White preadipocytes from wild type mice (Wt) and knockout (KO) mice lacking p38α (p38α−/−), p38β (p38β−/−), p38γ (p38γ−/−), p38δ (p38δ−/−), MKK3 (MKK3−/−) and MKK6 (MKK6−/−) kinases were immortalized by infection with SV40TpBABE-neo virus as previously described (Matesanz et al., 2017 (link)). The validity of these cell culture model has been previously supported (Matesanz et al., 2017 (link), 2018 (link)). In any case, we have reconfirmed the KO of MKK3 and MKK6 by western blot and those of p38 by qPCR (Supplementary Figure S1 ).
Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco),l -glutamine (2 mM, Gibco), streptomycin (100 μg/ml, Gibco) and penicillin (100 U/ml, Gibco) and incubated at 37°C under a 5% CO2/95% air atmosphere. Confluent cells were trypsinized and seeded in tissue dishes at a density of 6 × 105 cells/ml. After 8 h, the medium was aspirated and replaced with fresh medium without sera. After 4–5 h, the medium was replaced with fresh medium containing DMSO (control cells) or the indicated concentrations of BIRB 0796 (Cell Signaling), Box5 (EMD Millipore), JNK-IN-8 (Calbiochem), U0126 (Cell Signaling), PD184352 (Tocris), Microcystin L-R (Sigma) or recombinant Wnt5a (R&D Systems) and the incubation was continued for a further 8 h.
Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco),