Journal: Frontiers in immunology

Article Title: Aspartate Metabolism Facilitates IL-1β Production in Inflammatory Macrophages.

doi: 10.3389/fimmu.2021.753092

Figure Lengend Snippet: FIGURE 4 | Asparagine promotes the production of interleukin 1b (IL-1b) and the activation of signal pathways in macrophages. (A) IL-1b and tumor necrosis factor alpha (TNF-a) secretion in M1 macrophages and asparagine-treated M1 macrophages (n = 4–5). (B) mRNA expressions of IL-1b and TNF-a in M1 macrophages and asparagine-treated M1 macrophages (n = 6). (C–E) Protein abundance of IL-1b, IkB kinase (IKK), p-IKK, IkB, p-IkB, p65, p-65, hypoxia-inducible factor-1a (HIF- 1a), Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck like protein containing a caspase recruitment domin (ASC), and caspase-1 in macrophages (n = 4). (F) IL-1b secretion in M1 macrophages with asparagine combined with IKK16 or VX-765 (n = 3). Data were analyzed with unpaired t-test (A–E) or with one- way ANOVA (F). Error bars represent the mean ± SEM. *p ≤0.05, **p ≤0.01.

Article Snippet: PX-478, MCC-950, VX-765, and IKK16 were purchased from Selleck Chemicals (Houston, TX, USA).

Techniques: Activation Assay, Quantitative Proteomics

Journal: Frontiers in Immunology

Article Title: Nuclear Factor κB is Required for Tumor Growth Inhibition Mediated by Enavatuzumab (PDL192), a Humanized Monoclonal Antibody to TweakR

doi: 10.3389/fimmu.2013.00505

Figure Lengend Snippet: Inhibition of NFκB activation prevents the growth inhibitory activity of enavatuzumab . (A) BT549 cells were transfected with two different siRNAs against p65 or upstream kinases IKK1 (IκBKβ), IKK2 (CHUK). Transfection controls were non-targeting control (negative) or KSP (positive control for transfection efficiency). After 48 h, cells were treated with enavatuzumab or IgG1 control at 10 μg/mL for an additional 5 days in the presence of anti-human crosslinking antibody (3.5 μg/mL), and the cell viability was determined. siRNA significantly reduced growth inhibition by enavatuzumab, compared to mock or control siRNA transfected cells (horizontal line) (* p -value < 0.05). (B) Enavatuzumab sensitive lines were treated with IKK inhibitor IKK16 at 160 nM (black bars), enavatuzumab/crosslinker (gray bars), or IKK16 plus enavatuzumab/crosslinker (striped bars) and % survival was measured after 5 days. IKK16 significantly blocked growth inhibition by enavatuzumab compared to enavatuzumab alone (* p -value < 0.05). (C) Targeting individual NFκB subunits (p50, p65, RelB, p52) by siRNA reduced enavatuzumab activity in sensitive cell lines (MDA-MB-468 and BT549). Cells were transfected with pooled targeting siRNA or control siRNA for 48 h, prior to treatment with enavatuzumab or IgG1 control for 5 days in the presence of a crosslinking antibody. Percent survival was calculated from the relative viability of cells treated with enavatuzumab versus control-treated cells (* p -value < 0.05). (D) The effect of NFκB induced by enavatuzumab on cell division was investigated by pre-labeling HT3 cells with the Cell Trace™ reagent. Cells treated with enavatuzumab/crosslinker (green line) are compared to the IgG1 control-treated cells (red line). Cells transfected with two different siRNAs to p65, p50, p52, or RelB and treated with enavatuzumab/crosslinker (blue and brown lines) are also displayed.

Article Snippet: The IκB kinase (IKK) selective inhibitor IKK16 (Tocris Bioscience) was used at 160 nM ( ).

Techniques: Inhibition, Activation Assay, Activity Assay, Transfection, Control, Positive Control, Labeling

Journal: bioRxiv

Article Title: Transient efferocytosis-induced activation of IKKβ reprograms macrophages to promote tissue resolution

doi: 10.64898/2026.04.27.720940

Figure Lengend Snippet: a Mouse bone marrow-derived Mφs (BMDMs) ( n = 3) or b human monocyte-derived Mφs (HMDMs) ( n = 3) were incubated with or without apoptotic Jurkat cells (ACs) for 45 min and then immunoblotted for phospho- and total p65 and p38. c BMDMs were incubated with apoptotic HMDMs (ApMφs) for 45 min and then immunoblotted as in panel a ( n = 3). d BMDMs were incubated with ACs for 15, 30, or 45 min, or incubated with ACs for 45 min and chased for 1, 6, or 24 hours and then immunoblotted as in panel a ( n = 3). e Mice were injected with PBS or 250 μg dexamethasone (DEX) i.p., and thymi were collected 4 or 8 hours after injection. Tissue sections were labeled with TUNEL (blue) and immunostained for Mac2 (magenta) and phospho-p65 or phospho-p38 (green) and quantified for the MFI of phospho-p65 or phospho-p38 in cytoplasmic TUNEL + (AC + ) or cytoplasmic TUNEL − (AC − ) macrophages. Sections were imaged with a 20x objective. Scale bar, 20 μm ( n = 5). White arrows, AC - macrophages; gray arrows, AC + macrophages. Overlap of Mac2 and phospho-p65 or phospho-p38 appears white in the images. See Extended Data Fig. 1C for PBS and DEX images, with each channel shown separately. f BMDMs incubated with ACs and chased for different periods were immunoblotted for phospho-IKKβ and total IKKβ ( n = 3). g,h BMDMs pre-treated ± 500 nM IKK16 for 2 hours were incubated with or without ACs for 45 min, lysed, and immunoblotted for phospho-p65 and total p65 or phospho-p38 and total p38 ( n = 3). i BMDMs transfected with 50 nM scrambled or siIkbkg for 72 hours were incubated with or without ACs for 45 min and then immunoblotted for phospho-p65, total p65, phospho-p38, and total p38 ( n = 3). j BMDMs pre-treated ± 500 nM IKK16 for 2 hours were incubated with or without ACs for 45 min, chased for 1 hour, and then immunoblotted for p-STAT3 and total STAT3 ( n = 3). k BMDMs pre-treated ± 10 μM SB203580 (p38 inh) for 2 hours were incubated with or without ACs for 45 min, chased for 1 hour, and then immunoblotted as in panel k ( n = 3). Data are normalized to the first control group in each experiment. Bars represent means ± SEM. Statistics were performed using the Student’s t-test in panels a-c and two-way ANOVA in panels e and g-k. ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns = no significance.

Article Snippet: WT, Cd274 fl/+ , or Cd274 fl/+ ; LysMCre +/- BMDMs were plated on 12-well plates and pre-treated with or without 0.5 μM IKK16 (MedChemExpress) for 2 hours before incubating with unlabeled ACs at a 5:1 ratio for 45 min. After a 2-hour chase, the cells were incubated with 6-8-μm phosphatidylserine-coated polystyrene beads (Spherotech; SVP-60-5) for 45 min at a 5:1 ratio of beads:macrophages and then washed with PBS.

Techniques: Derivative Assay, Incubation, Injection, Labeling, TUNEL Assay, Transfection, Control

Journal: bioRxiv

Article Title: Transient efferocytosis-induced activation of IKKβ reprograms macrophages to promote tissue resolution

doi: 10.64898/2026.04.27.720940

Figure Lengend Snippet: a BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 were incubated with or without ACs for 45 min, chased for 1 hour, and assayed for Il10 mRNA ( n = 3). b HMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 were incubated with or without ACs for 45 min, chased for 1 hour, and assayed for IL-10 mRNA ( n = 3). c,d BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 10 μM p38 inhibitor or 2.5 μM Stattic were incubated with or without ACs for 45 min, chased for 1 hour, and assayed for Il10 mRNA ( n = 3). e BMDMs pre-treated with vehicle control (Ctrl) or 500 nM IKK16 for 2 hours were incubated with PKH67 (green) labeled ACs for 45 min, chased for 2 hours, and incubated with PKH26 (red) labeled ACs for 45 min. The cells were fixed, imaged by fluorescence microscopy, and quantified for the percentage of PKH67 + BMDMs that are also PKH26 + ( n = 3). f As in panel e, but the macrophages were treated with 5 µm cytochalasin D 20 min before the addition of the PKH26-labeled ACs ( n = 3). g BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 were incubated with ACs for 45 min, chased for 2 hours, and incubated with or without beads for 20 min before lysis and measuring Rac1 activity ( n = 6). h BMDMs pre-treated with isotype control IgG or anti-IL-10R antibody were used to measure continual efferocytosis ( n = 3). i BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 for 2 hours were incubated with or without ACs for 45 min, chased for 24 hours, and quantified for cell number ( n = 3). j BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 for 2 hours were incubated with or without ACs for 45 min, chased for 3 hours, and immunoblotted for Myc ( n = 3). Bars represent means ± SEM. Statistics were performed using the Student’s t-test in panels e, f, and h and two-way ANOVA in panels a-d and g, i, and j. ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns = no significance.

Article Snippet: WT, Cd274 fl/+ , or Cd274 fl/+ ; LysMCre +/- BMDMs were plated on 12-well plates and pre-treated with or without 0.5 μM IKK16 (MedChemExpress) for 2 hours before incubating with unlabeled ACs at a 5:1 ratio for 45 min. After a 2-hour chase, the cells were incubated with 6-8-μm phosphatidylserine-coated polystyrene beads (Spherotech; SVP-60-5) for 45 min at a 5:1 ratio of beads:macrophages and then washed with PBS.

Techniques: Control, Incubation, Labeling, Fluorescence, Microscopy, Lysis, Activity Assay

Journal: bioRxiv

Article Title: Transient efferocytosis-induced activation of IKKβ reprograms macrophages to promote tissue resolution

doi: 10.64898/2026.04.27.720940

Figure Lengend Snippet: a BMDMs were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Cd274 (PD-L1) mRNA ( n = 3). b BMDMs pre-treated with isotype control IgG or α-MerTK antibody for 2 hours were incubated with PKH26-labeled ACs for 45 min, chased for 6 hours, subjected to non-permeabilizing immunostaining for cell-surface PD-L1, and imaged by confocal microscopy ( n = 4). Scale bar, 20 μm. c BMDMs or HMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Cd274 or CD274 mRNA ( n = 3). d,e BMDMs pre-treated for 2 hours with vehicle control (Ctrl), 10 μM SB203580 (p38 inh), or 2.5 μM Stattic were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Cd274 mRNA ( n = 3). f BMDMs transfected with 50 nM scrambled or siMyc for 72 hours were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Cd274 mRNA ( n = 3). g BMDMs transfected with 50 nM scrambled or siCd274, or h Cd274 fl/+ and Cd274 fl/+ ;LysMCre +/- (PD-L1 het KO) BMDMs were incubated with PKH67-labeled ACs for 45 min, chased for 2 hours, and incubated with PKH26-labeled ACs for 45 min. The images were quantified for the percentage of PKH67 + BMDMs that were also PKH26 + ( n = 3). I As in panel g, but the macrophages were treated with 5 µm cytochalasin D 20 min before the addition of the PKH26-labeled ACs ( n = 3). j Cd274 fl/+ (Ctrl) or Cd274 fl/+ ;LysMCre +/- (PD-L1 Het) BMDMs were incubated with ACs for 45 min, chased for 2 hours, incubated with 6-8 μm polystyrene beads for 20 min, and assayed for Rac1 activity ( n = 6). k,l BMDMs pre-treated for 2 hours with vehicle control (Ctrl) or 500 nM IKK16 were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Tet2 mRNA or immunoblotted for TET2 protein ( n = 3). m,n BMDMs pre-treated for 2 hours with vehicle control (Ctrl), 10 μM SB203580 (p38 inh), or 2.5 μM Stattic were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Tet2 mRNA ( n = 3). o BMDMs transfected with 50 nM scrambled or siMyc for 72 hours were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Tet2 mRNA. p TET2 WT or KO BMDMs were incubated with or without ACs for 45 min, chased for 6 hours, and assayed for Cd274 mRNA ( n = 3). Bars represent means ± SEM. Statistics were performed using the Student’s t-test for panels a and g-i and two-way ANOVA for panels b-f and k-p. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns = no significance.

Article Snippet: WT, Cd274 fl/+ , or Cd274 fl/+ ; LysMCre +/- BMDMs were plated on 12-well plates and pre-treated with or without 0.5 μM IKK16 (MedChemExpress) for 2 hours before incubating with unlabeled ACs at a 5:1 ratio for 45 min. After a 2-hour chase, the cells were incubated with 6-8-μm phosphatidylserine-coated polystyrene beads (Spherotech; SVP-60-5) for 45 min at a 5:1 ratio of beads:macrophages and then washed with PBS.

Techniques: Incubation, Control, Labeling, Immunostaining, Confocal Microscopy, Transfection, Activity Assay

Journal: bioRxiv

Article Title: Reduced SH3RF3 may protect against Alzheimer’s disease by lowering microglial pro-inflammatory responses via modulation of JNK and NFkB signaling

doi: 10.1101/2024.06.23.600281

Figure Lengend Snippet: (A-D) iMGLs treated with either siSH3RF3 or control siRNA were subjected to poly(I:C) or vehicle for 2h. qPCR analysis of SH3RF3 (A), IL1β (B), IL6 (C) and TNFα (D) showed successful knockdown of SH3RF3 and induction of all cytokines upon poly(I:C) treatment which were significantly reduced by SH3RF3 KD. Percentage expression was calculated by normalizing gene of interest with respective loading control (GAPDH) for each sample and then all data were normalized to the poly(I:C) treated control siRNA condition and is presented as mean±SEM for N=3 biological replicates per experiment for three independent experiments (N=9). Comparisons between both control Si with poly(I:C) and SH3RF3 Si with poly(I:C) were made with student t test, *<0.5, **<0.01. (E-G) Multiplex ELISA analysis of inflammatory cytokines from conditioned media from iMGLs treated with poly(I:C) for 16h. IL1β (E), IL6 (F) and TNFα (G) all show significant induction by poly(I:C) which is reduced by SH3RF3 KD. Data is presented as mean ±SEM for N=3 biological replicates per experiment for three independent experiments (N=9). Statistical comparison among multiple groups were made using one-way ANOVA. (H-I) Western blot analysis of JNK and NFκB pathway activation from iMGLs treated SH3RF3 or control siRNA subjected to poly(I:C) for 6 hr. Representative blots for phosphorylated JNK and total JNK (H), phosphorylated p65 and total p65 (I) with densitometry quantification for 3 independent experiments. GAPDH is used as loading control. Data is presented as mean ±SEM for N=3 biological replicates per experiment for three independent experiments (N=9). Statistical comparison among multiple groups were made using one-way ANOVA. (J-L) qPCR analysis of IL1β (J), IL6 (K) and TNFα (L) in iMGLs treated with poly(I:C) for 2 hr, with the presence or absence of pathway inhibitors, all of which reduce transcription of all 3 cytokines. CEP-1347 is a JNK pathway (MLK/MAP3K) inhibitor, IKK16 is a NFκB pathway inhibitor, and NSC23766 is a Rac1 inhibitor. Data is presented as mean ±SEM for N=3 biological replicates per experiment for three independent experiments (N=9). Statistical comparison among multiple groups were made using one-way ANOVA. *<0.5, **<0.01, ***<0.001, ****<0.0001

Article Snippet: In some experiments, iMGLs were pre-treated with chemical inhibitors or DMSO vehicle for 2 hr before inflammatory stimuli, which included CEP-1347 (100 nM; Tocris), IKK16 (1 uM; Tocris), and NSC23766 (50 uM; EMD Millipore). iMGLs were then treated with either the dsRNA viral mimic poly(I:C) at (25 ug/mL) , oligomerized Aβ42 (5 uM; rPeptide), or vehicle control (dH 2 O or F12 media respectively).

Techniques: Control, Knockdown, Expressing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Comparison, Western Blot, Activation Assay

Journal: bioRxiv

Article Title: Reduced SH3RF3 may protect against Alzheimer’s disease by lowering microglial pro-inflammatory responses via modulation of JNK and NFkB signaling

doi: 10.1101/2024.06.23.600281

Figure Lengend Snippet: (A-B) Representative immunoblots and densitometry analysis of JNK (pJNK/JNK, A) and NFkB (phosho-p65/p65, B) pathway activation in iMGLs treated with siSH3RF3 or control siRNA subjected to oAβ42 or vehicle for 3h. Densitometry data is presented as mean ±SEM for N=3 biological per experiment for three independent experiments (N=9). Statistical comparison among multiple groups were made using one-way ANOVA. (C-E) qPCR analysis of inflammatory cytokines IL1β (C), IL6 (D) and TNFα (E) upon 5 μM oAβ42 treatment for 12h in iMGLs pre-treated with either DMSO vehicle, CEP-1347 (JNKi), or IKK16 (NFkBi ) . Data is presented as mean ±SEM for N=3 per experiment for two independent experiments (N=6). Statistical comparison among multiple groups was made using one-way ANOVA. (F-H) qPCR analysis of inflammatory cytokines IL1β (F), IL6 (G) and TNFα (H) upon 5 μM oAβ42 treatment or vehicle for 6h in isogenic control and PSEN1 G206A iMGLs. Data is presented as mean ±SEM for N=3 biological replicates per experiment for three independent experiments (N=9). Statistical comparison among multiple groups were made using one-way ANOVA. **<0.01, ***<0.001, ****<0.0001.

Article Snippet: In some experiments, iMGLs were pre-treated with chemical inhibitors or DMSO vehicle for 2 hr before inflammatory stimuli, which included CEP-1347 (100 nM; Tocris), IKK16 (1 uM; Tocris), and NSC23766 (50 uM; EMD Millipore). iMGLs were then treated with either the dsRNA viral mimic poly(I:C) at (25 ug/mL) , oligomerized Aβ42 (5 uM; rPeptide), or vehicle control (dH 2 O or F12 media respectively).

Techniques: Western Blot, Activation Assay, Control, Comparison

Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Chemical genetics screen identifies pathways that inhibit intracellular growth of B. neotomae. (A) Inhibitory effects of selected small molecules on intracellular replication in J774A.1 host cells at 48 h postinfection. Both B. neotomae (Bn) luminescence and CFU were measured in parallel. Data shown are means ± SD from at least two independent experiments, 3 replicates for luminescence data and 2 replicates for CFU data. Concentrations of the following tested compounds were in general above the IC50 (Table 1) and substantially below the CC50: arcyriaflavin A (3.6 μM), BX912 (3.6 μM), IKK16 (3.2 μM), I3M (3.6 μM), Ki20227 (2.0 μM), CYT387 (1.4 μM), Rhodblock6 (6.9 μM), SP600125 (10.2 μM), esomeprazole (10.2 μM), NS8593 (4.7 μM), and pinacidil (10.2 μM). (B) Inhibitory effects of selected small molecules on intracellular replication in primary murine bone marrow-derived macrophages (BMDM) at 48 h postinfection based on luminescence signals. Data shown are means ± SD from 4 replicates. Saponin (0.2%) and 0.3% DMSO in the absence of infection were used as positive and negative cytotoxicity controls, respectively. Significant suppression of luminescence was observed for all inhibitors in comparison with the DMSO control (P < 0.0001 by ANOVA and Dunnett’s post hoc multiple-comparison tests). (C) Effect of kinase target siRNA on intracellular growth of B. neotomae in J774A.1 host cells as assessed by luminescence. Data points represent means ± SD from at least three independent experiments. Significant suppression of luminescence (*, P < 0.001) was observed for siRNA knockdowns compared to the NTsi control. (D) Effect of siRNA knockdowns on mRNA expression assayed using same experimental protocol as the one described for panel C. Data points represent means ± SD from three independent samples and were normalized to the β-actin level within each sample and then to expression in untreated controls.

Article Snippet: IKK16 (IKK inhibitor VII) , APExBIO , B1586.

Techniques: Derivative Assay, Infection, Comparison, Control, Expressing

Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Compound potency, cytotoxicity, and selectivity

Article Snippet: IKK16 (IKK inhibitor VII) , APExBIO , B1586.

Techniques:

Journal: Infection and Immunity

Article Title: A Chemical Genetics Screen Reveals Influence of p38 Mitogen-Activated Protein Kinase and Autophagy on Phagosome Development and Intracellular Replication of Brucella neotomae in Macrophages

doi: 10.1128/IAI.00044-19

Figure Lengend Snippet: Strains and key resources used in this study a

Article Snippet: IKK16 (IKK inhibitor VII) , APExBIO , B1586.

Techniques: Bicinchoninic Acid Protein Assay, Transfection, Software

Journal: Molecular cancer therapeutics

Article Title: PIM kinase inhibitors block the growth of primary T-cell acute lymphoblastic leukemia: Resistance pathways identified by network modeling analysis

doi: 10.1158/1535-7163.MCT-20-0160

Figure Lengend Snippet: (A) HEK-293T-NF-κB-luc cells were incubated with conditioned media from HSB-2 naïve or AZDR1 and LGBR2 cells with or without IKK16 (2 μM) for 6h. (B & C) HEK-293T-NF-κB-luc cells were incubated with media containing TNF-α (20 ng/mL) alone or in combination with IKK16 (2 μM) or CM from HSB-2 LGBR2 or AZDR1 with or without IKK16 (2 μM) for 6h. NF-κB mediated luciferase activity was measured using One-Glo Luciferase Assay System. Cell lysates were Western blotted with the specified antibodies. LE: long exposure. (D) Cell viability of HSB-2 naïve and AZDR1 cells treated with indicated concentrations of AZD alone and/or in combination with IKK16 for 72h. (E) T-ALL PDX cells were incubated with the indicated concentrations of LGB321 (LGB) alone or in combination with IKK16 for 72h and then the percentage of viable cells was quantified by the ATPlite assay. For (D and E), the growth of DMSO control cells was considered 100% and percent cell growth after individual treatment is reported relative to the DMSO. The data shown are the average +/− S.D. of three independent experiments.

Article Snippet: LGB-321 (Cat # A14420), AZD1208 (Cat # A13203), and IKK16 (# A12836) were purchased from Adooq bioscience.

Techniques: Incubation, Luciferase, Activity Assay, Western Blot