small molecule sting antagonist h 151  (InvivoGen)

Journal: bioRxiv

Article Title: Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

doi: 10.64898/2026.01.07.694713

Figure Lengend Snippet: A) Graphical representation of methodology and findings adapted from Gulen et al. to identify genes induced by the innate immune pathway involving STING, herein referred to as STING-responsive genes (SRG). Cells taken from either skin of human healthy donors, which are then irradiated to promote aging, or brains from old mice were treated with STING inhibitor (H-151). RNAseq (validated by qPCR) was then performed to interrogate the expression of inflammatory genes induced through the activation of the STING pathway during aging. Using their combined data, a list of SRG comprising of 108 genes was compiled (full list shown in Supp. Table 2). B) Dot plot indicating the average expression of SRG across all conditions from the single-cell RNAseq dataset of human iPSC-derived peripheral myeloid cells wherein a larger view showcases key proinflammatory cytokines/chemokines and type I interferons within the SRG panel. The size of the dot determines the proportion of cells expressing the particular gene/gene set while the color scale dictates the normalized average expression. C) Representative immunofluorescence staining of macrophages from human (hiPSC-MDM) and mouse (BMDM) origins for DNA (red) and mitochondria (green) using anti-DNA antibody and AlexaFluor 647-labelled MitoTracker or anti-TOMM40 antibody, respectively. Scale bars: 10 μm and 2 μm (inset). The red-positive puncta dissociated from the mitochondria (green signal) was defined as cytosolic DNA in WT and PiKO stimulated cells, as well as non-stimulated controls. Open and closed squares represent data point for human and mouse experiments, respectively. Two-way ANOVA followed by Holm-Sidak’s multiple comparison test was applied. Mean ± SEM, n=5 independent experiment with each experiment comprised of 20-25 images analyzed. D, E) Representative immunoblots of human and mouse macrophages stained with anti-STING and anti-phospho NF-κβ (pNF-κβ) antibodies. β-actin was used as loading control. The relative protein expression of STING and pNF-κβ in WT and PiKO stimulated cells, as well as non-stimulated controls, were quantified and normalized to β-actin. Open and closed squares represent data point for human and mouse experiments, respectively. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=5 independent experiments.

Article Snippet: Matured macrophages were replated and co-stimulated with 500 ng/mL Ultrapure lipopolysaccharide (LPS) from E.coli O111:B4 (Invivogen, tlrl-3pelps) and 50 ng/mL mouse recombinant interleukin-1beta (IL-1β) (PeproTech, 211-11B) with or without 8 μg/mL small molecule STING antagonist H-151 (InvivoGen, inh-h151) for 6 h. For the exchange medium experiment, bone marrow-derived macrophages (BMDM) were treated with inflammatory stimuli for 24 h then cells were washed, and conditioned medium (CM) was collected 24 h later.

Techniques: Irradiation, Expressing, Activation Assay, Derivative Assay, Immunofluorescence, Staining, Comparison, Western Blot, Control

Journal: bioRxiv

Article Title: Myeloid PINK1 represses mtDNA release and immune signaling that impacts neuronal pathology in patient-derived idiopathic PD models

doi: 10.64898/2026.01.07.694713

Figure Lengend Snippet: A) Graphical representation of mtDNA-dependent STING/NF-κβ signaling pathway resulting in inflammation, underscoring H-151 as a STING inhibitor. B) Schematic diagram to describe cell fractionation using commercially available buffers and differential centrifugation. Shown are representative immunoblots of cytosolic (cyto) and mitochondrial (mito) fractions isolated from stimulated WT and PiKO BMDM indicating purity of subcellular fractions according to the expression of mitochondrial protein TOMM20 and cytosolic protein GAPDH. β-actin was used as loading control. Both fractions from stimulated BMDM of WT and PiKO mice were assessed for relative gene expression of mtDNA (MT-ND1). Relative ratios of cyto-to-mito MT-ND1 (cyto/mito) in stimulated PiKO with and without H-151 were normalized to corresponding WT groups. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=3 mice per group. C) Representative immunoblots of WT and PiKO BMDM stained with anti-pNF-κβ and anti-STING antibodies. β-actin was used as loading control. The relative protein expression of pNF-κβ and STING in stimulated cells with or without H-151, as well as non-stimulated controls, were quantified and normalized to β-actin. Two-way ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=4 mice per group. D) Quantification of inflammatory target genes downstream of STING innate immune pathway (IL6 and IFNB1) by qPCR across all conditions. Twoway ANOVA followed by Tukey’s multiple comparison test was applied. Mean ± SEM, n=6-7 mice per group.

Article Snippet: Matured macrophages were replated and co-stimulated with 500 ng/mL Ultrapure lipopolysaccharide (LPS) from E.coli O111:B4 (Invivogen, tlrl-3pelps) and 50 ng/mL mouse recombinant interleukin-1beta (IL-1β) (PeproTech, 211-11B) with or without 8 μg/mL small molecule STING antagonist H-151 (InvivoGen, inh-h151) for 6 h. For the exchange medium experiment, bone marrow-derived macrophages (BMDM) were treated with inflammatory stimuli for 24 h then cells were washed, and conditioned medium (CM) was collected 24 h later.

Techniques: Cell Fractionation, Centrifugation, Western Blot, Isolation, Expressing, Control, Gene Expression, Comparison, Staining