Normicin

All cell lines used were obtained from Invivogen (France). HEK-Blue hTLR2, HEK-Blue hTLR3, HEK-Blue hTLR4, HEK-Blue hTLR7, HEK-Blue hTLR9, HEK-Blue hNOD1, HEK-Blue hNOD2 as well as control cell lines HEK-Blue Null1 and HEK-Blue Null2 were cultured (37 °C, 5% CO₂) in Dulbecco’s Modified Eagles Medium (DMEM) (Gibco) supplemented with 100 µg/ml Normicin (Invivogen), 100 units penicillin, 100 units streptomycin, 2.92 mg/mL L-glutamine (Gibco), 10% heat-inactivated FBS (Gibco) and cell line-specific selective antibiotics (Invivogen) in a T75 flask. When 70–80% confluency was achieved, HEK-Blue cells were detached from culture flasks using PBS (Gibco) and sub-cultured. When passage 20 was achieved, cells were discarded.
HEK-Blue cells were plated in 96-well plates (flat-bottom) at 50.000 viable cells in 100 µL medium per well and cultured overnight (37 °C, 5% CO₂). Cells were stimulated the next day with 100 µL OMVs starting at 2 µg/mL in fivefold serial dilutions and TLR/NOD antagonists as positive controls. Subsequently, plates were incubated for 24 h (37 °C, 5% CO₂). Next, 20 µL culture supernatant of each well was transferred to wells in a 96-well plate containing 180 µL of pre-warmed Quanti-Blue detection reagent. Absorbance was read after 1 h at 630 nm with a microplate reader (Bio-Tek). TLR activation was determined by calculating the fold change over the unstimulated cells.

Pieces of approximately 100 mg of epidermal tissue were dissected from Epi Hras^G12R grafts and Epi WT grafts 28 days after transplantation and control skin from unaffected littermates. Similarly, tibias weighing 100 to 120 mg were extracted from bone HRAS^G12V and control mice 28 days after induction with doxycycline. All tissues were cut in pieces of approximately 1 to 8 mm³ and incubated in 12-well plates with 1 mL of α-MEM medium (Thermo Fisher Scientific) with 0.1% of Normicin (Invivogen, ant-nr-1) in a culture incubator. Twenty-four hours later, medium was replaced by fresh medium, which was collected 48 hours later and centrifuged at 15,000g for 5 minutes to remove solids. Protein was measured in samples diluted 1:10 in PBS using the Coomassie method (Coomassie Plus, Thermo Fisher Scientific, 23236). Protein standards were BSA solutions of 25 to 2000 μg/mL in 1:10 media:PBS (Thermo Fisher Scientific, 23209). FGF23 levels were normalized by protein content in the media.