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. 2016 Jan 26;35(5):479–495. doi: 10.15252/embj.201593428

Figure 3. Contribution of calcineurin to the activation of TFE3 in response to tunicamycin.

Figure 3

  1. ARPE‐19 cells infected with either adenovirus expressing TFE3‐WT‐Myc or TFE3‐S321A‐Myc for 16 h. Cells were fixed, permeabilized, and stained with antibodies against Myc. Scale bar, 10 μm. Quantification of the percentage of ARPE‐19 cells with nuclear TFE3‐Myc (means ± SD of 3 independent experiments, Student's t‐test, ****P < 0.0001; n > 400 cells per condition).
  2. Immunoblots of protein lysates from ARPE‐19 cells infected as indicated in (A). Data are representative of three independent experiments.
  3. Immunoblots of TFE3‐Ser321 phosphorylation state in nuclear fractions of ARPE‐19 cells incubated with DMSO (Ctrl.), tunicamycin (Tun.), or Torin‐1. Data are representative of three independent experiments.
  4. Immunoblots of protein of a GST pull‐down from nuclear fractions of MEF cells treated with DMSO or tunicamycin (5 μg/ml) (Tun.) for 16 h. Data are representative of three independent experiments.
  5. Immunoblots of protein lysates from ARPE‐19 cells incubated in the presence of DMSO (Ctrl.), tunicamycin (5 μg/ml) (Tun.), or starved in EBSS (Strv.) for 16 h. Data are representative of three independent experiments.
  6. Quantification of the percentage of ARPE‐19 cells with nuclear TFE3 upon tunicamycin (Tun.) or tunicamycin + FK506 treatments for 16 h (mean ± SD of three independent experiments, one‐way ANOVA versus tunicamycin‐treated cells, ***P < 0.001; n > 400 cells per condition).
  7. Quantification of the percentage of calcineurin‐depleted ARPE‐19 cells with nuclear TFE3 upon tunicamycin (Tun.) or starvation (Strv.) treatments for 16 h (mean ± SD of three independent experiments, one‐way ANOVA versus siNon‐target‐treated cells within the indicated treatment, ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001; n > 400 cells per condition).
  8. Immunoblots of protein lysates from ARPE‐19 cells depleted of calcineurin catalytic subunits PPP3CA and PPP3CB. Data are representative of three independent experiments.
  9. Relative quantitative real‐time PCR analysis of PPP3CA and PPP3CB mRNA transcript levels in ARPE‐19 cells depleted of calcineurin catalytic subunits PPP3CA and PPP3CB (mean ± SD of the RNA fold change of indicated mRNAs normalized to actin mRNA from three independent experiments, one‐way ANOVA versus the siNon‐Target (siNT)‐treated cells, ***P < 0.001).
  10. Quantification of the percentage of ATF4‐depleted ARPE‐19 cells with nuclear TFE3 upon tunicamycin treatment for 16 h (mean ± SD of two independent experiments, one‐way ANOVA versus siNon‐target‐treated cells within the indicated treatment, ***P < 0.001; n > 400 cells per condition).

Source data are available online for this figure.