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

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: Identification of Tbk1 gene as a direct target of BACH2 (A) The ChIP-seq binding profiles for BACH2 in Ebf1 KO pre-pro-B cell line (GSE87503) and those of histone H3 lysine 27 acetylation (H3K27Ac) and histone H3 lysine 4 trimethylation (H3K4me3) in mouse primary B cell (GSM1441280 and GSM1441328 in GSE60103 ). The ChIP-seq data were visualized using Integrative Genomics Viewer (IGV v2.11.1 ). Two BACH2-binding sites were found in the promoter (#1) and intron (#2) regions of the Tbk1 gene. Blue arrow indicates the transcription start site (TSS) of the Tbk1 gene. (B) The binding of BACH2 to sites #1 and #2 was demonstrated by ChIP-qPCR in the BAL17 mature B cell line. The results are shown as enrichment relative to samples immunoprecipitated by NRS. (C) The electrophoretic mobility shift assay showing the binding of BACH2 to the MERE-like sequences found in BACH2-binding sites #1 (left) and #2 (right). The 32 P-labeled probes were mixed with the BACH2 and MAFK proteins without any additive (lane 2) or in the presence of an anti-BACH2 antibody (lane 3, anti-BACH2), a normal rabbit serum (lane 4, NRS) or an anti-MafK antiserum (lane 5, anti-MafK). Competition assays using the unlabeled wild-type or mutant probes as competitors were also shown (lanes 6 and 7, no competitor, lane 8, wild-type competitor and lane 9, Mut competitor). The results shown here are representative of those from three experiments. (D) The RT-qPCR analysis of the TBK1 mRNA expression in wild-type and BACH2 KO mouse splenic B cell stimulated with LPS (20 μg/mL) for 1 or 2 days. (E) The western blotting results of wild-type and BACH2 KO mouse splenic B cell. BACH2 was revealed with an anti-BACH2 antibody. TBK1 was revealed with an anti-TBK1 antibody. GAPDH served as an internal control. (F) The effect of BX795 on the TBK1 mRNA expression in wild-type and BACH2 KO splenic B cell stimulated with LPS (20 μg/mL) in the presence or absence of 1 μM BX795 for 1 or 2 days. (G) HEK293T cells were transfected with the indicated reporter and effector plasmids. The amounts of plasmids were as follows: pGL4-4.28- Slc48a1 MARE luciferase reporter (2.0 μg), pEF seapansy (10 ng), pCMV BACH2 (200 ng), and pcDNA FLAG 3.1 TBK1 (200 ng).

Article Snippet: BX795 , CEM , CS-0259.

Techniques: ChIP-sequencing, Binding Assay, ChIP-qPCR, Immunoprecipitation, Electrophoretic Mobility Shift Assay, Labeling, Mutagenesis, Quantitative RT-PCR, Expressing, Western Blot, Control, Transfection, Luciferase

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: TBK1 promotes plasma cell differentiation by inactivating BACH2 (A) Upper left: a schematic representation of the experimental design. Lower left and right panels: BX795 repressed the Blimp-1 gene expression in mouse splenic B cells. Lower left: The expression of the Blimp-1 -EGFP reporter gene determined by a FACS analysis. B220-positive cells from the wild-type Blimp-1 -EGFP mice were stimulated with 20 μg/mL LPS in the presence or absence of 1 μM BX795 on day 1 or day 2. Each gate shows the percentage of EGFP-positive cells. Representative results of a triplication are shown in right panel. The experiment was performed with N = 3. Right: the percentage of EGFP-positive cells cultured with LPS+BX795 or LPS alone in wild-type B cells. The data are presented as the means ± SD of triplicate determinations. The statistical analyses were performed by the use of the Student’s t test. The experiment was performed with N = 3. (B) The binding of BACH2 to the Prdm1 promoter (left panel) and Prdm1 intron5 (right panel) regions was demonstrated by ChIP-qPCR in the BAL17 mature B cell line. BAL17 cells were treated with BX795 for 3 h. Results are shown as enrichment relative to sample immunoprecipitated by a normal rabbit serum (NRS). The experiment was performed with N = 3. (C) The precursor mRNA expression of Prdm1 in wild-type and BACH2 KO splenic B cells treated with BX795 for 3 h was quantified by RT-qPCR. After the stimulation by LPS, the cells were cultured for 12 h before the treatment. The quantities of Prdm1 were normalized to the β-actin mRNA. The data are mean ± SD of three independent experiments. The experiment was performed with N = 3. (D) The mammalian two-hybrid (M2H) assay showing the inhibitory effect of TBK1 and TBK1-S172A on the interaction of BACH2 with NCoR1 (left). The inhibition of the interaction by TBK1 was diminished by a TBK1 inhibitor (right). HEK293T cells were co-transfected with the pGL4.35-9×GAL4UAS-Luc reporter (Promega) vector, pFN26A-hBACH2 GAL4-DBD fusion expression vector and pFN10A-hNCOR1 VP-16 AD expression vector together with either pCAG-hTBK1 or pCAG hTBK1(S172A). After 24 h of incubation, the cells were treated with the TBK1 inhibitor MRT67307 at the indicated concentrations of 0–3.0 μM for 6 h. After that, the cells were lysed, and luciferase activity was measured. The results are shown as fold induction compared to the negative control (pGL4.35-9×GAL4UAS-Luc alone) and represent the mean of triplicates from a representative experiment, with error bars showing the standard deviation.

Article Snippet: BX795 , CEM , CS-0259.

Techniques: Clinical Proteomics, Cell Differentiation, Gene Expression, Expressing, Cell Culture, Binding Assay, ChIP-qPCR, Immunoprecipitation, Quantitative RT-PCR, Inhibition, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Negative Control, Standard Deviation

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet: The localization of BACH2 is regulated by TBK1 (A) Immunocytochemistry for the BACH2 protein in the presence of BX795 in M1 cells. The subcellular localization of BACH2 was evaluated by classification of 120 cells for each condition into two classes: cytoplasm dominant (N < C) and nucleus dominant (N > C). Blue represents nuclei stained by Hoechst, green represents BACH2 and red represents lamin B1. Scale bar represents 10 μm. (B) The subcellular localization of BACH2 in mouse splenic B cells cultured in the presence of BX795. Cells were cultured in the presence of LPS (20 μg/mL) for 12 h and then were treated with 1 or 5 μM BX795 for 3 h. BACH2 was revealed with an anti-BACH2 antibody (SE, short exposer; LE, long exposer). TBK1 was revealed with an anti-TBK1 antibody. p70S6K and p-p70S6K were revealed with anti-p70S6K and anti-p-p70S6K antibodies. Lamin B1 served as a control for the nuclear fraction and GAPDH was used as a control for the cytoplasmic fraction. W, whole-cell extract; C, cytoplasm; N, nucleus. (C) The dose-dependent effect of BX795 on the nuclear accumulation of BACH2 in mouse splenic B cells. Cells were cultured in the presence of LPS (20 μg/mL) for 20 h and then were treated with 1, 3 or 5 μM BX795 for 3 h. BACH2 was revealed with an anti-BACH2 antibody and lamin B1 served as a control for the nuclear fraction. (D) The western blotting results of the BACH2 protein before and after a CIAP-treatment of the whole-cell extracts from M1 cells. β-actin served as an internal control. (E) Namalwa cells were treated with the TBK1 inhibitors (2 μM) for the indicated periods, and BACH2 and actin were detected by western blotting.

Article Snippet: BX795 , CEM , CS-0259.

Techniques: Immunocytochemistry, Staining, Cell Culture, Control, Western Blot

Journal: iScience

Article Title: Heme regulates protein interactions and phosphorylation of BACH2 intrinsically disordered region in humoral response

doi: 10.1016/j.isci.2024.111529

Figure Lengend Snippet:

Article Snippet: BX795 , CEM , CS-0259.

Techniques: Virus, Recombinant, Protease Inhibitor, Transfection, Multiplex sample analysis, Protein Purification, Reporter Assay, Luciferase, Cell Isolation, Plasmid Preparation, Reverse Transcription, SYBR Green Assay, ChIP-qPCR, Sequencing, Negative Control, Software