biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsMarcin Rucinskitranscription profiling by arrayRattus norvegicusRattus norvegicushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16213This study investigated how serum composition modulates ACTH-induced gene expression in primary rat adrenocortical cells. Cells were cultured in standard or charcoal-stripped serum (which removes steroids along with other serum components such as growth factors and lipids), then stimulated with ACTH (10 nM) for 24 hours. Microarray analysis revealed that ACTH under serum-depleted conditions activated 532 differentially expressed genes (456 upregulated, 76 downregulated), compared to only 220 genes under standard conditions. Key steroidogenic genes including Cyp11a1, Cyp11b1, Star, and Scarb1 showed markedly enhanced upregulation in the serum-depleted condition. Functional enrichment analysis demonstrated that ACTH preferentially activated steroid biosynthesis and lipid metabolic pathways while suppressing cell cycle and extracellular matrix-related genes, particularly under serum depletion. These findings demonstrate that serum composition critically shapes ACTH responsiveness, revealing multifactorial regulation of adrenocortical cell function beyond steroid feedback alone.biostudies-arrayexpressGrowth Protocol - Primary adrenocortical cells isolated from 20 rats were seeded at a density of approximately 1-2 × 10⁴ cells/well in 24-well plates (NUNC) in complete DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich) and 1% antibiotic-antimycotic solution (Sigma-Aldrich). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Growth medium was replenished every 24 hours.Scaning - Post-hybridization processing, including washing and staining, was performed using an Affymetrix Fluidics Station according to standard protocols. Arrays were scanned on an Affymetrix GeneChip Scanner, and initial quality control parameters (hybridization efficiency and labeling integrity) were evaluated using Affymetrix Expression Console software (version 2.0). Raw intensity data were extracted as CEL files.Nucleic Acid Extraction - Total RNA was isolated from pooled cell pellets using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Briefly, cells were lysed in guanidinium-thiocyanate-containing buffer, and lysates were applied to spin columns. Following recommended wash steps, purified RNA was eluted in nuclease-free water. RNA concentration and purity were quantified using NanoDrop spectrophotometer (Thermo Fisher Scientific), confirming A260/A280 ratios greater than 1.8. RNA integrity was assessed by capillary electrophoresis using an Agilent Bioanalyzer.Hybridization - The labeled cDNA was hybridized overnight at 45°C onto Affymetrix GeneChip Rat Gene 1.1 ST Arrays according to the manufacturer's standard protocol.Sample Treatment - Four experimental groups were established based on serum type and hormone treatment. Twenty-four hours after initial seeding, half of the cultures were transitioned to DMEM/F12 medium containing charcoal-stripped FBS (designated as \""S\"" for serum starvation), while the remaining cultures were maintained in medium containing standard serum. After an additional 24-hour period, ACTH (1-24) (Sigma-Aldrich) was administered at a final concentration of 10 nM to the appropriate treatment groups. Control groups received an equivalent volume of vehicle solution (PBS or medium). Cultures were then incubated for a further 24-hour period. The final experimental groups were designated as follows: Control, ACTH, Control(S), and ACTH(S).Labeling - Total RNA (50-200 ng) from each experimental group was reverse-transcribed, amplified, and labeled according to the Affymetrix WT Plus Kit protocol following the manufacturer's instructions. Biotin-labeled fragmented cDNA was generated for array hybridization.Sample Collection - Primary rat adrenocortical cells were isolated from adrenal glands of 10-week-old male Wistar rats. Animals were euthanized by rapid decapitation, and adrenal glands were promptly removed and placed in ice-cold phosphate-buffered saline (PBS, pH 7.4). Under sterile conditions, excess fat and connective tissue were carefully dissected from each gland. Glands were minced into approximately 1 mm³ fragments and enzymatically digested in DMEM/F12 containing collagenase type I (1 mg/mL, Sigma-Aldrich) at 37°C for 30 min in a shaking water bath. Following digestion, the cell suspension was filtered through a sterile 70 µm nylon mesh filter to eliminate undigested tissue fragments. The filtrate was centrifuged at 200 × g for 10 min at room temperature. Cell pellets from 2-3 culture wells were pooled for each experimental replicate to obtain sufficient RNAMIAME ScoreRaw DataOrganizationAssays and DataProcessed DataMAGE-TAB FilesArray DesignsMarcin RucinskiData Transformation - Raw CEL files were imported into R (version 4.1.2 or later) and processed using the robust multiarray average (RMA) algorithm within the \""oligo\"" package of Bioconductor. Processing included background correction, quantile normalization, and summarization at the transcript (gene) level. Expression values were log₂-transformed. The resulting normalized expression matrix was used for all downstream differential expression analyses.falseSerum starvation affects the transcriptomic and proliferative response to ACTH in primary cultures of rat adrenocortical cellsThis study investigated how serum composition modulates ACTH-induced gene expression in primary rat adrenocortical cells. Cells were cultured in standard or charcoal-stripped serum (which removes steroids along with other serum components such as growth factors and lipids), then stimulated with ACTH (10 nM) for 24 hours. Microarray analysis revealed that ACTH under serum-depleted conditions activated 532 differentially expressed genes (456 upregulated, 76 downregulated), compared to only 220 genes under standard conditions. Key steroidogenic genes including Cyp11a1, Cyp11b1, Star, and Scarb1 showed markedly enhanced upregulation in the serum-depleted condition. Functional enrichment analysis demonstrated that ACTH preferentially activated steroid biosynthesis and lipid metabolic pathways while suppressing cell cycle and extracellular matrix-related genes, particularly under serum depletion. These findings demonstrate that serum composition critically shapes ACTH responsiveness, revealing multifactorial regulation of adrenocortical cell function beyond steroid feedback alone.2025-12-03T00:00:00Z2026-05-27T15:48:25.476Z2025-11-19T20:24:51.235ZE-MTAB-16213EFO_0002768EFO_0002944EFO_0003814EFO_0003813EFO_0003789EFO_0005518EFO_0003816EFO_0003815EFO_0003969