The ptch1-C727VfsX745 and ptch1-S733IfsX736 mutation constructs (Fig. 1B) were created using a QuikChange II XL Site-Directed Mutagenesis kit (Agilent Technologies, Inc., Beijing, China) and an N-terminal HA-tagged PTCH1 plasmid (EX-T0559-M06; GeneCopoeia Inc., Rockville, MD, USA) as the template, according to the manufacturer’s instructions. The primers for the mutants were designed online (www.genomics.agilent.com). The pCMX-cyclin B1 plasmid, containing an N-terminal Myc tag (Addgene, Cambridge, MA, USA), served as a template to construct the cyclin B1^Glu derivative with the 4 Ser phosphorylation sites in the CRS domain mutated to Glu to mimic a phosphorylated state. We synthesized CRS^Glu and its derivative modified by appending a nuclear localization signal (NLS, NLS-CRS^Glu), and then inserted it into the pcDNA3.1 vector (Invitrogen Life Technologies Corp., Carlsbad, CA, USA) (28,31). All constructs were verified by DNA sequencing.

Human embryonic kidney 293T cells, originally obtained from ATCC (Manassas, VA, USA) and maintained in our laboratory, were seeded at a density of 1.0×10⁵ cells/plate (60 mm) and transfected using Lipofectamine 2000 (Invitrogen Life Technologies Corp.) with 4.0 mg of empty HA vector (mock), ptch1 (wild-type), ptch1-C727VfsX745 or ptch1-S733IfsX736 DNA. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Invitrogen Life Technologies Corp.) with 10% fetal bovine serum (FBS; HyClone Laboratories, Inc., South Logan, UT, USA). Following transfection, we counted the cells every 24 h using a Coulter Counter (Beckman Coulter Inc., Brea, CA, USA). The results represent the means ± SD of 3 separate transfections.

NIH3T3 cells (preserved in our laboratory) grown to 80% confluence in 24-well plates were co-transfected with 4 mg of GLI1-luciferase reporter construct (Genentech Inc., San Francisco, CA, USA) and 2 mg of the empty HA vector (mock), ptch1, ptch1-C727VfsX745, or ptch1-S733IfsX736 constructs using Lipofectamine 2000. The cells were serum-starved with 0.2% FBS for another 24 h, after which the cells were lysed. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA) at 2 days after transfection. Each assay was performed multiple times, and the results shown represent the means ± SD of 3 independent experiments, presented as GLI1-luciferase reporter activity in the transfected cells.

At 48 h after transfection, the cells were rinsed with phosphate-buffered saline (PBS) and then lysed in TNE lysis buffer (10 mMTris-HCL, pH 7.5, 150 mMNaCl, 1 mM EDTA, 0.5% NP 40) containing PhosSTOP Phosphatase Inhibitor Cocktail (Roche Diagnostics, Indianapolis, IN, USA) and Complete Protease Inhibitor Cocktail (Roche Diagnostics) for 30 min on ice. Generally, cells from one 10-cm dish were lysed with 0.5 ml TNE buffer. The samples were spun at 14,000 × g for 15 min at 4°C, and the protein content was standardized using the Bradford protein assay kit (Applygen Technologies Inc., Beijing, China). The samples were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% gels, transferred to nitrocellulose membranes, and immunoblotted for Myc-cyclin B1, HA-tagged ptch1, and HA-tagged mutated ptch1 using anti-Myc-tag (9B11) antibodies (Cell Signaling Technology, Beverly, MA, USA) and anti-HA-tag (C29F4) antibodies (Cell Signaling Technology), each at a 1:1,000 dilution. All samples were probed with the appropriate fluorescein-conjugated secondary antibodies, either mouse or rabbit, at a dilution of 1:15,000. GAPDH was used as a loading control where appropriate.

The 293T cells were seeded at 80% confluence in 60-mm dishes. At 36 h after transfection with the empty vector, ptch1, ptch1-C727VfsX745 or ptch1-S733IfsX736 constructs, the 293T cells were harvested, washed twice with PBS, and fixed in 70% ethanol overnight at 4°C. The fixed cells were washed with PBS prior to incubation with 50 μg/ml RNase and 0.1% Triton X-100 in PBS for 30 min at 37°C. The cells were then stained with propidium iodide (PI) to facilitate the analysis of cell cycle distribution using a FACScan laser flow cytometer (FACSCalibur; Becton-Dickinson, Franklin Lakes, NJ, USA).

At 36 h after transfection, the 293T cells were rinsed with PBS and then lysed in TNE buffer. Following centrifugation (14,000 × g, 15 min, 4°C), the cell extracts were pre-cleared with protein A/G Plus-Agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and then incubated with Myc-tag (9B11) antibodies at a dilution of 1:1,000 and the appropriate Protein A/G Plus-Agarose (Santa Cruz Biotechnology) overnight at 4°C. The samples were washed with TNE buffer and resuspended in SDS-PAGE loading buffer. Finally, the samples were analyzed by SDS-PAGE on 10% gels, transferred to nitrocellulose membranes, and immunoblotted for Myc-cyclin B1 and HA-tagged ptch1 proteins using Myc-tag (9B11) or HA-tag (C29F4) antibodies, each at a 1:1,000 dilution. Western blot analysis was performed using standard procedures.

Two days after transfection using Lipofectamine 2000, COS-1 cells, obtained from ATCC, were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 and blocked with 5% FBS in PBS. Triple immunofluorescence labeling was performed as follows: i) Myc-tagged CRS^Glu and NLS-CRS^Glu derivatives were detected with Myc-tag (9B11) antibodies and Alexa Fluor 555 Conjugate (Cell Signaling Technology); ii) HA-tagged ptch1, HA-tagged ptch1-C727VfsX745, or HA-tagged ptch1-S733IfsX736 construct expression was detected using anti-HA-tag (C29F4) antibodies and Alexa Fluo 488 Conjugate (Cell Signaling Technology); iii) nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The cells were detected using the laser confocal scanning microscope imaging system.

Data were analyzed using SPSS software (version 13.0), and significant differences between numerical groups were determined using the independent t-test. P-values <0.05 were considered to indicate statistically significant differences. All statistical tests were two-sided.

We examined the effects of the 2 PTCH1 mutations, which introduce premature truncations within the large ICL of ptch1 protein (Fig. 1B), using cell proliferation assays in 293T cells transiently transfected with the appropriate constructs (Fig. 2A). Transfection with ptch1 (wild-type) significantly decreased cell numbers by 15–33% compared with the mock-transfected cells. Moreover, transfection with ptch1-C727VfsX745 or ptch1-S733IfsX736 markedly increased cell numbers by 32–64% and 30–49%, respectively, at 2–4 days following transfection, as compared with the ptch1-transfected cells, but only marginally increased cell numbers by 8–20% or 4–12%, respectively, compared with the mock-transfected cells. These data suggest that the overexpression of wild-type ptch1 inhibited cell growth, which is consistent with the hypothesis that ptch1 acts as a tumor suppressor (32). However, the expression of the mutant constructs, ptch1-C727VfsX745 and ptch1-S733IfsX736, did not lead to the inhibition of cell proliferation. Importantly, our results revealed that ptch1 and the mutant proteins were successfully expressed in the 293T cell lysates, as shown in the inset of Fig. 2A.

The overexpression of wild-type ptch1 did not increase GLI1 activity compared with the mock-transfected cells. By contrast, ptch1-C727VfsX745 and ptch1-S733IfsX736 expression markedly increased GLI1 reporter activity compared with the ptch1-transfected cells by 106 and 99%, respectively (Fig. 2B). These results demonstrated that the expression of these 2 ptch1 mutant proteins induced GLI1 reporter activity, thereby activating intracellular signaling associated with the Hh pathway.

To confirm the effects of these 2 mutations on the activity of cyclin B1, we performed flow cytometric analysis to determine whether these mutants affect the cell cycle distribution. In the 293T cells overexpressing ptch1-C727VfsX745 or ptch1-S733IfsX736, the percentage of cells in the G₂ phase decreased by approximately 5- or 3-fold compared to the ptch1-transfected cells (Fig. 2C). These results suggested that the ptch1 mutants promoted the rapid progression of the 293T cells through the G₂/M phase of the cell cycle.

In order to determine whether ptch1-C727VfsX745 or ptch1-S733IfsX736 are associated with cyclin B1, we performed co-immunoprecipitation experiments. We found that neither ptch1 mutant constructs co-immunoprecipitated with cyclin B1^Glu well. By contrast, wild-type ptch1 bound to cyclin B1^Glu immunoprecipitates (Fig. 3). These results indicated that the prematurely truncated mutants, ptch1-C727VfsX745 and ptch1-S733IfsX736, exhibited reduced binding with phosphorylated cyclin B1 as compared with wild-type ptch1.

The ptch1, ptch1-C727VfsX745 and ptch1-S733IfsX736 proteins were localized to the membrane (Fig. 4A). The CRS^Glu entered the nucleus (Fig. 4Ba and b), and NLS-CRS^Glu was constitutively localized to the nucleus (Fig. 4Bc and d).

As endogenous ptch1 expression levels in the COS-1 cells are low, we co-transfected these cells with an empty vector (mock), HA-tagged ptch1, HA-tagged ptch1-C727VfsX745 or HA-tagged ptch1-S733IfsX736 constructs with the NLS-CRS^Glu construct to determine whether the ptch1 mutations in the C-terminus of ICL alter its subcellular localization. NLS-CRS^Glu exhibited altered localization to the membrane when co-expressed with ptch1 (Fig. 4Ce-h). By contrast, NLS-CRS^Glu retained its nuclear localization in the presence of ptch1-C727VfsX745 and ptch1-S733IfsX736 (Fig. 4Ci-p). Thus, these data suggest that ptch1-C727VfsX745 and ptch1-S733IfsX736 do not alter the localization of phosphorylated cyclin B1.