PM 100

Primary mouse fibroblast and NIH-3T3 mouse fibroblasts were cultured in DMEM supplemented with 10% serum. Cells were exposed to 100 pM TGF-β or to the ethanol carrier for 4 hours before RNA extraction. Total RNA was extracted from confluent 75 cm² culture dish (approx 2 million cells) using Trizol reagent (Invitrogen) according to the manufacturer's protocol and resuspended in 20 μl RNAse-free water. Reverse transcription was performed with the GeneAmp Gold RNA PCR Core kit (PE Applied Biosystem) using 5 μl (approx 2.5 μg) of RNA in a 25 μl final volume using oligo-dT as a primer. The resulting cDNA solution was diluted 10-fold in deionized water and the solution thus obtained was considered as the undiluted sample (1-fold dilution) for the qPCR measurements. This final dilution step was found to be necessary to prevent inhibitory effects on the PCR efficiency that likely result from contaminant carry-over (data not shown).

Lipids were extracted and partially purified as previously described in mammalian tissues [23] (link), [25] (link), [29] , [30] (link). In brief, 40∶1 volumes of methanol were added to each sample followed by 2-AG-d₈ and NAGly-d₈ (10 µl of 100 pM). These deuterium-labeled compounds were used as internal standards to determine the extraction efficiency. Methanolic samples were covered with parafilm and left on ice and in darkness for ∼2 h. Samples on ice were then homogenized using a polytron for ∼1 min followed by centrifugation at 19,000 g at 24°C for 20 min. Supernatants were then collected and placed in polypropylene tubes. HPLC-grade water was added, making the final supernatant/water solution 25% organic. To isolate the compounds of interest partial purification of the 25% solution was performed on a Preppy apparatus assembled with 500 mg C18 solid-phase extraction columns. The columns were conditioned with 5 ml of HPLC-grade methanol immediately followed by 2.5 ml of HPLC-grade water. The supernatant/water solution was then loaded onto the C18 column, and then washed with 2.5 ml of HPLC grade water followed by 1.5 ml of 40% methanol. Elutions of 1.5 ml of 70%, 75%, 80%, 85%, 90%, 95%, and 100% methanol were collected in individual autosampler vials and stored at −20°C until analysis by mass-spectrometry.

The protocol for biotinylated proteins purification was modified from Opitz et al. ^{43 (link)}. LMY52 wild type and LMY52-BirA*-dCas9 with or without sgRNA mutants generated (Supplementary Table 3) were inoculated in 4 L of SDC medium without inositol plus D-biotin (B4639, Sigma-Aldrich) added to final concentration of 10 μM. Cells were grown to OD₆₀₀ of 0.8 and harvested by filtration using a Kontes™ Ultra-Ware™ Microfiltration Assembly with Fritted Glass Support (90 mm) and MF-Millipore™ Membrane Filters, 0.45 µm pore size (HAWP0900 Millipore). Pellets were immediately frozen in liquid nitrogen and kept at −80 °C. Cell lysis was performed under cryogenic conditions using solid phase milling in a planetary ball mill (Retsch PM 100) producing a fine cell grindate^{80 (link)}. Grindate samples were stored at −80 °C until processed. Biotinylated proteins were purified using streptavidin-sepharose beads (GE Healthcare, 17-5113-01). For each sample, 0.7 g of crude extract was resuspended in 6.3 mL of buffer A: commercial buffer W (100 mM Tris-Cl, 150 M NaCl, pH 8, 2-1003-100, IBA) containing 0.1 % SDS, 0.5 % Sodium Deoxycholate, and protease inhibitors mixture (cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail, 11836170001, Roche). Samples were homogenized with a vortex agitator, polytron (PT1200E, for 30 s, one time), and incubated at 65 °C for 5 min in a water bath. Subsequent clarification was performed by two rounds of centrifugation at 2465 × g, 4 °C, 5 min, and 20,292 × g, 4 °C, 20 min, respectively. Triplicates for each sample were prepared; clear extracts were incubated with 30 µL of beads previously equilibrated with buffer A. Samples with beads were incubated for 3 hours at 4 °C with gentle mixing (VWR® Nutating Mixer). Beads were washed with buffer A and transferred to 1.5 mL Protein LoBind tubes (Eppendorf, 0030108422) and three consecutive washes with buffer A were performed, followed by four quick washes with 50 mM Ammonium Bicarbonate pH 8 (ABC). 30 µL of 50 mM ABC pH 8 containing 6 mM fresh DTT were added to each tube followed by 30 min incubation at 37 °C with gentle agitation in a ThermoMixer (Eppendorf) at 350 rpm. Samples were spun quickly and 5μL IAA 126 mM dissolved in ABC 50 mM pH 8 were added to each tube and incubated for another 30 min in the dark at room temperature. Tubes were quickly spun again and 5 µL Trypsin/LysC Mix (Promega, V5073) dissolved in ABC 50 mM pH 8 at 200 ng/µL were added to each one. Samples were digested on-beads overnight at 37 °C, 350 rpm. The next day, after a quick spin, 60 µL of MS grade H₂O were added to the beads, mixed, and centrifuged at low speed (1 min, 400 × g). Eighty (80) µL of the peptides digests were transferred to new labeled Protein LoBind tubes before adding formic acid (FA) to a final concentration of 4% v/v. The peptides were dried in a SpeedVac at room temperature and then stored at −80 °C.

Plasma levels of 25-OH-D and 1α,25-(OH)₂-D were analyzed using commercially available EIA kits that determine total (bound and free) levels of vitamin D metabolites (Immunodiagnostic Systems, Ltd., Scottsdale, Arizona). The sensitivity of the 25-OH-D ELISA kit is 5 nM, the intra- and inter-assay variability is ≤8.7% coefficient of variation (CV), and the specificity is 100% for 25-OH-D₃, 75% for 25-OH-D₂, 100% for 24,25-(OH)₂-D, and less than 0.3% for the remaining vitamin D metabolites. The sensitivity of the 1α,25-(OH)₂-D ELISA kit is 6 pM with a specificity of 100% for 1α,25-(OH)₂-D₃, 39% for 1α,25-(OH)₂-D₂, less than 0.05% for the remaining vitamin D metabolites, and the intra- and inter-assay variability is estimated at ≤20% CV.
Vitamin D-binding protein (VDBP) was determined with a commercial ELISA kit (Aviva Systems Biology LLC, San Diego, CA, USA) using the manufacturer’s instructions.