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. 2020 Jun 2;39(14):e103454. doi: 10.15252/embj.2019103454

Figure 2. The aberrant release of IL‐1β by Gata6‐KO‐deficient pMФ follows classical inflammasome activation.

Figure 2

  • A
    Normalized expression of Tlr and Cd14 genes following microarray analysis performed on unstimulated cell‐sorted Gata6‐WT and Gata6‐KOmye pMФ. Data are shown as mean ± SEM from three biological replicates. Statistical significance was determined using empirical Bayesian statistic corrected for false discovery rate by the Benjamini–Hochberg procedure. n.d = non‐detectable.
  • B
    Mean fluorescence intensity (MFI) of extracellular TLR2, TLR4 and CD14 expression on naïve Gata6‐WT and Gata6‐KOmye pMФ. n = 4–7 individual mice per group.
  • C, D
    Il1b and Tnf mRNA relative expression (C) and IL‐1β Western blot protein analysis (D) of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates.
  • E
    Representative dot plot, percentage and mean fluorescence intensity (MFI) analysis of pro‐IL‐1β+ Gata6‐WT and Gata6‐KOmye pMФ flow cytometry analysis 3 h after stimulation with 100 ng/ml LPS. n = at least three independent experiments.
  • F
    Nlrp3 mRNA relative expression of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 3 h. Data shown are pooled from three independent experiments.
  • G
    Western blot protein analysis of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 6 h. Data shown are representative of at least three independent experiments. Western blot was performed on whole cell lysates.
  • H, I
    IL‐1β ELISA (H) and Western blot protein analysis (I) of supernatants collected from Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or 10 μM MCC950 for 24 h (n = 5). Data shown in (H) are pooled from five independent replicates.
  • J, K
    Caspase1 (Casp1) mRNA relative expression (J) and Western blot protein analysis (K) of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 3 and 6 h respectively. Data shown are pooled from three independent experiments.
  • L
    IL‐1β ELISA of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS and either vehicle control (Vh, DMSO) or Ac‐YVAD‐cmk for 24 h. Data shown are pooled of five independent replicates.
  • M
    IL‐1β ELISA of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with either vehicle control (Vh), 5 mM ATP or 20 μM nigericin. Data shown are pooled of five independent replicates.
  • N
    Representative picture of confocal immunofluorescence analysis of Gata6‐WT and Gata6‐KOmye pMФ stimulated with 100 ng/ml LPS for 3 h, followed by a 30 min pulse with 5 mM ATP. The white arrows show ASC specks. Scale bar = 10 μm.
Data information: Data are expressed as mean ± SEM and analysis were performed using two‐way ANOVA analysis Tukey's multiple comparison post‐test unless otherwise stated. *P < 0.05, **P < 0.01, ***P < 0.001.