Figure 2.

Depletion of STING or cGAS abolishes 5-FU/oxaliplatin mediated cGAS/STING activation in colon cancer cells. (A-D) Sting1 knockout (Sting1^KO ) was performed by CRISPR/Cas9 in MC38 cells using two guide RNAs (g1 and g2). Cells were treated with 2 μM 5-FU or 20 μM oxaliplatin (OXA) for 24 hours. Western blot analysis was performed to examine expression of STING, P-STING (S365), TBK1, P-TBK1 (S172), STAT1, P-STAT1 (Y701) and ISG15 (A, C). IFNβ expression was determined by Q-PCR analysis (B, D). (E-H) Cgas knockout (Cgas^KO ) was performed by CRISPR/Cas9 in MC38 cells using two guide RNAs (g1 and g2). Cells were treated with 2 μM 5-FU or 20 μM oxaliplatin (OXA) for 24 hours. Western blot analysis was performed to examine expression of TBK1, P-TBK1 (S172), STAT1, P-STAT1 (Y701) and ISG15 (E, G). IFNβ expression was determined by Q-PCR analysis (F, H). (I, J) Cgas or Sting1 knockout (Cgas^KO or Sting1^KO ) was performed by CRISPR/Cas9 in CT26 using two guide RNAs. Single cell clones (#9 and #11) of Cgas^KO cells were isolated. Other knockout cells were pools. Cells were treated with 2 μM 5-FU plus 10 μM oxaliplatin (OXA) for 24 hours. Western blot analysis was performed to examine expression of STING, P-STING (S365), TBK1, P-TBK1 (S172), STAT1, P-STAT1 (Y701), cGAS and ISG15 expression (I). IFNβ and CXCL10 expression was determined by Q-PCR analysis (J). (K, L) STING1 was knocked out in HT29 cells by two gRNAs (g1 and g2). Cells were treated with 5 μM 5-FU plus 20 μM oxaliplatin (OXA) for 48 hours. Western blot analysis was performed to examine expression of STING, P-STING (S366) and ISG15 (K). IFNβ expression was determined by Q-PCR analysis (L). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.