Journal: NPJ Precision Oncology
Article Title: Loss of MicroRNA-29b promotes DNMT3b-mediated STING downregulation to attenuate radiotherapy-induced antitumor immunity in KRAS-mutated colorectal cancer
doi: 10.1038/s41698-026-01290-8
Figure Lengend Snippet: A WiDr cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. WiDr-Vec. and WiDr-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. B CoLo320 cells were infected with lentivirus carrying pBabe-puro-vector (Vec.) or pBabe-puro-KRAS G12D and selected for three days. CoLo320-Vec. and CoLo320-KRAS G12D cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05. C HCT116 cells were infected with lentivirus carrying pLKO-scramble shRNA (shNC) or pLKO-shKRAS (shKRAS) and selected for three days. HCT116 shNC and HCT116 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. D SW620 shNC and SW620 shKRAS cells were irradiated (5 Gy), and then harvested for qRT-PCR analysis after 24 hr ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. E WiDr and Colo320DM (endogenous wild-type KRAS) cells were individually infected with vector (Vec.) and KRAS G12D . Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. F CT26, HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shNC and shKRAS. Cells were selected by puromycin for three days. The level of DNMT1, DNMT3a and DNMT3b was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. ** p < 0.01. G KRAS G12D -tranduced WiDr (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. H . KRAS G12D -tranduced CoLo320 (endogenous wild-type KRAS) cells were treated with 2.5 μmol/L and 5.0 μmol/L AZA for two days. The level of STING was evaluated by immunoblotting ( n = 3). One-way ANOVA t test. * p < 0.05 and ** p < 0.01. I HCT116 and SW620 (endogenous mutant KRAS) cells individually infected with lentivirus carry shDNMT1, shDNMT3a and shDNMT3b. Cells were selected by puromycin for three days. The mRNA level of STING was evaluated by qRT-PCR ( n = 3). One-way ANOVA t test. * p < 0.05. J The protein level of STING was evaluated by immunoblotting ( n = 3).
Article Snippet: The retroviral pBabe-puro vector (#1764) and pBabe-puro-KRAS G12D (#58902) were purchased from Addgene.
Techniques: Infection, Plasmid Preparation, Irradiation, Quantitative RT-PCR, shRNA, Western Blot, Mutagenesis
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