Journal: Neoplasia (New York, N.Y.)

Article Title: A G-quadruplex stabilizer, CX-5461 combined with two immune checkpoint inhibitors enhances in vivo therapeutic efficacy by increasing PD-L1 expression in colorectal cancer

doi: 10.1016/j.neo.2022.100856

Figure Lengend Snippet: CX-5461 enhances interferon signaling in CRC cells. a, b Venn diagrams of the potential upstream regulators in HT-29 cells after treatment with low (1 μM) or high (10 μM) doses of CX-5461 and control group provided by IPA with a twofold change cutoff. c Ranking of the candidate upstream regulators of cytokines and group from CX-5461 treatment and vehicle control groups predicted using IPA. d Core analysis of the IFNA2 and IFNB1 signatures in CX-5461-treated and control HT-29 cells. e IFN-α and IFN-β levels in the supernatant of HT-29 and CT-26 cells collected from different posttreatment timepoints analyzed using ELISA. f Total RNAs (5 μg) isolated from HT-29 and DLD-1 cells treated with 5 μM CX-5461 for 24 h and then subjected to RT-qPCR analyses to determine the mRNA levels of the seven predicted putative downstream targets: MYC, CAV1, IFIT1, IFIT2, IFIT3, PLAAT4 , and CDKN1A . Data are presented the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.005, compared with the respective untreated cells at 0 h, Student's t test.

Article Snippet: After centrifugation, the cGAMP levels in the supernatant were determined using a 2′,3′-Cyclic GAMP ELISA Kit (ArborAssay #K067-H1).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Isolation, Quantitative RT-PCR

Journal: Neoplasia (New York, N.Y.)

Article Title: A G-quadruplex stabilizer, CX-5461 combined with two immune checkpoint inhibitors enhances in vivo therapeutic efficacy by increasing PD-L1 expression in colorectal cancer

doi: 10.1016/j.neo.2022.100856

Figure Lengend Snippet: CX-5461 activates IRF-3, instead of NF-κB, through the cGAS-STING pathway. a Relative cGAMP levels in HT-29 cells treated with 5 μM CX-5461 at different posttreatment timepoints analyzed using ELISA. Data are present as the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.005, compared with the untreated cells at 0 h, Student's t test. b Total lysates (30 μg) were prepared from HT-29, DLD-1, and CT26 cells incubated with 5 μM CX-5461 for different durations and subjected to Western blot analyses using p-IRF3, IRF3, and STING antibodies as probes. β-Actin was used as the loading control. c Total lysates (30 μg) prepared from HT-29 and DLD-1 cells were subjected to Western blot analyses using primary antibodies against p-NF-κB (ser536) and total NF-κB. d Nuclear fractions (30 μg) prepared from HT-29 and DLD-1 cells were subjected to Western blot analyses to examine the nuclear levels of NF-κB. Cytoplasmic fractions were analyzed using p- NF-κB (ser536) and total NF-κB. Lamin A/C was used as the nuclear loading control. β-Actin was used as the cytoplasmic fractions loading control. e The expression levels of CCL5, CXCL10, TNF-α, and IL-6 in the supernatants of HT-29 and DLD-1 cells collected after exposure to different dosages were analyzed using ELISA. Data are present as the means ± SDs of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.005, compared with the respective untreated cells, Student's t test.

Article Snippet: After centrifugation, the cGAMP levels in the supernatant were determined using a 2′,3′-Cyclic GAMP ELISA Kit (ArborAssay #K067-H1).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Expressing