Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

This work focuses on the impact of oxidizing and reducing ash chemistries on the modifications of two porous SiOCH films with varied porosities (8% [low porosity (lp)-SiOCH] and 45% [high porosity (hp)-SiOCH]). The ash processes have been performed on SiOCH blanket wafers in either reactive ion etching (RIE) or downstream (DS) reactors. The modifications of the remaining film after plasma exposures have been investigated using different analysis techniques such as x-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy (FTIR), x-ray reflectometry, mercury probe capacitance measurement $\left(C\text{‐}V\right)$ , and spectroscopic ellipsometry (SE). FTIR analyses show that the lp-SiOCH film is not significantly altered by any of the ash processes investigated ($\mathrm{DS}\text{‐}{\mathrm{H}}_2∕\mathrm{He}$ , $\mathrm{RIE}\text{‐}{\mathrm{O}}_2$ , and $\mathrm{RIE}\text{‐}{\mathrm{NH}}_3$ ), except by downstream oxidizing plasmas ($\mathrm{DS}\text{‐}{\mathrm{O}}_2$ or $\mathrm{DS}\text{‐}{\mathrm{N}}_2∕{\mathrm{O}}_2$ ) which induce some carbon depletion and moisture uptake, resulting in a slight increase of the $k$ value. The porosity amplifies the sensitivity of the material to plasma treatments. Indeed, hp-SiOCH is fully modified (moisture uptake and carbon depletion) under oxidizing downstream plasma exposures ($\mathrm{DS}\text{‐}{\mathrm{O}}_2$ and $\mathrm{DS}\text{‐}{\mathrm{N}}_2∕{\mathrm{O}}_2$ ), while it is partially altered with the formation of a denser and modified layer ($40–60\mathrm{nm}$ thick), which is carbon depleted, hydrophilic, and composed of $\mathrm{Si}{\mathrm{O}}_x{\mathrm{N}}_y{\mathrm{H}}_z$ with $\mathrm{RIE}\text{‐}\mathrm{N}{\mathrm{H}}_3$ and $\mathrm{DS}\text{‐}{\mathrm{N}}_2∕{\mathrm{H}}_2$ plasmas and $\mathrm{Si}{\mathrm{O}}_x{\mathrm{H}}_y$ with $\mathrm{RIE}\text{‐}{\mathrm{O}}_2$ plasma. In all the cases, the $k$ value increase is mainly attributed to the moisture uptake rather than methyl group consumption. hp-SiOCH material is not altered using reducing DS chemistries (${\mathrm{H}}_2∕\mathrm{He}$ and ${\mathrm{H}}_2∕\mathrm{Ar}$ ). The porous SiOCH film degradation is presented and discussed with respect to chemistry, plasma parameters, and plasma mode in terms of film modification mechanism.