Dexamethasone (dex)

Thirty-six adult male C57BL/6 mice were divided into six groups according to a computer-based randomization in experiment 1 (n = 12) as follows: 1) sham + vehicle, 2) ICH + vehicle, and 3) ICH + Dex. Autologous blood injection in the basal ganglia was used to establish a mild ICH model in mice. Based on previous studies (Gu et al., 2011 (link); Liang et al., 2017 (link)) and our preliminary experiments, 25 μg/kg of Dex (Jiangsu Hengrui Medicine Co., Ltd., Jiangsu, China) was administered using intraperitoneal injection post-ICH daily for 3 days. Mice in sham + vehicle and ICH + vehicle groups received an equal volume of saline via intraperitoneal injection (Figure 1A).
Twenty-four adult male C57BL/6 mice were divided into two groups according to a computer-based randomization in experiment 2 (n = 12) as follows: 1) ICH + Dex + GSK and 2) ICH + Dex + vehicle. GSK1016790A (GSK, 1 μmol/2 μl, HY-19608, MedChemExpress, United States), an agonist of TRPV4, was dissolved in 10% dimethyl sulfoxide (DMSO) (D8418, Beyotime, China) and 90% corn oil (HY-Y1888, MedChemExpress, United States), and injected into the left lateral ventricle (stereotaxic coordinates: 1.0 mm lateral to the midline, 0.3 mm posterior to the bregma, and 2.5 mm below the skull) 30 min before Dex administration. Vehicle was administered with an equivalent volume of DMSO plus corn oil as a control (Figure 1B).

C2C12 cells were treated with dexamethasone (DEX; catalogue no.: HY-N2609) and TNF-α (catalogue no.: HY-P1825), both obtained from MedChemExpress (Monmouth Junction, NJ, USA), to induce insulin resistance and inflammation, respectively. All cell experiments were conducted in a blinded fashion.
miR-193b mimic/inhibitor transient transfections were also conducted in C2C12 cells. miR-193b mimic and inhibitor sequences have been described in a previous study [24 (link)]. The miR-193b mimic was a duplex RNA, with the sense sequence 5′-aacuggcccacaaaguccc-3′ and the antisense sequence 5′-gacuuugugggccaguuuu-3′. Nontargeting negative control sequences (sense 5′-uucuccgaacgugucacgutt-3′, antisense 5′-acgugacacguucggagaatt-3′) were used as controls. The inhibitor of miR-193b (5′-gggacuuugugggccaguu-3′) was a single RNA sequence that was exactly complementary to miR-193b. A nontargeting negative control sequence (5′-caguacuuuuguguaguacaa-3′) was used as a control. Transient transfections were performed using Lipofectamine 3000 (Invitrogen), according to the protocol provided by the manufacturers.
After treatment, cells were harvested for myosin immunostaining (see below), or PCR analysis of miR-193b, PDK1 and Dnmt1 expression, and western blotting for analysis of PDK1, Akt, p-Akt, Myh, mTOR and p-mTOR content, as described above (see ESM Methods and ESM Table 1).

LiCl (Sigma, 203637) was used at 25 mM. FH‐535 (Tocris, 4344) and ICG‐001 (Tocris, 4505) stocks were diluted in dimethyl sulfoxide (DMSO, Sigma) and used at the indicated concentrations. Vincristine (Pfizer, 1 mg ml⁻¹) and dexamethasone (Fortecortin, 4 mg ml⁻¹) were kindly provided by Hospital del Mar. L‐Asparaginase was obtained from MedChemExpress (HY‐P1923).

The H1944 or A549 cells were trypsinised and resuspended in PBS at a density of six (H1944) or two (A549) million cells/50 µl and mixed with an equal volume of BME (#3533-005-02, Sigma-Aldrich) NOD-scid-γ (NSG) mice (±7 weeks old) were anesthetised before injection of six million cells subcutaneously into one of the flanks. Once the tumour size reached between 100 and 300 mm³ the mice were treated with 4 mg/kg dexamethasone (D2915-100MG, Sigma-Aldrich; dissolved in water), 75 (H1944) and 20 (A549) mg/kg Linsitinib (HY-10191, MedChemExpress; dissolved in 25 mM tartaric acid), 25 mg/kg GSK1838705A (MedChemExpress; dissolved in 20% sulfobutyl ether β-cyclodextrin (ISP; pH 3.5)), combination, or vehicle by I.P. injections (dexamethasone) and orally (IGF-1R inhibitors) on a daily basis. Tumour volume was monitored by calliper measurements every 2 days. Mice were kept under standard temperature and humidity conditions in individually ventilated cages, with food and water provided ad libitum. The NKI Animal Experiments Committee approved all in vivo experiments (project number 9139 and 9907).

HEI-OC-1 cells were cultured at 33°C with 10% CO₂ in DMEM containing 1.0 g/L of glucose, 10% FBS, and 1% N-2 (Gibco, New York, NY, United States). The middle cochlea basilar membranes were then dissected from P3 rats and laid onto a glass sheet (coated with ornithine and laminin) that had been placed in a dish. The HEI-OC-1 cells, and the HCs, were cultured with different concentrations of dexamethasone (0.5, 5 μM) for 24 h, or with 0.5 nM rapamycin, or 5 mM 3-Ma, for 6 h before exposure to H₂O₂. HEI-OC-1 cells were then treated with H₂O₂ (2, 5, and 10 mM) while the HCs were treated with H₂O₂ (0.5, 0.8, 1 mM) for 1 h. Both cell types were then allowed to recover in culture medium for another 24 h, together with corresponding concentrations of dexamethasone, rapamycin, or 3-MA. The dexamethasone, rapamycin, and 3-MA were all obtained from Med Chem Express (NJ, United States).