New insights into the molecular actions of Grosheimin, Costunolide, α- and β- Cyclocostunolide on primary cilia structure and Hedgehog signaling
New insights into the molecular actions of Grosheimin, Costunolide, α- and β- Cyclocostunolide on primary cilia structure and Hedgehog signaling
RPE cells from two 12-well plates per experimental condition were carefully harvested using a sterile cell scraper. The collected cells were immediately lysed in RLT buffer supplied with the RNeasy Mini Kit (Qiagen, catalog number 74104) to ensure optimal RNA stabilization and preservation for subsequent extraction and downstream molecular analyses. Not applicable. RPE cells were grown at 37 °C in 5% CO 2 in DMEM/F-12. Media was supplemented with L-glutamine, sodium pyruvate, 10% FBS (Fetal Bovine Serum, Gibco), 100 U/mL penicillin, 100 µg/mL streptomycin, and 3 µg/mL ciprofloxacin (NORMON laboratories). RPE cells were seeded at 70,000 cells/well in 12-well plates, and experiments were performed after 24h. Cilia were induced by incubating cells for 24 h in media supplemented with 0.5% FBS (starvation).  SLs were administered concurrently with the 0.5% FBS-containing media to the cells, achieving a final working concentration of 10 µM for each compound. As the test compounds were initially prepared as DMSO stock solutions, an equivalent volume of DMSO alone was included as the control condition to account for any potential effects of the solvent. Raw sequencing data was generated in BCL (binary base call) format on the Illumina NextSeq 500. These BCL files were converted into FASTQ format using Illumina's BaseSpace Seq Hub. This process included base calling, filtering by quality and length, trimming of adapters, and de-multiplexing based on the unique indices of each sample. Samples suspended in RLT buffer were thoroughly vortexed to ensure complete cell lysis and homogenization. The lysates were then purified using column-based RNA extraction according to the manufacturer's protocol. Following the first cleanup step, samples were treated with RNase-free DNase (Qiagen, catalog number 79254) to remove genomic DNA contamination, then subjected to a second column cleanup to ensure highly pure RNA. RNA quality and integrity were carefully assessed to confirm suitability for sequencing. A total of 150 ng of RNA from each sample was used to prepare libraries following the Illumina Stranded mRNA Prep Ligation protocol. Libraries were quality-checked using TapeStation DNA High Sensitivity D1000 assay and quantified using Qubit™ DNA HS Assay. Library sizes were approximately 270-290 bp.