Lipopolysaccharides (lps)

Immortalized bone marrow-derived macrophage cells (iBMDM cells [14 (link)]) were grown in complete Dulbecco’s Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA). Lipopolysaccharide (1 µg/mL) from E. coli (LPS, AdipoGen Life Sciences, Inc., San Diego, USA) was added to the iBMDM cells and human macrophages for the time points described in the figure legends. Human macrophages were treated with 100 µg/mL lipoteichoic acid from Staphylococcus aureus (LTA, Sigma-Aldrich, St Louis, MO, USA). Where indicated, the cells were treated with 5 μM SB-204990 (a gift from GlaxoSmithKline, Brentford, UK) or 2 μg/mL actinomycin D (Sigma-Aldrich).

Mice were challenged with LPS (2 mg/kg, Sigma, Supplementary Table 1) dissolved in saline through postauricular (p.a.) injection^{84 (link)} once a day for 3 days to establish the EH mouse model. The control groups were injected p.a. with an equivalent of 0.9% physiological saline.
To evaluate the effect of SGK1 inhibition, we treated the EH mice model with SGK1 inhibitor GSK650394 (10 mM, 40 μL/d/mouse, p.a., MCE)^{85 (link)}. According to the instructions, GSK650394 was dissolved in dimethyl sulfoxide (DMSO, Sigma) and the corresponding control group mice were treated with an equal amount of DMSO.
To evaluate the roles of NLRP3 in LPS-induced EH, sgk1^−/− mice were intraperitoneally (i.p.) injected with NLRP3 inhibitor MCC950 (10 mg/kg, AdipoGen) or saline. MCC950 or saline was injected for 3 consecutive days before LPS challenge^{86 (link)}.
In all cases, hearing and vestibular function were tested on day 5. Thereafter, the mice were euthanized, blood samples were collected in a tube containing ethylenediaminetetraacetic acid, plasma was separated immediately, and inner ears were subjected to protein or mRNA expression and EH analysis.