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retroviral pbabe puro vector  (Addgene inc)
96
Addgene inc retroviral pbabe puro vector
A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
Retroviral Pbabe Puro Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral vector pbabe puro sv40 lt  (Addgene inc)
94
Addgene inc retroviral vector pbabe puro sv40 lt
A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
Retroviral Vector Pbabe Puro Sv40 Lt, supplied by Addgene inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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retroviral expression vector pbabe puro mtert  (Addgene inc)
93
Addgene inc retroviral expression vector pbabe puro mtert
A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
Retroviral Expression Vector Pbabe Puro Mtert, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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pbabe blast retroviral vector  (Addgene inc)
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Addgene inc pbabe blast retroviral vector
A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
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retroviral vector pbabe encoding sv40 lt puro antigen  (Addgene inc)
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Addgene inc retroviral vector pbabe encoding sv40 lt puro antigen
A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
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retroviral pbabe puro htert vector system  (Addgene inc)
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A WiDr cells were infected with lentivirus carrying <t>pBabe-puro-vector</t> (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).
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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).

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