Mes koh

For sterile culture, seeds were surface sterilised (30% (v/v) bleach, 0.02% (v/v) Triton X‐100), washed three times in sterile water and sown on modified Hoagland media (HM) (Haydon et al., 2012 (link)) or half‐strength Murashige & Skoog media (½MS) (Sigma‐Aldrich), 3 mM MES‐KOH (pH 5.7), solidified with 0.8% (w/v) agar Type M (Sigma‐Aldrich). Seeds were chilled for 2 d at 4°C and grown under 12 h light (80–100 μmol m⁻² s⁻¹) : 12 h dark at constant 20°C (L : D).

The iISGs and mISGs were purified following the previous protocol with minor modifications [33 (link),82 (link)]: Briefly, MIN6 cells were treated with HG for 1 h, and then homogenized in homogenization buffer [0.3 mol/L sucrose (Sigma, SLCH3216), 1 mmol/L EDTA (Merck, 654,833), 1 mmol/L MgSO₄ (Macron avantor, 6066–04), 10 mmol/L MES-KOH (Sigma, M8250), 1 mmol/L PMSF (Thermo Scientific, 36,978), pH 6.5]. The nuclear debris was removed by centrifugation at 1000 g for 5 min. The supernatant was loaded on top of a discontinuous Optiprep (Sigma, D15561) gradient, prepared by 5 layers of 30%, 23.4%, 17.6%, 13.2%, and 8.8% of Optiprep. The Optiprep gradient was centrifuged for 75 min at 100,000 g in an SW41 rotor (Beckman), and the gradients were divided into twelve fractions. Both fraction 6 (interface between 13.2% and 17.6%) and fraction 8 (interface between 17.6% and 23.4%) containing insulin were iISGs and mISGs, respectively.