Cell Culture, Patient Samples and Transfection
Human embryonic kidney 293T (HEK293T) cells were procured from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM medium (#SH30243, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (#SH30070, Hyclone, Logan, UT, USA) and 1% antibiotic/antimycotic (#SV30010, Hyclone, Logan, UT, USA) at 370C with 5% CO2. All lung adenocarcinoma lines were procured from ATCC and cultured in RPMI (#SH30027, Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic/antimycotic. siRNA transfections were performed as described previously [17]. Human primary tumor and adjacent normal lung tissue samples were obtained from tissue bio-repository facility of James Graham Brown Cancer Center, at University of Louisville. Local IRB committee of the University of Louisville approved the proposed human study.
Immunoprecipitation, Protein estimation and Western Blot
Immunoprecipitation (IP) was performed as described previously [18]. Briefly, HEK293T cells were transiently transfected in triplicates with FLAG-UBR5 and FLAG alone plasmids. Protein pull-down experiments were performed using anti-FLAG beads, washed and then competition assays were performed using FLAG peptides in molar excess. The samples were then sent for mass spectrometry (MS) and FLAG only samples were used as the control for all data analysis. Harvested cells for each procedure (IP, transfection, infection) were lysed with 1% CHAPS lysis buffer and total protein was estimated as described previously [19]. Western blots were performed in Bolt Bis-Tris gels (#BG4120BOX, Life Technologies, Grand island, NY, USA) as per manufacturer’s protocol using antibodies from Santa Cruz, Dallas, TX, USA (GAPDH # sc47724); Bethyl, Montgomery, TX, USA (UBR5 # A300-573A, GCL1N1 # A301-843A) and Cell Signaling, Danvers, MA, USA (FLAG # 14793, DNA-PK # 4602, mTOR # 2972, RAPTOR # 2280, RICTOR # 2114, AKT # 4691, pAKTS473 # 4060).
Cell viability and Clonogenic assay
Cell viability and clonogenic assays were performed as described earlier [17]. Briefly, A549 cells were cultured in 60 mm culture plates. After 24 hrs of infection with shRNA, cells were trypsinized, counted and 2000 cells were reseeded per well in 96-well plates. Cell viability was analyzed for four successive days using Alamar blue (#DAL1100 Invitrogen detection technologies, Eugene, OR. USA). At the same time following infection, 1000 cells were seeded in 6-well plates in triplicate for each condition. Cells were allowed to grow on 6-well plates for 10 days and supplemented with fresh media every two days. After 10 days, formed colonies were washed once with PBS, fixed with ethanol and stained with crystal violet for imaging and analysis.
In Vivo Xenograft Studies
As previously described NRGS (nude mice, NOD/RAG1/2−/−IL2Rγ−/−Tg[CMV-IL3,CSF2,KITLG]1Eav/J, stock no: 024099) mice were obtained from Jackson laboratories (Bar Harbor, ME, USA) and bred and maintained under standard conditions in the University of Louisville Rodent Research Facility (Louisville, KY 40202, USA) on a 12-h light/12-h dark cycle with food and water provided ad libitum [19]. For xenograft studies, A549 cells were infected with virus particles containing shRNAs targeting UBR5 and a non-targeting (NT) control. 24hrs post-infection, cells were harvested, washed with sterile PBS and 1.25 × 105 cells were suspended in 200µl PBS and delivered by subcutaneous injection in each flank of 8 male mice. For each mouse the Left flank served as the control, receiving cells treated with non-targeting shRNAs, and the Right flank received cells treated with shRNAs targeting UBR5. Four weeks post-injection mice were euthanized as per protocols, including carbon dioxide asphyxiation followed by either cervical dislocation or bilateral thoracotomy, as approved by the Institutional Animal Care and Use Committee (IACUC) of University of Louisville. The tumors were resected, and measurements made. Wilcoxon paired t-test was used to calculate the significance of difference between tumor volume.
Reverse Phase Protein Arrays (RPPA)
A549 cells stably expressing Cas9 were transfected in triplicate as described above using two synthetic gRNAs of each (sgNon-targeting and sgUBR5) which were purchased Synthego (Redwood City, CA, USA). 72hrs post-transfection, the cells were harvested, washed with PBS then frozen until further processing. Frozen cell pellets were then shipped in triplicates to MD Anderson Cancer Center, Houston, TX, USA for protein isolation and RPPA analysis. The core facility of Cancer Center, isolated protein and performed analysis on validated targets. The RPPA data were the averages of six readings.
Statistical Analysis
All statistics were performed using GraphPad Prism 8 software. Unless otherwise specified, significance was determined by one-way ANOVA, using a cut off of p < 0.05.