Mouse gene 1.0 st array

For gene array analysis, we mated heterozygous (TAM67+/−) and heterozygous (rTA+/−) mice and treated the pregnant dams with doxycycline (2 mg/ml) in drinking water beginning on embryonic days E15.5. At E20 the pups were euthanized and RNA was collected from whole skin. RNA samples were pooled using epidermis from three separate mice. Approximately 5 to 8 μg of total RNA was reverse transcribed, amplified and labeled as described (Li et al., 2001 (link)) and labeled cRNAs were hybridized to Affymetrix Mouse Gene 1.0 ST Arrays (#901168) and 0.5 fold or greater change in expression was considered significant. For annotation we used the DAVID program [http://david.abcc.ncifcrf.gov] (Dennis et al., 2003 ).

Kidney RNA was labeled and hybridized to Affymetrix Mouse Gene 1.0 ST arrays following the standard WT protocol (GeneChip® Whole Transcript (WT) Sense Target Labeling Assay, rev. 5, www.affymetrix.com). Arrays were scanned and data were imported into Partek Genomics Suite version 6.2 (Partek, Inc., St. Louis, MO). Robust Multichip Average signals (RMA)^{40 (link)} were generated for the core probe sets using the RMA background correction. The log₂ transformed signals were used for principal components analysis, hierarchical clustering and signal histograms to determine if there were any outlier arrays^{41 (link)}; none were found. The probe sets were analyzed using a 1-way ANOVA by treatment (WT with saline, WT with 1h FGF23 injection). False discovery rates (FDR) were calculated using q-value^{42 (link)}. The gene array data was deposited with GEO accession number GSE138992.

Forebrains were rapidly dissected from skulls and snap frozen in liquid nitrogen. Total RNA was isolated using the NucleoSpin^™RNA/protein kit (Macherey-Nagel, Düren, Germany). RNA purity, integrity, and quantity were assessed using the NanoDrop ND-8000 (NanoDrop, Wilmington, DE) and the Bioanalyzer system (Agilent 2100; Agilent Technologies Inc, Palo Alto, CA).
RNA samples were processed using the Affymetrix GeneChip WT PLUS kit and hybridized to Mouse Gene 1.0 ST Arrays (Affymetrix, Santa Clara, CA). Ten arrays per sex per experimental group were used, with each array corresponding to one animal. Four to six litters were represented in each diet group to minimize litter effects. Quality control and normalization were performed using the pipeline at the www.arrayanalysis.org website (Maastricht University, the Netherlands).^{14 (link)} Normalization was performed using the Robust Multichip Average (RMA) algorithm^{15 (link)} and the default Affymetrix Chip Description File (CDF) for this chip. The 27619 main probe sets were used for further analysis; probe sets corresponding to Affymetrix controls or unmapped sequences were discarded after normalization.

A total of 23 Affymetrix Mouse Gene 1.0 ST Arrays were used for gene expression analysis (Affymetrix, Santa Clara, CA). This procedure was performed as described previously (HogenEsch et al., 2016 (link)). Ear skin samples collected in RNAlater were homogenized in TRIzol (ThermoFisher Scientific). After total RNA isolation, quality assessment was performed using a 2100 Bioanalyzer. Subsequently, cDNA was synthesized using the GeneChip Expression 3′– Amplification One-cycle kit (Affymetix). Following cDNA synthesis, it was amplified, labeled with biotinylated nucleotides (Affymetrix), and hybridized onto 1.0 ST GeneChip arrays. Slides were scanned and quantified using GeneChip Scanner 3000 and GCOS 1.0 software, respectively.

We plated mouse embryonic fibroblasts (MEFs) on six 10-cm culture dishes in DMEM containing 10% fetal bovine serum (FBS), and incubated them at 37 °C with 5% CO₂. After 16 h, we removed the cell medium and washed the cells twice with DMEM containing 1% FBS. We then added 10 ml of 1% FBS DMEM with DMSO or celastrol (250 nM). After 7 h of incubation, we removed the medium from the plates, and snap froze the culture dishes in liquid nitrogen. For RNA extraction, the plates were placed on ice for 15 s. We then added 1 ml of TRIzol reagent to the plates, and incubated at room temperature for 15 min. The cells in TRIzol were collected in 1.5 ml tubes. We then added 200 μl of chloroform, followed by vortexing for 20 s. The tubes were centrifuged at 4 °C for 15 min at 13,400 × g. We transferred the clear top phase to new vials containing 400 μl isopropanol. After 10 min of incubation at room temperature, the tubes were centrifuged at 4 °C for 12 min at 13,400 × g. RNA pellets were washed with 75% ethanol twice. RNA pellets were air dried for 10 min at room temperature and dissolved in 30 μl of RNase free water. We further purified the RNAs using RNeasy MinElute Cleanup Kit according to the manufacturer’s protocols. 1 μg of total RNA was used for microarray analysis using Affymetrix Mouse Gene 1.0 ST arrays.