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. 2016 Jul 14;35(16):1766–1778. doi: 10.15252/embj.201694696

Figure 1. GSDMD Nterm induces the formation of a large plasma membrane pore.

Figure 1

  • A
    Cell viability as assessed by GFP expression in HEK293T cells transfected with the pRetroX TetOne3G‐eGFP plasmid only (vector) or pRetroX TetOne3G‐eGFP harboring the N‐terminal fragment of GSDMD. Cells were treated with the indicated concentrations of doxycycline 24 h post‐transfection, and the percentage of GFP‐positive cells was determined 16 h later by flow cytometry.
  • B
    LDH release from HEK293T cells transfected with the pRetroX TetOne3G‐eGFP plasmid only (vector) or pRetroX TetOne3G‐eGFP harboring the N‐terminal fragment of GSDMD. At 24 h post‐transfection, cells were treated with the indicated concentrations of doxycycline for 8 h and the percentage of LDH release was determined. Graphs show mean and s.d. of quadruplicate wells.
  • C, D
    PI staining of and LDH release from HEK293T cells transfected with pRetroX TetOne3G‐eGFP harboring the N‐terminal fragment of GSDMD in the presence of osmoprotectants. At 24 h post‐transfection, PEGs of the indicated molecular weights were added to a final concentration of 30 mM, cells were treated with 250 ng ml−1 doxycycline for 8 h, and the level of PI staining (C) or LDH release (D) was determined.
  • E, F
    PI staining of and LDH release from LPS‐primed primary BMDMs infected with log‐phase S. typhimurium for the indicated time points in the presence of PEGs of the indicated molecular weight (numbers on the x‐axis, 30 mM final concentration).
Data information: Graphs show mean and s.d. of quadruplicate wells (B–F) or the mean and s.d. of duplicate wells (A). *P < 0.05 as determined by Student's t‐test. Data are representative of at least three independent experiments.