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: 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