Journal: Molecular Medicine Reports

Article Title: Baicalein protects against endothelial cell injury by inhibiting the TLR4/NF-κB signaling pathway

doi: 10.3892/mmr.2017.8266

Figure Lengend Snippet: BAI reduced LPS-induced damage via TLR4/NF-κB signaling. The protein expressions of TLR4/NF-κB signaling were established by (A-D) western blotting. (A) Blots for LPS stimulation demonstrated that (B) TLR4, p-TAK1, p-TBK1 and p-p65 were upregulated by LPS, then downregulated by BAI treatment. (C) MRT67307 treatment and (D) inhibited the effect of BAI on p-TBK1. (E) The immunofluorescent staining of p65 (magnification, ×200) revealed that (F) there was more cell nuclear translocation with LPS stimulation and less with BAI treatment. The data are expressed as the mean ± standard deviation. *P<0.05 vs. control (0 µM BAI/LPS); # P<0.05 vs. LPS group (10 µM LPS only); **P<0.05 vs. LPS+BAI (10 µM LPS, 6.25 µM BAI). BAI, baicalein; LPS, lipopolysaccharide; HUVECs, human umbilical vein endothelial cells; TLR4, Toll-like receptor 4; p, phosphorylated; TAK1, transforming growth factor β-activated kinase 1; TBK1, tumor necrosis factor receptor-associated family member associated nuclear factor-κB activator-binding kinase 1.

Article Snippet: MRT67307 (S7948) was bought from Selleck Chemicals (Houston, TX, USA).

Techniques: Western Blot, Staining, Translocation Assay, Standard Deviation, Control, Binding Assay

Journal: Nucleic Acids Research

Article Title: IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

doi: 10.1093/nar/gkaa271

Figure Lengend Snippet: IKBKE is required for AR-target gene expression and cell growth in treatment-resistant PC cells. ( A ) LNCaP-EnzR cells were reverse transfected in full media with either N/S or two independent siRNAs against IKBKE and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 3). ( B ) LNCaP-EnzR cells were treated as in (A) after 96 h, AR, TMPRSS2, FKBP5 and IKBKE expression was determined by immunoblotting and ( C ) AR, TMPRSS2, FKBP5 and IKBKE mRNA expression determined by qPCR ( n = 3). ( D ) LNCaP-EnzR cells were transfected in full media with either N/S or three pooled siRNAs against IKBKE. After 72 h, cells were re-seeded at a density of 2000 cells per well and cultured for 2 weeks. Colony forming efficiency was assessed by fixing and staining colonies with crystal violet ( n = 3). ( E ) LNCaP-EnzR cells were seeded and left to adhere and grow for 24 h, prior to treatment with either vehicle (DMSO) or the IKBKE antagonists, CAY10576 (5 μM), BX795 (5 μM), MRT67307 (5 μM), or CYT387 (5 μM) and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 2). ( F ) CWR22Rv1 cells were seeded and left to adhere and grow for 24 h, prior to treatment with either vehicle or CAY10576 (5 μM), BX795 (5 μM), MRT67307 (5 μM), or CYT387 (5 μM) and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 2). ( G ) CWR22Rv1 cells were reverse transfected with either N/S or three pooled siRNAs against IKBKE, in full media supplemented with 10 μM Enz and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 120 h ( n = 3). 2way ANOVA, Dunnett's multiple comparisons test used in A, E, F and G **** P < 0.0001. One-way ANOVA paired Dunnetts multiple comparison test used in C and D * P < 0.05, ** P < 0.001.

Article Snippet: CAY10576 (Santa Cruz Biotechnology), MRT67307 (Stratech Scientific), BX795 (Cambridge BioScience), CYT387 (Adooq Biosciences and Cambridge BioScience), Ruxolitinib (Cambridge BioScience) and R1881 (Sigma) were stored at −80°C for no more than 6 months.

Techniques: Targeted Gene Expression, Transfection, Expressing, Western Blot, Cell Culture, Staining, Comparison

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: MRT67307 , Merck , 506306.

Techniques: Cell Differentiation, Expressing, Cell Culture, Binding Assay, 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: Heme-dependent interaction of BACH2 with FBXO22 (A) A comparison of the Heavy/Light ratio of BACH2 and FBXO22 in the SILAC experiment. (B) The interaction of mouse FBXO22 with BACH2. FLAG-FBXO22 and BACH2 were overexpressed in HEK293T cells. FLAG-FBXO22 was immunoprecipitated with an anti-FLAG antibody. The resulting samples were analyzed by immunoblotting using anti-FLAG or anti-BACH2 antibodies. (C) The M2H assay with HEK293T cells in the presence or absence of the TBK1 inhibitor MRT67307 at 0 to 3.0 μM. HEK293T cells were co-transfected with the indicated combinations of the pFN26A-hBACH2 GAL4 DBD fusion expression vector, pFN10A-hFBXO22 VP-16 AD fusion expression vector, pGL4.35-9×GAL4UAS-Luc reporter vector (Promega), pCAG-hTBK1 and pCAG-hTBK1(S172A). After 24 h of incubation, the cells were treated with the TBK1 inhibitor MRT67307 at 0 to 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. (D) The M2H assay showing the interaction of the BTB domain of BACH2 with FBXO22. pFN26A-hBACH2 BTB and pFN10A-hFBXO22 plasmids were transfected into HEK293T cells. After that, each sample was used for the M2H assay. The results are shown as in (C). (E and F) The western blotting analysis indicating that the SCF FBXO22 E3 ligase complex promotes HA-BACH2 polyubiquitination in a heme-dependent manner. Anti-HA or anti-ubiquitin antibodies were used. (G) Namalwa cells were transfected with control or FBXO22 siRNAs and were treated with hemin for the indicated periods in the presence of cycloheximide. Proteins were detected using the indicated antibodies.

Article Snippet: MRT67307 , Merck , 506306.

Techniques: Comparison, Immunoprecipitation, Western Blot, Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Negative Control, Standard Deviation, Control

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: MRT67307 , Merck , 506306.

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