Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Enhanced IFN Sensing by Aggressive Chronic Lymphocytic Leukemia Cells.

doi: 10.4049/jimmunol.2200199

Figure Lengend Snippet: FIGURE 1. IFN production and response of CLL cells in vitro. Purified CLL cells with U-CLL (n 5 26) or M-CLL (n 5 27) IGHV loci were cultured in AIM V media (A). CLL cells from nine additional patients were stimulated with the TLR7 agonist resiquimod (S28; 1 mg/ml) in the presence or absence of ibrutinib (400 nM) (B). After 24 h, IFN-a levels in the supernatants were measured as described in the Materials and Methods. Freshly purified CLL cells were stimulated with the supernatant of a 7-d-old culture from an unrelated sample for 30 min (C) or with S28 for 24 h (D) in the presence or absence of ani- frolumab (ANI). p-STAT2 levels were then measured by immunoblotting and densitometry using b-actin as a loading control. CLL cells in AIM V were cul- tured with inhibitors of TBK1 (BX795 [1 mM] and GSK8612 [5 mM]) or STING (H151 [1 mM]) for 24 h (E). Other samples were cultured with the TBK1 inhibitors or the IRAK4 inhibitor (1 mM) for 24 h and stimulated with S28 for another 24 h (F). p-STAT2, -IRF3, and -IRF7 levels were then measured by immunoblotting and quantified by densitometry using b-actin as a loading control. Representative immunoblots are shown. Averages and SEs are indicated in the graphs with the dots and lines representing different patient samples. p values were calculated with one-way ANOVA with multiple comparisons, paired and unpaired Student t tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The TBK1 inhibitor BX795 and PTPN2 inhibitor SF1670 were from Tocris Bioscience (Toronto, ON, Canada).

Techniques: In Vitro, Purification, Cell Culture, Western Blot, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Hepatocyte GPCR signaling regulates IRF3 to control hepatic stellate cell transdifferentiation

doi: 10.1186/s12964-023-01416-6

Figure Lengend Snippet: PKA induces tyrosine phosphorylation of IRF3. A HEK293 and HEK293T cells were co-transfected with 200 ng of Flag-IRF3 and 200 ng of HA-Gαs, respectively. 24 h post transfection, the migration shift of IRF3 was determined by Phos-tag gel electrophoresis. B MIHA and HEK293 cells were co-transfected with 200 ng of HA-IRF3 and 400 ng of Flag-TBK1 (K38M), respectively. 24 h post transfection, cells were treated with or without forskolin (30 µM, 1 h). C MIHA and HEK293 cells were treated with or without forskolin (30 µM, 1 h) in the presence or absence of BX795 (1 µM) for 1 h. The migration shift of IRF3 was determined by Phos-tag gel electrophoresis. D HEK293 cells were treated with or without forskolin (30 µM, 1 h) in the presence or absence of BX795 (10 µM) for 3 h. RT-qPCR was used to detect expression of ISG15 . E HEK293 cells were transfected with 400 ng of Flag-IRF3 (WT) or Flag-IRF3 (5 A) or Flag-IRF3 (5D), respectively. 24 h post transfection, cells were treated with or without forskolin (30 µM, 1 h). F HEK293 cells were transfected with 400 ng of Flag-IRF3 (WT) or Flag-IRF3 (1-375), respectively. 24 h post transfection, cells were treated with or without forskolin (30 µM, 1 h). G HEK293 cells were transfected with 400 ng of Flag-IRF3 (WT) or Flag-IRF3 (5 A) or Flag-IRF3 (8 A) or Flag-IRF3 (9 A), respectively. 24 h post transfection, cells were treated with or without forskolin (30 µM, 1 h). H LATS1/2 knockout or wild-type HEK293 cells were transfected with 200 ng of Flag-IRF3 (WT), respectively. 24 h post transfection, cells were treated with or without forskolin (30 µM, 1 h). Statistical data were expressed as mean ± SEM; * p < 0.05 and ** p < 0.01

Article Snippet: Glucagon (#24204), Forskolin (#11018) and BX795 (#14932) were from Cayman.

Techniques: Phospho-proteomics, Transfection, Migration, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Expressing, Knock-Out