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. 2022 Dec 12;24(2):e55472. doi: 10.15252/embr.202255472

Figure EV5. Macrophage polarization is affected in THP1 TFEB‐S401A mutant cells in response to LPS. Related to Fig 6 .

Figure EV5

  1. Immunoblot analysis of protein lysates from PMA‐differentiated (Rested) THP1‐WT or TFEB‐S401A knock‐ins (clones I11 and M17) cells incubated with 1 μg/ml LPS for the indicated times.
  2. Quantification of immunoblot data shown in (A). Data are presented as mean ± SD using one‐way ANOVA (unpaired) followed by Tukey's multiple comparisons test, *P < 0.05 and ***P < 0.001 as compared to the same treatment condition in THP1‐WT cells from three independent experiments.
  3. Immunoblot analysis of proteins from nuclear and cytosolic fractions from PMA‐differentiated (Rested) THP1‐WT or TFEB‐S401A knock‐in (clone I11) cells incubated with 1 μg/ml LPS for the indicated times or 250 nM Torin‐1 for 1 h.
  4. Quantification of immunoblot data shown in (C). Data are presented as mean ± SD using one‐way ANOVA (unpaired) followed by Tukey's multiple comparisons test, (ns) not significant as compared to the same treatment condition in THP1‐WT cells from three independent experiments.
  5. Immunofluorescence confocal microscopy of PMA‐differentiated (Rested) THP1‐WT or TFEB‐S401A knock‐in (clones I11 and M17) cells incubated with 0.1 μg/ml LPS for 4 h prior to the addition of 15 μM Nigericin for 45 min. Arrows point to the position of ASC specks indicative of inflammasome activation. Scale bars, 10 μm.
  6. Quantification of the percentage of cells with ASC specks shown in (E). Data are presented as mean ± SD using one‐way ANOVA (unpaired) followed by Tukey's multiple comparisons test, ***P < 0.001 and ****P < 0.0001 as compared to THP1‐WT cells treated with LPS + Nigericin with > 200 cells counted per trial from three independent experiments.

Data information: n ≥ 3 biological replicates (each dot represents a biological replicate).

Source data are available online for this figure.