ikkβ inhibitor ikk 16  (MedChemExpress)

Journal: bioRxiv

Article Title: An APP-centered molecular gateway integrates innate immunity and retinoic acid signaling to drive irreversible metamorphic commitment

doi: 10.64898/2026.01.22.700939

Figure Lengend Snippet: (A) Settlement rate of inhibitor-treated larvae. The box plots with superimposed jitter plots display the larval settlement rate under various inhibitor treatments. The biofilm stimulus condition is used as the positive control. The concentration of each inhibitor is indicated on the horizontal axis. The data represent the settlement rate of larvae remaining attached out of 10 larvae across six independent biological replicates (total n = 60). Statistical significance among treatment groups was assessed using one-way ANOVA followed by Tukey’s HSD post hoc test, with grouping letters indicating significant differences ( p < 0.05); treatments sharing a letter are not significantly different. (B) Quantitative assessment of the functional hierarchy. The box plots with superimposed jitter plots show the Metamorphic Progression Scores (MPS) for larvae treated with various pharmacological inhibitors with or without all-trans retinoic acid (RA). The MPS was calculated based on the metamorphic stage reached by the larvae in the identical assays used for the settlement rate analysis in (A). The concentration of each inhibitor is indicated on the horizontal axis. The MPS represents the average metamorphic stage reached (0 = brachiolaria; 1 = early; 2 = middle; 3 = late; 4 = pre-juvenile; 5 = juvenile). Statistical significance among the treatment groups was assessed using one-way ANOVA followed by Tukey’s HSD post hoc test ( *p < 0.05; n.s., not significant). A significant RA-dependent rescue condition (a statistically significant increase in MPS compared with the inhibitor-alone condition) is highlighted in grey, establishing the functional hierarchy of the pathways relative to the RA commitment signal. (C) Representative image illustrating pathway functional hierarchy. Images show representative larval morphology under the control, inhibitor-only, and inhibitor + RA conditions. These images specifically represent the high-concentration inhibitor treatments (MyD88 inhibitor: 50 µM; MAPK inhibitors: 10 µM; IKKβ and HSP90AA1 inhibitors: 1 µM). MyD88 inhibition completely blocks the behavioral decision of settlement. JNK and p38 inhibition caused a distinct early-stage arrest (low MPS), and the effects of their inhibition were significantly rescued by RA co-treatment. In contrast, ERK inhibition arrested metamorphosis at the middle stage, and this block was not rescued by exogenous RA. Similarly, IKKβ and HSP90AA1 inhibition arrested metamorphosis at later stages, and this block was not rescued by exogenous RA, functionally placing all three pathways (ERK, IKKβ, and HSP90AA1) downstream of the RA commitment signal. Scale bar: 200 µm. Inhibitors used: T6167923 (MyD88 inhibitor), IKK-16 (IKKβ inhibitor), U0126 (ERK inhibitor), SP600125 (JNK inhibitor), SB202190 (p38 inhibitor), and Luminespib (HSP90AA1 inhibitor).

Article Snippet: The inhibitors were dissolved in DMSO and applied at the indicated concentrations: the MyD88 inhibitor T6167923 (5 or 50 μM; MedChemExpress), the IKKβ inhibitor IKK-16 (0.1 or 1 μM; MedChemExpress), and MAPK inhibitors SP600125 (JNK), SB202190 (p38), and U0126 (ERK) (1 or 10 μM; MedChemExpress or FUJIFILM Wako Pure Chemical Corporation), and the HSP90AA1 inhibitors luminespib (0.1 or 1 μM; Chemscene).

Techniques: Positive Control, Concentration Assay, Functional Assay, Control, Inhibition, Blocking Assay

Journal: Advanced Science

Article Title: Regulation of Neuroinflammation by Microglial DUBA‐IRAK1‐IKKβ Signaling Loop

doi: 10.1002/advs.202503972

Figure Lengend Snippet: LPS inhibits proteasome degradation by regulating the formation of DUBA oligomers. A) BV2 cells were transfected with FLAG‐DUBA and HA‐K48 Ub plasmids for 24 h, followed by stimulation with LPS (500 ng mL −1 ) or MG132 (5 µM) for 6 h. Proteins were immunoprecipitated from whole‐cell lysates with anti‐FLAG antibodies and then analyzed by Western blot. B) BV2 cells were left untreated or stimulated with LPS (500 ng mL −1 ) for 30 min before lysis. Whole‐cell lysates were treated with or without BS3 (3 mM) and then analyzed by Western blot. C) BV2 cells were transfected with FLAG‐DUBA and MYC‐DUBA plasmids for 24 h, followed by stimulation with LPS (500 ng mL −1 ) for indicated periods of time. Proteins were immunoprecipitated from whole‐cell lysates and then analyzed by Western blot. D) BV2 cells were transfected with FLAG‐DUBA and MYC‐DUBA plasmids for 24 h. The subcellular distribution of indicated proteins was determined by immunofluorescence. Scale bar, 10 µm. E,F) Schematic diagram (E) and representative immunoblots (F) of the in vitro deubiquitination assay. G) BV2 cells were transfected with indicated plasmids for 24 h. Whole‐cell lysates were analyzed by Western blot. H) BV2 cells were left untreated or stimulated with LPS (500 ng mL −1 ) in the absence or presence of IKK‐β inhibitor (10 µM) for 6 h. Whole‐cell lysates were analyzed by Western blot. Representative immunoblots (left panel) and DUBA quantification (right panel) are shown. Mean ± SEM. ** P < 0.01. I) Schematic diagram of LPS‐induced DUBA stabilization.

Article Snippet: Cells were treated with LPS (CAT#P1010, Solarbio), MG132 (CAT#HY‐13259, MedChemExpress), chloroquine (CAT#HY‐17589A, MedChemExpress), cycloheximide (CAT#HY‐12320, MedChemExpress), actinomycin D (CAT#HY‐17559, MedChemExpress), IKKβ inhibitor (CAT#HY‐13802, MedChemExpress), IKK inhibitor (CAT#HY‐13453, MedChemExpress), p38 inhibitor (CAT#HY‐10578, MedChemExpress), ERK inhibitor (CAT#HY‐12028, MedChemExpress), and/or JNK inhibitor (CAT#HY‐13319, MedChemExpress) as described in figure legends.

Techniques: Transfection, Immunoprecipitation, Western Blot, Lysis, Immunofluorescence, In Vitro

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: ( A ) ATAC-seq profiles depicting chromatin accessibility of the top 10 genes with inflammation-induced (left) and inflammation-independent PU.1 occupancy (right). ATAC-seq profiles in hi-77 −/− cells without inflammation were mined from published data ( GSE201968 ) at loci harboring PU.1 occupancy (CUT&Tag replicate 2) in vehicle-treated (−) and inflammation-treated (+) hi-77 −/− cells. ( B ) Comparison of average ATAC-seq signals from four biological replicates between genes with inflammation-induced PU.1 occupancy and genes with inflammation-independent PU.1 occupancy in (A). ( C ) Dose-response curve of three representative genes from inflammation-induced PU.1 occupancy and inflammation-independent PU.1 occupancy to IKK inhibitor BMS-345541. hi-77 −/− progenitors were pretreated with increasing concentrations of BMS-345541 for 1 hour and treated with both IFN-γ (1 ng/ml) and Pam 3 CSK 4 (100 ng/ml) for 4 hours ( n = 6 biological replicates). hi-77 +/+ treated with or without both agents served as negative controls. Median inhibitory concentration (IC 50 ) for each gene was calculated using nonlinear regression. Cd69 IC 50 was uncalculated. ( D ) The IC 50 of genes with inflammation-induced PU.1 occupancy was compared to those of genes with inflammation-independent occupancy. Individual values in (B) and (D) and means ± SEM in (B) to (D) were shown. Unpaired t test in (B) and (D) and multiple unpaired t test in (C). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: IKKβ inhibitors are from Selleckchem [BMS-345541 (S8044)] or a gift from S. Miyamoto (UW-Madison) [IKK16 (S2882)].

Techniques: Comparison, Concentration Assay

Journal: Science Advances

Article Title: Dual mechanism of inflammation sensing by the hematopoietic progenitor genome

doi: 10.1126/sciadv.adv3169

Figure Lengend Snippet: The hematopoietic progenitor genome uses a dual PU.1-dependent mechanism to sense and respond to inflammation. A gene cohort has inaccessible chromatin in the steady state, and inflammation increases chromatin accessibility and occupancy of the hematopoietic transcription factors GATA2 and PU.1 to activate transcription via an IKKβ-dependent mechanism. At another inflammation-activated gene cohort, GATA2 and PU.1 occupancy precede inflammation, and this mechanism is not compromised by IKKβ inhibition. RUNX1 co-occupies chromatin with GATA2 and PU.1, yet GATA2 and RUNX1 differentially control inflammation-activated transcription, and transcriptional responses involving distinct TLR signaling pathways activated by unique pathogen-associated molecular patterns .

Article Snippet: IKKβ inhibitors are from Selleckchem [BMS-345541 (S8044)] or a gift from S. Miyamoto (UW-Madison) [IKK16 (S2882)].

Techniques: Inhibition, Control, Protein-Protein interactions

Journal: bioRxiv

Article Title: NF-κB signaling directs a program of transient amplifications at innate immune response genes

doi: 10.1101/2025.03.11.641929

Figure Lengend Snippet: ( A ) and ( B ) Volcano plots summarizing transcriptome changes (q<0.1) in fibroblasts following 24-hour transfection of pIC (31.25 ng/mL, A ) or dsDNA (250 ng/mL, B ). ( C ) Heatmap of gene-set enrichment analysis (GSEA) of Hallmarks gene sets. Asterisks in the box denote significance (q-value < 0.1) after Benjamini-Hochberg adjustment of p values. ( D, F ) Percentage of cells undergoing IFNR TSSG in fibroblasts transfected with pIC (D) and dsDNA (F) in the presence or absence of the JAK1/2 inhibitor ruxolitinib (5 μM). Cells were treated with ruxolitinib 1 hour prior to transfection (n =3). ( E, G ) Immunoblot analysis of immune response activation and inhibition for samples in D and F. GAPDH was used as a loading control. ( H, J ) Percentage of cells undergoing IFNR TSSG in fibroblasts transfected with non-targeting (NT) or TBK1 (H) and IRF3 (J)-targeting siRNA for 48 hours prior to pIC transfection. Results represent 3 independent transfections of 2 different siRNAs. ( I, K ) Immunoblot analysis of siRNA target knockdowns and immune response activation from H, and J. GAPDH was used as a loading control. ( L, N ). Percentage of cells undergoing IFNR TSSG in fibroblasts transfected with pIC (H) and dsDNA (J) in the presence or absence of the IKKβ inhibitor TPCA1 (2 μM). Cells were treated with TPCA1 1 hour prior to transfection (n =3). ( M, O ) Immunoblot analysis of immune response activation and inhibition for samples in L and N. GAPDH was used as a loading control. ( P, R ) Percentage of cells undergoing IFNR TSSG in fibroblasts transfected with non-targeting (NT) or IKKβ ( IKBKB ) (P), and RELA (R)-targeting siRNA for 48 hours prior to pIC transfection. Results represent 3 independent transfections of 2 different siRNA. ( Q, S ) Immunoblot analysis of siRNA target knockdowns and immune response activation from P, and R. GAPDH was used as a loading control. Statistical significance was assessed by a 2-tailed student’s t-test. Black dots represent independent biological replicates. Error bars indicate SD.

Article Snippet: The IKKβ inhibitor TPCA1 (Selleck, cat# S2824) (dissolved in DMSO) was used at a final concentration of 2 µM, added to overnight culture media.

Techniques: Transfection, Western Blot, Activation Assay, Inhibition, Control

Journal: bioRxiv

Article Title: NF-κB signaling directs a program of transient amplifications at innate immune response genes

doi: 10.1101/2025.03.11.641929

Figure Lengend Snippet: ( A to L ) CUT&RUN tracks for two biological replicates treated with TNFα (5 ng/mL) for 24 hours for H3K9me3, H3K37me1, H3K9me1, H3K36me3, H4K20me1 and H3K27me3. For each mark, a zoomed 45kb view of the RelA binding site and a 14Mb view are provided. Vertical red line indicates location of the 45kb region. ( M ) H3K4me1 CUT&RUN tracks from two biological replicates of fibroblasts treated with TNFα (5 ng/mL) for 24 hours, with or without TPCA1 pretreatment. ( N ) Scatter plot representing -log 2 p-values of H3K4me1 peaks from both biological replicates of CUT&RUN following TNFα treatment. ( O ) H3K27ac CUT&RUN tracks from two biological replicates of fibroblasts treated with TNFα (5 ng/mL) for 24 hours, with or without TPCA1 pretreatment. ( P ) Scatter plot representing -log 2 p-values of H3K27ac peaks from both biological replicates of CUT&RUN following TNFα treatment. ( Q ) Percentage of fibroblasts undergoing IFNR TSSG from treated for 24 hours with TNFα in the presence or absence of the p300/CBP inhibitor SGC-CBP30 (2 μM). Cells were treated with the inhibitor 1 hour prior to TNFα treatment (n = 3). ( R ) Immunoblot analysis of immune response activation and inhibition for samples in Q. GAPDH was used as a loading control. Unless otherwise noted, statistical significance was assessed by a 2-tailed student’s t-test. Black dots represent independent biological replicates. Error bars indicate SD.

Article Snippet: The IKKβ inhibitor TPCA1 (Selleck, cat# S2824) (dissolved in DMSO) was used at a final concentration of 2 µM, added to overnight culture media.

Techniques: Binding Assay, Western Blot, Activation Assay, Inhibition, Control