Figure 1

Primary cilia downregulate NRF2 activity via mTOR-dependent autophagy. (a) Representative immunofluorescence images of ARL13B, acetylated α-tubulin (AcTub) and γ-tubulin (γTub) in control (Kif3a^+/+, WT), Kif3a^−/− and Ift88^−/− MEFs starved 24 h to induce ciliogenesis. Nuclei were counterstained with DAPI. Scale bar, 5μm. (b) Expression of NRF2 target genes Hmox1 and Gclc was analyzed by RT-qPCR in Kif3a^+/+ and Kif3a^−/− MEFs starved for 24 h and then treated for 6 h in starvation medium with vehicle (DMSO) or 20 μM dimethyl fumarate (DMF), as indicated. Data are β-actin (Actb)-normalized relative mRNA levels (mean ± SEM, n = 4 independent experiments). (c) Same as in (b) using Ift88^+/+ and Ift88^−/− MEFs (mean ± SEM, n = 3 independent experiments). (d) Kif3a MEFs treated as in (b) were analyzed by Western blot with antibodies against NRF2 and α-tubulin, as loading control. Molecular weight markers in kilodaltons are on the right. Quantitation of NRF2/tubulin band intensity ratio is shown below. The compared bands were in the same gel and blot. See methods for details. Uncropped blots are shown in Supplementary Fig. S12. (e,f) Hmox1 (e) and Gclc (f) were analyzed as in (b) in Kif3a MEFs treated with DMF (20 μM), Torin-1 (250 nM) or Rapamycin (200 nM), as indicated. Data shown as in (b) (mean ± SEM, n = 6 independent experiments). (g) Kif3a MEFs were serum-starved for 24 h and then incubated for 4 h in Earle’s balanced salt solution (EBSS) with or without 10 μM Chloroquine and the indicated drugs, as in (e-f). Cell lysates were analyzed by Western blot with anti-LC3B antibodies. Each sample’s LC3B-II/LC3B-I ratio is shown below, together with relative autophagy flux (LC3B ratio increase by Chloroquine, normalized to control). (h) Kif3a MEFs treated as in (e-f) were analyzed by Western blot and quantitated as in (d). Statistical analysis in (b-c) and (e-f): two-way ANOVA followed by Tukey’s multiple comparisons tests. Asterisks indicate *p < 0.05, **p < 0.01 or ***p < 0.001.