Determination of TNF‐α and IL‐6 in culture media

MPMs were cultured at a density of 700 000 per well of six‐well plates in normal growth media for 24 h. Cells were then treated with shikonin at 0.4, 1 or 2.5 μM for 30 min. DMSO was used as vehicle control. Following pretreatment, cells were exposed to 0.5 μg·mL⁻¹ LPS for 24 h. Culture media were collected and used to measure TNF‐α and IL‐6 levels using elisa. Data were normalized to amount of total proteins from lysates of the same culture and expressed as a percentage of the LPS group in cell‐based experiments (or were expressed as a percentage of one of mice in LPS group in animal‐based experiments).

To test shikonin's effect on Pam3CSK4‐induced TLR2 activation and inflammatory cytokine release, which is not dependent on MD2, MPMs were pretreated with shikonin for 30 min and exposed to the TLR2 agonist Pam3CSK4 at 0.1 μg·mL⁻¹ for 12 h. TNF‐α and IL‐6 levels were measured as indicated above.