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: Clinical and Translational Medicine

Article Title: Single‐cell landscape of the tumour immune microenvironment in human gynaecologic malignancies

doi: 10.1002/ctm2.70538

Figure Lengend Snippet: Validation of the regulatory function of NFKB1 and the prognostic relevance of IFN‐Mac_CXCL9 and Angio‐Mac. (A and B) Bar plots showing VEGFA secretion and expression in THP‐1 derived M2 macrophages after NFKB1 knockout (A) or treatment with an IKK inhibitor–BAY 11‐7082 (B). (C‐D) Representative images of mIHC staining for IFN‐Mac_CXCL9 (C) using CD68, CXCL9 and PITX1 and Angio‐Mac (D) using CD68, VCAN and NFKB1. The arrows indicate cells positive for all three markers. Scale bars, 100 µm.

Article Snippet: M2 macrophages were derived from M0 cells via IL‐4 (20 ng/mL, 200‐04; Sigma–Aldrich) and IL‐13 (20 ng/mL, 200‐13; PeproTech) treatment for 48 h, followed by treatment with the IKK inhibitor BAY 11‐7082 (20 μM, HY‐13453, MCE) for another 48 h. VEGFA levels in the culture supernatants were measured via ELISA (ABclonal RK00023), and total RNA was extracted for qPCR analysis.

Techniques: Biomarker Discovery, Expressing, Derivative Assay, Knock-Out, Staining

Journal: eLife

Article Title: Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IκBα through a phosphoenzyme intermediate

doi: 10.7554/eLife.98009

Figure Lengend Snippet: ( A ) A schematic of the IKK-complex (pre- and post-stimulation) showing binding of NEMO helping activation of IKK as well as channelizing its recognition of substrate IκBα. The mechanism of specific phosphorylation at Ser32 and Ser36 of IκBα is unclear ( B ) Domain organization of IKK2 based on the X-ray structures highlighting its functional kinase domain (KD), ubiquitin-like domain (ULD), scaffold dimerization domain (SDD), and NEMO-binding domain. Serine residues in the activation loop - substitution of which to glutamate renders IKK2 constitutively active, and those in the SRR region known to be phosphorylated are marked. Tyrosine residues in the activation loop and the conserved ATP-interacting Lys44 are also marked. ( C ) In vitro kinase assay showing autophosphorylation of wild-type full-length IKK2 (FL IKK2WT) upon incubation with γ 32 P radiolabeled ATP for different time periods. ( D ) Similar in vitro kinase assays performed to assess the effect of NEMO on autophosphorylation of FL IKK2WT (left panel), and the effect of NEMO and IκBα on autophosphorylation and substrate phosphorylation (right panel) activities of FL IKK2WT. ( E ) In vitro kinase assay (schematic depicted in ) showing the effect of different concentrations of the Inhibitor VII on FL IKK2WT autophosphorylation and IκBα substrate phosphorylation (this assay was performed twice). ( F ) Kinase assay with radiolabeled ATP displaying auto- and substrate-phosphorylation of full-length and deletion constructs of the constitutively active form of IKK2 harboring phosphomimetic Ser177Glu and Ser181Glu substitutions. Figure 1—source data 1. Original unedited autoradiograph and Coomassie-stained gel files used in . Figure 1—source data 2. Original autoradiographs and Coomassie-stained gel files used in with sample labels.

Article Snippet: Chemical compound, drug , IKK Inhibitor VII , Calbiochem , CAS 873225-46-8 , IKK inhibitor.

Techniques: Binding Assay, Activation Assay, Phospho-proteomics, Functional Assay, Ubiquitin Proteomics, In Vitro, Kinase Assay, Incubation, Construct, Autoradiography, Staining