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. 2020 Feb 13;18(2):e3000620. doi: 10.1371/journal.pbio.3000620

Fig 4. PTCH1 is required for NRF2-mediated inhibition of ciliary translocation of SMO but not the suppression of primary ciliogenesis by NRF2.

Fig 4

(A) Immunoblot analysis of PTCH1, GLI2FL/R, and GLI3FL/R protein levels in a PTCH1−/− H1299 cell line. Relative quantification of immunoblot results is shown in S7A Fig. (B) GLI luciferase assay in PTCH1+/+ and PTCH1−/− H1299 cells. (C) Immunoblot analysis of PTCH1, Ac-Tub, ARL13B, and NQO1 protein levels following treatment with bixin (40 μM) for 0, 12, 24, 36, or 48 h in PTCH1+/+ and PTCH1−/− H1299 cells. Relative quantification of immunoblot results is shown in S7B Fig. (D) Immunoblot analysis of GLI2FL/R and GLI3FL/R, as well as SMO protein levels, following treatment with bixin (40 μM) for 0, 12, 24, 36, or 48 h in PTCH1+/+ and PTCH1−/− H1299 cells. Relative quantification of immunoblot results is shown in S7C Fig. (E) GLI luciferase assay in the cells treated with bixin for 48 h. (F) IF analysis of Ac-Tub (green) and SMO (red) colocalization in PTCH1+/+ and PTCH1−/− H1299 cells treated with bixin (40 μM) for 48 h. (Scale bar = 5 μm.) (G) % ciliated cells in PTCH1+/+ and PTCH1−/− H1299 cells treated with bixin (40 μM) for 48 h. (Scale bar = 10 μm.) Results are expressed as mean ± SD. A t test was used to compare the various groups, and p < 0.05 was considered statistically significant. *p < 0.05 compared between the two groups. Ac-Tub, acetylated tubulin; ARL13B, ADP-ribosylation factor-like protein 13B; FL, full-length activator; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IF, immunofluorescence; NQO1, NAD(P)H Quinone Dehydrogenase 1; NRF2, nuclear factor-erythroid 2-like 2; PTCH1, Patched 1; R, repressor; SMO, smoothened.