Fig 2: Immunocytochemical analysis of Sonic hedgehog (SHH)-signaling proteins in BCB- and BCB+ porcine cumulus-oocyte complexes (COCs). Representative images of porcine COCs of each group stained with antibodies directed against SHH, PTCH1, SMO, and GLI1 and comparison of expression in cumulus cells and oocytes after 0 h (a–c) and 44 h (d–f) of in vitro maturation, respectively. Within each category, groups marked with different letters are significantly different (p < 0.05). Bar = 100 μM.

Fig 3: SLITRK5 regulates SMO ciliary enrichment upon SHH stimulation.a Immunostaining for SLITRK5 (anti-Flag antibody, Green) and acetylated tubulin (Red) in osteoblasts treated with BSA or SHH for 4 h. Data are representative of three independent experiments, scale bar = 5 µm. b Quantification of the relative fluorescence intensity of ciliary SLITRK5-flag. n = 40–50 cells per group. Data are presented as mean ± s.d, two-tailed unpaired t test. c, d Ciliary localization of SMO-GFP (c) and PTCH1- Flag (d) in WT and Slitrk5−/− osteoblasts treated with BSA or SHH for 4 h. Data are representative of three independent experiments, Scale bar = 5 µm. e, f Quantification of the relative fluorescence intensity of ciliary SMO-GFP (e) and PTCH1-Flag (f) in WT and Slitrk5−/− osteoblasts treated with BSA or SHH for 4 h. n = 100–300 cells per group. Data are presented as mean ± s.d, One-way ANOVA (P < 0.0001) followed by a Tukey’s post hoc test.

Fig 4: Sonidegib treatment reduces taste buds (TB), SHH ligand and proliferation in rat fungiform papilla (FP) while innervation is retained. (a) Immunofluorescent antibody detection of SHH ligand (red) and K19 (green) for TB cells; K18 (red) for TB cells and Ki67 (green) for cell proliferation, after Vehicle, 16d or 28d Sonidegib treatments. SHH is reduced in association with TB, K19+ cell loss. Asterisks (*) indicate nonspecific SHH immunoproduct in cornified surface cells in 16d Sonidegib image. The Vehicle, K18/Ki67 image shows 3 regions positive for Ki67+ cells (Apical, Basal and Perigemmal). Proliferating cells are lost in Apical FP region after 16–28d Sonidegib. (b) Number of Ki67+ cells in Apical and Basal regions of FP in Vehicle- and Sonidegib-treated mice. Numbers of tongues analyzed are in parentheses. For each tongue 8–10 FP were analyzed. Sonidegib treatment reduces apical epithelial cell proliferation in FP compared to Vehicle. Statistical analysis was one-way ANOVA with Tukey HSD posthoc comparisons (*p ≤ 0.05, compared to Vehicle, APICAL). (c) Immunofluorescent antibody detection of K19 or K18 (red) for TB cells and NF (green) for lingual and CT innervation or P2X3 (green) for CT nerve fibers. Innervation was retained after Sonidegib exposure. Asterisks (*) indicate nonspecific P2X3 immunoproduct in surface layer in Vehicle image. (d) Enlarged images from 28d Sonidegib papillae. Arrows point to NF+ or P2X3+ fibers in the FP epithelium. Throughout, white dotted lines indicate the basal lamina. Yellow dotted lines indicate surface of epithelium. (a,c) Scale bar: 50 μm, applies to all images. (d) Scale bar: 25 μm, applies to both images.

Fig 5: Palmitoyl-CoA Uptake and Shh Palmitoylation in Cells Expressing Hhat(A) COS-7 cells expressing EV, Hhat WT, Hhat H379A, or Hhat Y351A were incubated on ice with 65 μg/ml digitonin for 10 min to permeabilize the plasma membrane, then incubated for 60 min at room temperature with 8 μM NBD-palmitoyl-CoA. 30 min prior to imaging, 1 μM ER Tracker Red was added to the cells. Cells were washed and then imaged by confocal microscopy. Scale bar, 25 μm.(B) NBD-palmitoyl-CoA uptake was monitored in microsomal membranes from HEK293FT cells expressing EV, Hhat WT, Hhat H379A, or Hhat Y351A as described in Figure 2; n = 3.(C) Shh palmitoylation was monitored in COS-1 cells expressing Shh and either pcDNA, WT, or mutant (H379A, Y351A) Hhat. Cells were incubated with 15 μCi [125I] IodoPalmitate for 4 h, lysed, and Shh immunoprecipitated from cell lysates was analyzed by SDS-PAGE. [125I] CPM incorporated into Shh was quantified by phosphorimaging; n = 2.(D) Shh palmitoylation was monitored in microsomal membranes from (B). Membranes were incubated with biotinylated Shh peptide and [125I] IodoPalmitoyl-CoA for 60 min prior to the addition of streptavidin-agarose beads. Beads were washed, and [125I] CPM incorporated into the Shh peptide were determined in a gamma counter; n = 2. See also Figure S2.(E) Shh palmitoylation activity was assayed using purified Hhat as in (D); n = 2.