Mice and in vivo studies
KrasLSL-G12D/+Trp53flox/flox mice, referred to as KP, were bred on a mixed genetic background of C57BL/6-129/Sv. Tumors in these mice were produced using intratracheal instillation of Lenti-Cre or Ad5mSPC-Cre viral particles under general anesthesia, focusing on both young (KP-Young; 2-3 months) and older (KP-Old; 18-19 months) mice.
NOD Xenograft Gamma (NXG) mice, aged 6-10 weeks at the study’s start, were also used, with all transplantation experiments utilizing age-matched cohorts. For subcutaneous implants, GFP-luciferase-expressing cells were injected into the flanks of these mice. Tumor measurements were taken regularly, ensuring none exceeded the maximum size limit.
For intravenous injections, specific cell counts were administered into the tail vein of NXG mice, performed in young immunodeficient hosts to avoid complications related to age or immunity.
Regarding CB-839, mice received either the drug at 200 mg/kg or a vehicle twice daily, post-tumor establishment. Vehicle control was a specific solution prepared for consistency.
In terms of Atf4 silencing via doxycycline, a concentrated solution was provided in drinking water. ISRIB studies involved cell pretreatment before injections, with strict controls over the environment and housing for the mice throughout the study.
IVIS imaging
Mice received an intraperitoneal injection of d-luciferin, followed by organ imaging to quantify luminescence ex vivo. The luminescence data was computed using specialized software, normalizing the results against background lighting.
Histology and IHC analyses
Lungs were processed for histological analysis by fixation and staining. The tumor area was quantified using image analysis software, following previously established grading protocols. For immunohistochemistry (IHC) analyses, sections underwent deparaffinization and epitope retrieval before antibody incubation and slide scanning.
Cells
Primary tumor cultures from KP-Young and KP-Old mice were derived months after tumor induction and maintained in specific culture conditions. A549 human lung adenocarcinoma cells were also used, with all maintained cell lines verified to be free from contamination.
Proliferation and viability assays
Cell population doublings involved specific cell lines seeded in batches and counted over several days. For viability assays, cells were plated, treated with various drugs, and assessed for viability after a set timeframe using a luminescent assay.
Clonogenic assay
Clonogenic assays utilized different concentrations of a specific nutrient over several days, with colonies fixed and counted for analysis.
3D cultures and anoikis resistance assay
Cells were seeded in ultra-low attachment plates for spheroid formation, and viability was assessed after a set period, normalizing between conditions.
Spheroid formation and collagen invasion assays
For further analysis, cells were embedded in collagen and images were taken to monitor growth and invasion over time.
Caspase activity assay
The assay involved measuring the activity of caspases in cells post-seeding and normalizing between different conditions.
Cell trace proliferation assay
Labelling for proliferation involved using a specific kit and flow cytometry to analyze data throughout the experimental timeline.
Lentiviral production and transduction
Lentiviruses were produced by transfecting specific cells and subsequently selected for study after validating expression through various techniques.
shRNA-mediated knockdown
Knockdown of Atf4 was performed using specialized cloning techniques, followed by verification of successful gene silencing.
Western blotting
Proteins were extracted and analyzed through standard methods, with detection relying on specific antibodies and validation through imaging systems.
Real-time quantitative PCR
RNA isolation and cDNA synthesis were coupled with gene expression analysis using established kits and systems.
Reagents
Mice received a range of drugs in specified concentrations, aimed at investigating the metabolic and cellular responses within experimental contexts.
Mitochondrial respiration
Cellular respiration rates were measured in a controlled format, providing insights into metabolic activity under various conditions.
GC–MS analysis of polar metabolites and stable isotope tracing
Metabolite analysis involved seeding cells in specialized media, followed by sampling and preparation for gas chromatography to assess metabolite presence and ratios.
RNA-seq
RNA was collected and sequenced through established procedures, allowing for comprehensive gene expression profiling to be carried out.
ATAC-seq
Library construction for ATAC-seq involved meticulous sequencing and data analysis protocols to ensure accuracy.
Western Sweden patient cohort
Patients diagnosed with NSCLC from specific hospitals were included for study, gathering data for further analysis regarding age, tumor characteristics, and genetic profiling.
TMA cohort
Tissue microarrays from the NSCLC cohort were analyzed to study the correlation of specific expressions with patient outcomes, all under regulatory compliance.
Statistics and reproducibility
Statistical analyses adhered to strict guidelines with all findings subjected to rigorous validation across several methodologies.
Reporting summary
Further research design information is accessible via linked summaries.
