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: Neurotoxicology

Article Title: Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro.

doi: 10.1016/j.neuro.2021.07.004

Figure Lengend Snippet: Fig. 3. IKK-β inhibition attenuates Mn-induced YY1 activation. (A) Astrocytes were transfected overnight with a YY1 promoter vector, followed by pre-treatment with IKK16 (200 nM, 1 h) and Mn exposure (250 μM, 3 h). YY1 promoter activity was measured via the luciferase assay. Astrocytes were pre-treated with IKK16 (200 nM, 1 h) and Mn (250 μM, 6 h), and cell lysates were analyzed for YY1 (B) mRNA and (C) protein expression via qPCR and western blotting, respectively. (*p < 0.05, **p < 0.01, ***p < 0.001, compared to the control; #significantly different # p < 0.05, ## p < 0.01; ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: The TNFα inhibitor (E)-4-2-4-chloro-3-nitrophenyl (C-87) and the IKK-β inhibitor IKK16 were purchased from Tocris (Littleton, CO).

Techniques: Inhibition, Activation Assay, Transfection, Plasmid Preparation, Activity Assay, Luciferase, Expressing, Western Blot, Control

Journal: Neurotoxicology

Article Title: Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro.

doi: 10.1016/j.neuro.2021.07.004

Figure Lengend Snippet: Fig. 2. Mn induces canonical NF-κB activation via phosphorylation of IKK-β. (A) Human H4 astrocytes were pre-treated with the selective IKK-β inhibitor IKK16 (200 nM, 1 h), followed by Mn exposure (250 μM, 3 h), and cell lysates were analyzed for phosphorylated IKK-β protein expression by western blotting. (B) After overnight transfection with an NF-κB reporter vector, astrocytes were treated with IKK16 and Mn for the indicated time periods, followed by the luciferase assay. (C) Astrocytes were treated with IKK16 and Mn as previously described, then cell lysates were fractionated and analyzed for NF-κB p65 protein expression in the nuclear fraction by western blotting. Histone H4 was used as a loading control. (*p < 0.05, **p < 0.01, ***p < 0.001, compared to the control; #significantly different # p < 0.05, ## p < 0.01, ### p < 0.01; ANOVA followed by Tukey’s post hoc test; N = 3).

Article Snippet: The TNFα inhibitor (E)-4-2-4-chloro-3-nitrophenyl (C-87) and the IKK-β inhibitor IKK16 were purchased from Tocris (Littleton, CO).

Techniques: Activation Assay, Phospho-proteomics, Expressing, Western Blot, Transfection, Plasmid Preparation, Luciferase, Control

Journal: Neurotoxicology

Article Title: Manganese-induced reactive oxygen species activate IκB kinase to upregulate YY1 and impair glutamate transporter EAAT2 function in human astrocytes in vitro.

doi: 10.1016/j.neuro.2021.07.004

Figure Lengend Snippet: Fig. 8. IKK-β phosphorylation mediatestheMn-induced repression of EAAT2. (A) Astrocytes were transfected overnight with an EAAT2 promoter vector, then incubated with IKK16 (200 nM, 1 h) and Mn (250 μM, 6 h), followed by the luciferase assay. Astrocytes were treated with IKK16 and Mn for the indicated time periods, and EAAT2 mRNA and protein levels were measured via real-time (B) qPCR and (C) western blotting, respectively. (# p < 0.05, ## p < 0.01, ### p < 0.001, compared to the control; @significantly different @ p < 0.05; ANOVA followed by Tukey’s post hoc test; N=3).

Article Snippet: The TNFα inhibitor (E)-4-2-4-chloro-3-nitrophenyl (C-87) and the IKK-β inhibitor IKK16 were purchased from Tocris (Littleton, CO).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation, Incubation, Luciferase, Western Blot, Control