plasmid construction
The pcDNA3-UBE2S-flag plasmid was obtained as described in our earlier report [28]. pCMV4-3 HA/IκBα was purchased from addgene (Cambridge, MA, USA). The coding region of IκBα was amplified by a KAPA HiFi PCR Kit (Roche) and cloned to the pcDNA3-HA vector as the pcDNA3-IkBa-HA plasmid. The pcDNA3-IkBa-flag plasmid was digested with BamHI and SalI and inserted into the PQE30 vector as the PQE30-IkBa plasmid. The pcDNA3-UBE2S-flag plasmid was digested with BamHI and XhoI and inserted into the pGEX-4T-2 vector as the pGEX-UBE2S plasmid. The following primer pairs were used for the PCR: IκBα-forward (5’-ATG TTC CAG GCG GCC G-3’) and IκBα-reverse (5’-TAA CGT CAG ACG CTG G-3’).
Cell viability assay
H460 and PC-9 cells (105 cells/well) were seeded in 48-well plates overnight. PC-9 cells were treated with DMSO, SC514, PS1145, and Ly294002 for 24 h. Cells were refreshed with culture medium with 5 mg/mL of MTT (Merck), and incubation continued at 37 °C for 3 h. The culture medium was removed, and 200 μl DMSO was added to resolve precipitation. The absorbance was measured at 570 nm using a Spark multimode microplate reader (TECAN, Männedorf, Switzerland).
Preparation of cell lysates
Cultured cells were washed three times with phosphate-buffered saline (PBS). Gold lysis buffer (20 mM Tris-HCl at pH 7.9, 1 mM EGTA, 0.8 % NaCl, 0.1 mM β-glycerylphosphate, 1 mM sodium pyrophosphate, 10 mM NaF, 1 mM Na4P2O7, 1 mM Na3VO4, 10% glycerol, 1% Triton X-100, 1 mM PMSF, 10 μg ⁄mL aprotinin, and 10 μg ⁄mL leupeptin) was used to lyse cells. The total cell lysate was collected by centrifugation at 14,000 g for 20 min at 4 °C and quantified by a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Cultured cells in 10-cm cultural plates were harvested, and nuclear extracts were collected using a mini-preparation method [53-56].
Western blotting
Protein samples were heated to 100 °C with sample buffer (250 mM Tris-HCl at pH 6.8, 10% sodium dodecylsulfate (SDS), 30% glycerol, 5% β-mercaptoethanol, and 0.02% bromophenol blue) for 5 min. SDS-polyacrylamide gel electrophoresis (PAGE) was used to separate proteins, which were then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 3% bovine serum albumin (BSA) for 30 min at room temperature, a specific primary antibody diluted in 3% BSA was incubated with the membrane on a shaker at 4 °C overnight. The membrane was washed with PBST (0.25% Tween-20 in PBS) three times, and the secondary antibody conjugated with horseradish peroxide (Millipore) was incubated for 1 h at room temperature. After washing with PBST three times, proteins were detected using an enhanced chemiluminescence (ECL) reagent kit (Millipore) followed by exposure to Amersham Imager 600 imagers (GE). Protein images were quantified using ImageJ software (http://rsb.info.nih.gov/ij/index.html, National Institutes of Health, Bethesda, MA, USA).
Immunostaining
COS-1 cells (105 cells/well) were seeded onto 12-well plates overnight. The pcDNA3-UBE2S-HA and pcDNA3-IkBa-flag plasmids were transfected into COS-1 cells by PolyJet (SignaGen) for 24 h. PFA at 4% was used to fix COS-1 cells for 20 min, and PBST (0.1% Triton X-100 in PBS) was used to permeabilize them for 10 min. After being washed with PBS three times and blocking with 3% BSA for 1 h at room temperature, anti-HA and anti-flag monoclonal antibodies were incubated at 4 °C overnight. Cy3 AffiniPure goat anti-mouse immunoglobulin G (IgG) was added, incubated for 30 min, and then stained with 0.1% DAPI for 5 min. Images were captured with an Olympus IX70-FLA inverted fluorescence microscope (Olympus, Tokyo, Japan) and SPOT system (Diagnostic Instruments, Sterling Heights, MI, USA).
Knockdown of UBE2S by small interfering (si)RNA
UBE2S and control siRNAs were purchased from ThermoFisher Scientific and prepared following the manufacturer’s instructions. In total, 106 cells were seeded onto six-well plates overnight and then transfected with 40 nM of siRNA using the GenMute siRNA transfection reagent (SignaGen Laboratories, Rockville, MD, USA) following the manufacturer’s instructions. All assays were performed 24 h after transfection.
Luciferase assay
Cells (105) were seeded onto 12-well plates overnight. The pGL3-5xκB-Luc and pcDNA3-UBE2S-flag plasmids were obtained as described in our earlier reports [28, 57]. The pGL3-Basic, pGL3-5xκB-Luc, control siRNA, UBE2S siRNA, and pcDNA3-UBE2S-flag plasmids were transfected into H460 and PC9 cells using the PolyJet transfection reagent (SignaGen Laboratories) according to the manufacturer’s instructions. Total cell lysates were collected at 48 h post-transfection. Luciferase activity was monitored with the Luciferase Assay Reagent (Promega) and detected by a Spark multimode microplate reader (Tecan, Mannedorf, Switzerland).
Expression and purification of GST-UBE2S and His-IκBα protein in Escherichia coli
PQE30-IκBα and pET4T2-UBE2S were transformed into E. coli DH5a strands and expressed by 0.2 M IPTG induction. Cells grown overnight were re-grown in 1/10 dilution of lysogeny broth (LB) for 1 h and inducted by 0.2 M IPTG for 3 h. Cells were collected by centrifugation and resuspended in cold GST buffer (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 1 mM EDTA, and a proteinase inhibitor cocktail) and His binding/wash buffer (0.5 M NaCl, 100 mM HEPES, 10 mM Imidazole at pH 8.0, 0.5% NP-40, 1 mM PMSF, and a proteinase inhibitor cocktail). Cells were processed by an Untrasonic Processors (Chrom Tech, Apple Valley, MN, USA) on ice. The lysate was centrifuged at 13,000 g for 10 min at 4 °C. The supernatant was applied to a glutathione Sepharose 4B GST-tagged protein purification resin (GE) and Ni Sepharose high-performance histidine-tagged protein purification resin (GE). After being washed with GST wash buffer (0.5% Triton X-100 and 1 mM EDTAU in PBS) and His binding/wash buffer, GST-UBE2S was re-suspended in PBS. His-IκBα proteins were eluted with elution buffer (100 mM HEPES and 0.5 M imidazole at pH 8.0), followed by changing the buffer to PBS by Amicon Ultra Centrifugal Filters (GE).
Pull-down and in vitro binding assays
For the pull-down assay, GST-UBE2S proteins on beads were added to the total lysate of H460 cancer cells and incubated at 4 °C overnight on a rolling shaker. After centrifugation at 500 g for 2 min and washing three times with PBS, the beads were resuspended in PBS and analyzed by Western blotting. The in vitro binding assay was performed by mixing equal volumes of GST-UBE2S and the IκBα protein to interact at 4 °C overnight on a rolling shaker. After centrifugation at 500 g for 2 min and washing three times with PBS, the beads were resuspended in PBS and analyzed by Western blotting.
Immunoprecipitation (IP)
Cultured cells in 10-cm plates were collected and treated with IP-lysis buffer (150 mM NaCl, 20 mM NaCl, 20 mM HEPES at pH 7.2, 10 mM NaF, 1 mM EDTA, 1% NP-40, 1 mM Na3V04, 1 mM PMSF, 1 DTT, and a proteinase inhibitor cocktail) for 10 min. Total cell lysate was collected by centrifugation at 13,000 rpm for 10 min. Pretreatment with protein G Mag Sepharose magnetic beads (GE) for 1 h was used to collect total proteins. An anti-UBE2S (GeneTex) or anti-IκBα (Abcam) monoclonal antibody was coated onto protein G Mag Sepharose magnetic beads (GE) for 1 h and washed with PBS three times. Coated protein G Mag Sepharose magnetic beads (GE) were added to total cell lysates, and the complex was pulled-down overnight. After washing with PBS three times, the protein G Mag Sepharose complex was immunoblotted with an anti-UBE2S (GeneTex) or anti-IκBα (Abcam) monoclonal antibody.
Cell migration assay
PC9 cells (5x105 cells/well) were plated onto six-well culture plates overnight in RPMI-1640 containing 10% FBS. Monolayer cells were scratched with a pipette tip. After washing with PBS, the monolayers were incubated at 37 °C for 24 h. Images of the monolayers were captured at 0 and 24 h using a phase-contrast Zeiss Axio Vert.A1 inverted microscope (Zeiss, Jena, Germany) and a Leadview 2800AM-FL camera (Leadview, Taipei, Taiwan). Migrating cells were calculated from triplicate determinations for each treatment group.
Zebrafish metastasis model
Zebrafish (Danio rerio) and embryos were maintained at 28 °C. All animal procedures were approved by the Institutional Animal Care and Use Committee or Panel (IACUC/IACUP) (protocol # LAC-2019-0355). The methods were carried out in accordance with the approved guidelines. The migration assay of cancer cells in zebrafish was analyzed following our earlier report [30, 44]. Briefly, GFP expressing PC9 cells were transfected with control or UBE2S siRNA for 24h. The 0.25 % trypsin (ThermoFisher) was used to detach cancer cells and following stained with 1 μM CM-DiI (ThermoFisher) for 20 min. After 3 times wash with PBS, tumor cells were diluted as 2x106 cells/mL with PBS. Tumor cells were microinjected into the yolk of 3 days post-fertilization (dpf) zebrafish larvae by IM300 Microinjector (Narishige, Tokyo, Japan) at 30 psi. Zebrafish larvae were then incubated at 28 °C for 1 h and then transferred to 32 °C for further incubation. The migrative tumor cells in zebrafish larvae were analyzed at 7 dpf zebrafish larvae using a phase-contrast Olympus IX70 microscope (Olympus, Tokyo, Japan) and a SPOT camera (Sterling Heights, MI, USA).
Statistical analysis
Three independent experiments were used to analyze the mean ± standard deviation (SD). Statistical significance was analyzed by a one-way analysis of variation (AVOVA) followed by Tukey’s test. Statistical significance was indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001.