Journal: BMC Cancer

Article Title: IΚΚε cooperates with either MEK or non-canonical NF-kB driving growth of triple-negative breast cancer cells in different contexts

doi: 10.1186/s12885-018-4507-2

Figure Lengend Snippet: IKKε supports viability and MEK activation. a ) 30 μg of protein was analyzed in whole cell lysates of breast cancer cells grown to 70% confluence. Cells were treated with vehicle control (Ctl), 2 μM MEK inhibitor (MEKi), 2 μM IKKβ inhibitor (IKKβi), or 2 μM BX795 for inhibition of IKKε (IKKεi) for 6 h before lysate collection. b ) 30 μg of protein was analyzed in whole cell lysates of breast cancer cells grown to 70% confluence after siRNA mediated knockdown of IKBKE (siIKKε). Quantification of three independent replicates reveals increased non-canonical NF-κB proteins with IKBKE knockdown. c ) 30 μg of protein was analyzed in whole cell lysates of MDA MB 468 cells after 1 h exposure to 2 μM BX795 for IKKε inhibition. Quantification of three independent replicates confirms pharmacological inhibition of IKKε activity significantly increased non-canonical NF-κB protein levels. *significantly different from corresponding vehicle control P < 0.05, unpaired T-test ( b - c )

Article Snippet: MEK inhibitor, AZD6244, and a non-selective inhibitor of Ser/Thr kinases, BX795, that inhibits IKKε, among others were purchased from Selleck Chemicals (Houston, TX).

Techniques: Activation Assay, Control, Inhibition, Knockdown, Activity Assay

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