Fig. 7: Gmnc suppression by NOTCH mediates multiciliation defects in CP development and tumorigenesis.
A Median FKPM (fragments per kilobase of exon per million reads mapped) values of genes in NOTCH-driven CP tumors and wild type CPs (n = 3 specimens per time point, mean ± s.e.m., two-tailed unpaired t-test, *P < 0.05; **P < 0.01). B RT-qPCR analysis of NOTCH-driven CPP and wild type CP (n = 3 animals per time point, mean ± s.e.m., two-tailed unpaired t-test, ***P < 0.001, ****P < 0.0001). Three independent experiments were conducted. C The expression of ARL13B (red) is shown in tumor cells infected with viruses expressing GMNC-myc, MCIDAS-myc, or control only. GMNC-myc (green), or MCIDAS-myc (green) labels infected cells. DAPI staining (blue) labels nuclei. Scale bar, 20 µm. Quantification of the percentage of MCCs in infected cells is shown on the right (n = 4 per treatment, mean ± s.e.m., one-way ANOVA, *P < 0.05). Results were obtained from three independent experiments. D The expression of Ki-67 (red) is shown in tumor cells from Lcre;NICD1 mice infected with viruses expressing GMNC-myc. GMNC-myc (green) labels infected cells. DAPI staining (blue) labels nuclei. Scale bar, 20 µm. Quantification of Ki-67 expression in tumor cells from Lcre;NICD1 mice infected with viruses expressing GMNC-myc or control vectors is shown in the lower panel (n = 5 per treatment, mean ± s.e.m., paired t-test, ****P < 0.0001). Data represent at least three independent experiments. E Representative images of Gmnc expression (green) by RNAscope are shown in tumor cells treated with vehicle or IMR-1. DAPI staining (blue) labels nuclei. Scale bar, 20 µm. Quantification of Gmnc transcript is shown (n = 7 per treatment; mean ± s.e.m., two-tailed unpaired t-test, ***P < 0.001). Three independent experiments were conducted. F The expression of ARL13B (red) is shown in Gmnc-deficient tumor cells treated with vehicle or IMR-1/IMR-1A. GFP (green) labels tumor cells. DAPI staining (blue) labels nuclei. Scale bar, 20 µm. Data represent five independent experiments. G RT-qPCR analysis of Foxj1 expression in tumor cells treated with vehicle or IMR-1 (tumors from Lcre;NICD1 mice: n = 6 per treatment; tumors from Lcre;NICD1;Gmncflox/− mice: n = 4 per treatment, mean ± s.e.m., paired t-test, *P < 0.05, NS, not significant). Results were obtained from three independent experiments. H Quantification of Ki-67 expression is shown in Gmnc-deficient tumor cells treated with vehicle or IMR-1 (n = 6 per treatment, mean ± s.e.m., two-tailed unpaired t-test, NS not significant). Three independent experiments were conducted.
