*4.6. Biological Assays*

Cell density was determined using Trypan Blue (Stemcell Technologies, Vancouver, BC, Canada) and a hemocytometer. Shh-Light II cells were seeded in a 96-well plate with 10,000 cells per well, and grown to complete confluence in the media described above. When cells were confluent, the media was replaced with DMEM supplemented with 0.5% bovine calf serum, and treated with 0.1 ng of N-terminal mouse recombinant Shh (R&D Systems, Minneapolis, MN, USA) dissolved in DMEM, and select alkaloid treatment. In each experiment, the controls and treatment wells contained all vehicles, with a final ethanol concentration of 0.05%. Gli activity in the Shh-Light II cell line was assayed 48 h after treatment with Shh protein and select compounds using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA). The Gli-activity was measured by luminescence emitted from cells using a Synergy H1m Microplate reader (BioTek, Winooski, VT, USA). The Gli-activity determined in the biological assay is presented as a relative response ratio (RRR) as described in the Dual-Luciferase Reporter Assay System manual. Each experiment was performed three times.

**Supplementary Materials:** The following are available online; Supplementary S1–S2, Table S1.

**Author Contributions:** Conceptualization, O.M.M., M.W.T.; Biomass Acquisition, J.M., R.C., J.E.; Alkaloid Extractions, M.W.T., R.C., J.E.; LC-MS Analysis, M.W.T.; Bioactivity Assay, M.W.T., J.F.; Methodology, O.M.M., M.W.T.; Resources, O.M.M.; Data Curation, M.W.T.; Writing—Original Draft Preparation, M.W.T.; Writing—Review & Editing, O.M.M., M.W.T.; Supervision, O.M.M.; Funding Acquisition, O.M.M.

**Funding:** This research was funded by Institutional Development Awards (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grants #P20GM103408 (INBRE) and P20GM109095 (COBRE in Matrix Biology), the National Science Foundation, Grants # 0619793 and #0923535; the MJ Murdock Charitable Trust, Idaho State Board of Education, and Research Corporation. Contents are solely the responsibility of the authors and do not necessarily represent the official views of NIH.

**Acknowledgments:** We also acknowledge support from the Biomolecular Research Centre at Boise State University.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Sample Availability:** Samples of the compounds are not available from the authors.

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