Fig 2: Gene and protein expression of HH-GLI pathway components in HNSCC. (A) Relative gene expression of PTCH1, GLI1, GLI2, and GLI3 determined by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Gene expression is shown relative to the level of the housekeeping gene RPLP0. (B) Protein expression determined by Western blot. FL refers to the full-length protein (activator form), while R refers to the repressor form.

Fig 3: Clinically achievable plasma concentrations of MBZ effectively inhibit GLI signaling. (A) Two patients with refractory AML were treated with MBZ monotherapy with a daily dose of up to 50 mg/kg. Patient blood samples taken at different time points over the period of 16 to 17 days. Isolated plasma samples were incubated GLI luciferase reporter cell line OCI-AML3 for 24 h before GLI promoter activity was measured. A healthy volunteer ingested MBZ at a dose of 50 mg/kg divided into two doses at time 0 h and 12 h. Blood was drawn at 4 h and at 24 h. In a modified Plasma Inhibitory Assay (PIA) MV4-11 GLI luciferase reporter cells were incubated with isolated plasma samples and GLI promoter activity measured after 24 h using the Dual-GLO Luciferase Assay Kit and the Infinite F200 PRO reader. Patient 1 (B) and patient 2 (C) with refractory AML were treated with MBZ monotherapy as described above. Under MBZ therapy, the blast counts in the peripheral blood of the AML patients were measured. Peripheral mononuclear cells (PBMC) of patient 1 and patient 2 were isolated from several samples throughout the entire treatment period and GLI2 analyzed by western blot analysis. ß-Actin was used as a loading control. Error bars represent the mean values ± standard deviation.

Fig 4: GLI2 dot disappears from the mother centriole in CEP164-mutated Panc1 cells. (A–C) The indicated cells were cultured in serum-fed medium for 48 h and immunostained with anti-glutamylated tubulin (green) and anti-GLI2 (red) antibodies. (A) DNA was stained with Hoechst (blue). Scale bar, 2.5 µm. (B) The percentage of cells with GLI2 dot at the non-ciliated centrosome was determined. The average of three independent experiments is shown; >100 cells were scored each time. (C) The quantified fluorescence intensity of GLI2 at centrosome is shown. n = 31 (WT + EV), 25 (Cep164-1 + EV), 35 (Cep164-1 + Cep164). (D) Panc1 cells were cultured in serum-fed medium for 48 h and immunostained with anti-CP110 (red), anti-CEP164 (blue), and anti-GLI2 (green) antibodies. Two representative images are shown. Scale bar, 2.5 µm. (B,C) All data are shown as mean ± SEM. **p < 0.01 compared with Cep164-1 + EV (B) (Chi-squared test), compared with Cep164-1 + EV (C) (two-tailed Student’s t-test).

Fig 5: Nek2A attenuates transcriptional activity of Gli2. (A) Nek2A regulates Hh components. HEK293T cells were transfected with miR-Nek2A-80, -202 and -603 for 48 h. The components of Hh signaling were detected by western blotting with indicated antibodies. GAPDH serves as a loading control. (B) Quantitation of the data in (A) by ImageJ. GAPDH level is used for data normalization. Error bars represent the standard deviation of three independent experiments. *P<0.05 and **P<0.01 compared with the control groups. (C) mRNA levels of Gli1 and Gli2 in miR-Nek2A treated cells. Total mRNA was extracted and quantified by real-time PCR. Error bars represent the standard deviation of three independent experiments. (D) Overexpression of Nek2A attenuates transfection activity of Gli2. HEK293T cells were co-transfected with Nek2A-Myc and Gli2, together with Bcl-2 luciferase reporter plasmid using Lipofectamine 2000 reagent. TK-Renilla construct was introduced to normalize firefly luciferase activity. The reporter gene activity was assayed by the Dual Luciferase assay system. All experiments were performed in triplicate. Error bars represent the standard deviation of three independent experiments. **P<0.01 compared with the control groups.