Fig 2: CMKLR1/Gi reinforces SHH signaling through repression of PKA.A Daoy cells were transfected with indicated siRNAs and subjected to measurement of cellular PKA activity. B Daoy cells were treated with or without MG132 (10 μM) and/or Forskolin (FSK, 50 μM) for 12 h, and were subjected to Western blot analysis. C Daoy cells were transfected with indicated siRNAs, and treated with cycloheximide (CHX, 20 μM) for indicated times. Cell lysates were then prepared for Western blot analysis. D Daoy cells were subjected to knockdown of endogenous Gli2 by siRNA, followed by transfection with the construct of wild-type Gli2 or an S234A mutant. Cells were then treated with FSK (50 μM) and CHX (20 μM), and the lysates were then used for Western blot analyses. E Daoy cells were transfected with the indicated siRNAs and/or constructs for HA-tagged Ubiquitin and Flag-conjugated Gli2 in the presence of MG132. Cell lysates were then prepared and subjected to immunoprecipitation and Western blot analyses. F, G Daoy cells were transfected with indicated siRNAs, and were subjected to Western blot analysis. H ONS-76 cells were incubated with Chemerin and the indicated siRNAs, followed by Gα(i) activity assay. I, J Daoy cells were transfected with indicated siRNAs, and were subjected to CCK-8 assay for cell growth (I) or wound-healing assay for cell migration (J). Bar, 200 μm. Data are representative images or expressed as the means ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig 3: HOXB4 transcriptionally activates SMO and enhances the Hedgehog signaling pathway. (A and B) SMO protein and mRNA after HOXB4 perturbation were measured by immunoblotting and qPCR; β‐actin was used as the loading control. (C) Nuclear and cytoplasmic fractions from 5637 (shNC, shHOXB4 1#, shHOXB4 2#) and RT4 (vector, HOXB4) cells were immunoblotted for GLI1 and GLI2; Lamin B1 and GAPDH were used as nuclear and cytoplasmic markers, respectively. (D) Quantification of nuclear‐to‐cytoplasmic (N/C) ratios of GLI1 and GLI2 in 5637 and RT4 cells based on the immunoblotting results in (C), normalized to the respective control groups. (E) HOXB4 binding motif predicted from JASPAR (matrix MA1499.1) (F) Schematic of the HOXB4 site on the SMO promoter, ChIP‐qPCR primer locations, and the wild‐type (SMO‐WT) and motif‐mutant (SMO‐MUT) promoter–reporter constructs. (G) Luciferase reporter assays in BCa cells transfected with SMO‐WT or SMO‐MUT; firefly luciferase activity normalized to Renilla and expressed relative to control. (H) ChIP‐qPCR analysis of HOXB4 occupancy at the SMO promoter (top panels) and a negative‐control HPRT1 promoter region (bottom panels) in 5637 cells with stable HOXB4 knockdown (left) and RT4 cells with HOXB4 overexpression (right). ChIP was performed with anti‐HOXB4 or normal IgG; enrichment quantified using the percent‐input method. The results are shown as the mean ± standard deviation (SD) from triplicate experiments. Two‐group comparisons (B) were analyzed using a two‐tailed unpaired Student's t‐test. Multiple‐group comparisons (B, D, G, H) were analyzed using one‐way ANOVA. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001, Abbreviation: ns, not significant.

Fig 4: TGF-β1/SMAD3 promotes the nuclear translocation and expression of GLI2. (A, B) Representative immunofluorescence images showing α-SMA (red) and GLI2 (green) in primary PSCs treated with TGF-β1 (A) or SIS3 (B) for 12 hours (n = 4). Nuclei were stained with DAPI (blue). Scale bar: 25 µm. The percentage of GLI2 in the nuclear area, as well as GLI2 and a-SMA fluorescence intensities, were quantified (n = 4). (C, D) Representative immunofluorescence images showing α-SMA (red) and GLI2 (green) in primary PSCs treated with TGF-β1 (C) or a combination of TGF-β1 and SIS3 (D) for 4 days (n = 4). Nuclei were stained with DAPI (blue). Scale bar: 25 µm. The percentage of GLI2 in the nuclear area, as well as GLI2 and α-SMA fluorescence intensities were quantified. (E, F) Protein levels of GLI2, SMAD3, p-SMAD3, and α-SMA were assessed by Western blot in primary PSCs treated with single or combined treatments of TGF-β1 and SIS3 for 4 days. β-actin was used to normalize protein levels. GLI2, α-SMA and the p-SMAD3/SMAD3 ratio were quantified across treatment groups (n = 3). (G) Co-IP of endogenous GLI2 and p-SMAD3. Primary PSCs were treated with 10 ng/mL TGF-β1 for 48 hours. (H) Representative immunofluorescence images showing colocalization of GLI2 and p-SMAD3 in TGF-β1-induced PSCs. D2 PSCs were treated with 10 ng/mL TGF-β1 for 48 hours (n = 4). Scale bar: 25 μm. Pearson's correlation coefficient was quantified (n = 4). Western blot lanes were reconstructed, and original blots are provided in Figure S5. All data are presented as mean ± SEM. Statistical significance: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.