Shh light 2 cells

HEK 293T cells were transfected using Lipofectamine 2000 with empty vector control, Hhip mutants or ShhN DNA. One day after transfection, cells were washed with PBS and the medium was switched to Optimem. After another 48 hours, cell medium was collected and used in the coculture assay. For the co-culture assay, MIA PaCa-2 cells (ATCC CRL-1420) were cultured together with Shh-LIGHT II cells (ATCC CRL-2795) at 10⁶ cells in 5 mL DMEM containing 0.5% FCS in 60 mm dishes on rotary shaker at 55 rpm. After spheroid cultures formed (typically 3d), Hhip conditioned medium or 5E1 antibody was added for an additional 24h. Cells were lysed and Gli-luciferase activity was assayed using the Dual Luciferase Reporter Assay Kit (Promega). Relative luminescence units were measured on a Victor plate reader (Perkin Elmer, Waltham MA) and corrected for an internal CMV-driven Renilla luciferase control.

Tumor-associated astrocytes were isolated from MB tissues from Math1-Cre/Ptch1^fl/fl/GFAP-GFP mice at 8 weeks of age. Briefly, MB tissues were digested using papain dissociation system to obtain a single cell suspension as mention above, the cells were suspended in DPBS plus 0.5%BSA, and stained with anti-ACSA-2-APC (1:500, Miltenyi Biotec), TAA were collected by harvesting GFP⁺/ACSA2-APC⁺ cells using fluorescence-activated cell sorting (FACS). For co-culture of TAA with GNPs and MB cells, isolated TAA were culture in PDL-coated wells for 3 days, then purified GNPs or MB cell were added on top of TAA at a ratio of 5 to 1, and co-cultured for indicated time points.
For detection of Shh ligand by ELISA assay, the culture medium for astrocytes was replaced with serum-free DMEM medium after being cultured with serum for 3 days. Conditioned culture medium was harvested 2 days later, the concentration of Shh ligand was measured using the mouse Shh-N ELISA kit (Sigma, MO).
For Luciferase assay, shh-light II cells (ATCC® CRL-2795™) were cultured in DMEM with 10% FBS. After the cells reached 70–80% confluence, the medium was replaced with DMEM without FBS (naïve culture medium), 50% Shh-CM or TAA-CM for 2 days, then luciferase levels in shh-light II cells were measured using Dual-Luciferase® Reporter Assay (Promega).