Flow dependence of gene expression in cultured mIMCD cells

Bioinformatic analysis of data from Mohammed SG, Arjona FJ, Verschuren EHJ, Bakey Z et al. Primary cilia-regulated transcriptome in the renal collecting duct. FASEB J 2018 Jul;32(7):3653-3668. PMID: 29452568. https://doi.org/10.1096/fj.201701228r Authors' methods: IMCD3 cells were cultured in DMEM/F-12 1:1 v/v medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 1 mM sodium pyruvate, 10% v/v fetal calf serum, and 10 µg/ml ciproxin, at 37°C and 5% v/v CO2. Cells were seeded into 6-channel ibiTreat-coated μ-Slide chambers (ibidi, Martinsried, Germany) at a cell suspension density of ~0.7 × 106 cells/ml. The medium was refreshed twice a day. To induce ciliogenesis, cells were grown to confluency for 2 d, followed by an additional 1 d in serum-deprived medium prior to the flow experiment (Fig. 1A). See: https://faseb.onlinelibrary.wiley.com/doi/epdf/10.1096/fj.201701228R#f1 (DOI: 10.1096/fj.201701228R) Application of Flow. After 1 d of culture in serum-deprived medium, cells were incubated under static conditions or exposed to fluid flow for 3 h at 37°C. To apply fluid flow, the ibidi μ-Slide chamber was connected to a peristaltic pump (Ismatec, Wertheim, Germany) with a series of tubes and connectors extending to a reservoir (see Supplemental Fig. S1A for schematic representation). Each well was perfused with serum-deprived medium at a physiologic rate of 0.45 ml/min. The fluid flow rate was set to generate a physiologic stress of 0.6 dyn/cm2, a force within the ciliary bending profile reported in previous studies (16, 17). For static condition, medium was refreshed. Next, cells were lysed for RNA isolation and RNA-seq analysis. See original paper. Knepper lab curated the data to make it accessible for the community at a user friendly web site: https://esbl.nhlbi.nih.gov/Databases/IMCD3_flow_effect/RD.htm