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. 2019 Jan 7;38(4):e99430. doi: 10.15252/embj.201899430

Figure 3. Reduction in BECN1 influences the IL‐1beta processing pathway.

Figure 3

  1. The expression of NLRP3 protein in non treated and LPS/ATP‐treated wild‐type and Becn1 +/− microglia was determined by Western blot and normalized to ACTIN. Reduction in BECN1 significantly enhanced expression of NLRP3; mean ± SEM, wild‐type n = 4, wild‐type LPS/ATP n = 9, Becn1 +/− n = 4, Becn1 +/− LPS/ATP n = 23; ANOVA with Tukey's post hoc test *P < 0.05.
  2. The expression of Nlrp3 mRNA in non treated and LPS/ATP‐treated wild‐type and Becn1 +/− microglia was determined by qPCR. No significant differences can be detected between wild‐type and Becn1 +/− microglia; mean ± SEM, wild‐type: n = 7, Becn1 +/−: n = 3; ANOVA with Tukey's post hoc test, ns for LPS/ATP‐treated wild‐type versus Becn1 +/−.
  3. LPS/ATP‐treated and non treated wild‐type and Becn1 +/− microglia were immunolabeled for ASC (red) and imaged by confocal microscopy to investigate the assembly of inflammasomes. In non‐stimulated cells, ASC is distributed diffusely in the cytoplasm (upper panels). However, after stimulation, formation of inflammasomes (arrowheads) can be detected in a subpopulation of cells as well as release of inflammasomes after pyroptosis/ejection (arrow). Quantification showed a significantly increased percentage of inflammasomes in or around Becn1 +/− microglia; mean ± SEM, wild‐type LPS/ATP n = 4, Becn1 +/− LPS/ATP n = 7; two‐tailed t‐test *P < 0.05; scale bar: 50 μm, insert scale bar: 10 μm.
  4. The presence of cleaved CASP1 (p10) protein in the supernatant of LPS/ATP‐treated wild‐type and Becn1 +/− microglia was determined by Western blot and normalized to ACTIN. Reduction in BECN1 significantly enhanced release of CASP1; mean ± SEM, wild‐type LPS/ATP n = 8, Becn1 +/− LPS/ATP n = 18; two‐tailed t‐test *P < 0.05.

Source data are available online for this figure.