Effects of combining inhibition of IKKβ with GLI1 on viability. OCI-LY19 cells were transduced with lentiviral particles expressing shRNA-targeting control (Luci.) and GLI1 (shGLI1#2). Luci. or stable GLI knockdown cells were treated with TPCA1 for indicated time periods. After 24 hours, cells were subjected to (A) immunoblotting and (B) cell viability assays. (C) Coculture of DLBCL cell lines with stromal cells (HS-5) in trans-well experiments. Cells were treated with or without TPCA1 and GANT61 or a combination of both for 24 hours. Cell lysates were then subjected to qRT-PCR analysis. Results are normalized to GAPDH mRNA level and expressed as fold changes in mRNA expression compared with control. Data represent the mean and standard deviation of 3 independent experiments. A, DLBCL cells alone; CC, DLBCL cells cocultured with HS-5 cells; G, GANT61; T, TPCA1; T+G, TPCA1+GANT61. (D) For isobologram graph analysis, DLBCL cells were cocultured with stromal cells (HS-5), treated with or without TPCA1 and GANT61 for 48 hours, and subjected to MTS assays. (E) NOD/SCID mice were inoculated subcutaneously in the flank with 5 × 10⁶ OCI-LY10 cells. When the tumor was palpable, the mice were randomly subdivided into 4 groups (control, TPCA1, GANT61, and combined TPCA1 + GANT61), and the average tumor size for each group was determined. The value for each group was set to 0% (Day 0), and all subsequent changes in tumor size for each group will be expressed as a percentage change compared with the starting tumor mass. Control, GANT61, TPCA1, or combined (TPCA1 + GANT61) regimens were administered subcutaneously several centimeters away from the tumor site 3 times per week. Tumor sizes was assessed by standard calipers, and tumor volumes were determined weekly. Data represent the mean and standard deviation of 2 independent experiments (*P < .05). (F) Kaplan-Meier survival curves of mice treated with control, GANT61, TPCA1, or combined (TPCA1 + GANT61) regimens. The log-rank (Mantel Cox) test was used in survival curve analysis (*P < .05). (G) Proposed working model for noncanonical activation of GLI1 in response to TNFα-mediated activation of IKKβ.