human thp1 asc gfp monocyte cell line  (InvivoGen)

Journal: bioRxiv

Article Title: Cell autonomous inflammation in VEXAS is mediated by cGAS-STING

doi: 10.64898/2026.05.26.727520

Figure Lengend Snippet: ( A ) Schematic workflow of proteomic and transcriptomic analyses across both model THP1 monocytes ( left ), and patient derived control and VEXAS monocytes ( right ) to identify disease initiating pathways. ( B ) Loss of cytoplasmic UBA1 function in THP1 model cells leads to ERAD dysregulation, UPR activation, and inflammatory signaling without evidence for cell death. UBA1 WT and UBA1 M41V THP1 cells were treated with dox for 9 d and subjected to immunoblotting using the indicated antibodies. Results are representative of n ≥ 3 biological replicates. ( C ) THP1 VEXAS model cells exhibit type I interferon signatures, confirming the results of the transcriptomic analyses. Parental, UBA1-/-, UBA1 WT and UBA1 M41V/L THP1 monocytes or macrophages (M ϕ) were treated with dox for 9 d and analyzed by qPCR for an Interferon Score by averaging the fold change of 6-hallmark ISGs (IFI44L, IFI27, IFIT1, ISG15, RSAD2, and SIGLEC1). n ≥ 3 biological replicates, error bars = s.e.m., **** = p < 0.0001, student’s t-test. ( D ) Loss of cytoplasmic UBA1 function in THP1 model cells leads to multi-cytokine inflammation. UBA1 WT and UBA1 M41V THP1 macrophages (Mϕ) were treated with dox for 9 d and ELISA for IL-6, TNFα, IL-1b, and CXCL-10. n = 3 biological replicates, ** = p < 0.01, error bar = s.e.m, student’s t-test. ( E ) Proteomic and transcriptomic analyses reveal overlapping and unique altered pathways in VEXAS and nominate ERAD and Golgi transport dysregulation as initial signals in disease pathogenesis. Pie charts depict the top 25 gene ontology biological pathways (GOBPs) significantly enriched in both patient-derived VEXAS over control monocytes (CD14 + ) and the VEXAS model cells (THP1). While in the transcriptomic comparison ( upper chart ) the majority of the top 25 enriched GOBPs are related to the immune response, the proteomic comparison ( lower chart ) reveals predominantly GOBPs associated with ERAD and Golgi transport. ( F ) Dot plots show gene set enrichment scores for the top 25 dysregulated GOBPs as identified by proteomic comparison of patient-derived control and VEXAS monocytes (CD14 + ). For each CD14 + top dysregulated GOBP, the mean normalized enrichment score (NES) and the Benjamini-Hochberg-adjusted p-values are shown for the two indicated comparisons (CD14 + protein, THP1 protein). n=3 biological replicates for each condition. ( G ) The proteomes of patient derived CD14 + and THP1 model VEXAS cells are highly similarly dysregulated and exhibit altered immune responses and ERAD dysregulation. The volcano plot depicts differentially expressed proteins (DEPs) of healthy donor and VEXAS CD14+ monocytes. DEPs colored in black are significantly regulated in both VEXAS CD14+ and THP1 model cells. Within this overlap, DEPs related to immune responses, ERAD and Golgi transport are highlighted. ( H ) Immunoblot analysis of THP1 model cells with indicated antibodies reveals that loss of cytoplasmic ubiquitylation causes ERAD and Golgi dysregulation.

Article Snippet: Human THP1 cells (ATCC, TIB-202) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 Medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/mL Penicillin-Streptomycin (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate.

Techniques: Derivative Assay, Control, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: bioRxiv

Article Title: Cell autonomous inflammation in VEXAS is mediated by cGAS-STING

doi: 10.64898/2026.05.26.727520

Figure Lengend Snippet: ( A ) ERAD alterations precede inflammation in THP1 VEXAS models, shown via heatmap representation of gene ontology biologic processes (GOBPs) across indicated time periods after doxycycline induction. Representative GOBPs and proteins were selected from those significantly dysregulated in patient CD14 + cell proteomics. Color scale indicates the mean normalized enrichment score (NES) of UBA1 M41V versus UBA1 WT THP1 monocytes with asterisks representing Benjamini-Hochberg-adjusted p-value of -log 10 (p-value) ≥ 1.3. ( B ) ERAD is impaired in VEXAS model cells. UBA1 WT and UBA1 M41V THP1 macrophages expressing the ERAD reporter, CD3d-GFP, were induced with doxycycline for 10 days, treated with cycloheximide (CHX) for indicated time periods, and subjected to immunoblotting with indicated antibodies. CD3d-GFP quantifications were normalized to Actin. N ≥ 3 biologic replicates, error bars = s.e.m., * = p < 0.05, ** = p < 0.01, *** = p < 0.001, students t-test. ( C ) Loss of cytoplasmic UBA1 activity leads to a preferential loss of ubiquitin charging to the ERAD-associated E2 enzymes UBE2J1 and UBE2G2. Schematic depicts the workflow of the biochemical screen to identify E2 charging defects associated with VEXAS mutations. Chinese hamster ovary (CHO) cells with a temperature sensitive Uba1 allele (ts20) and complemented with human UBA1 WT , UBA1 M41V , or UBA1 M41L were incubated at the restrictive temperature and subjected to immunoblotting using antibodies against the 28 detectable E2s in CHO cells. Heatmap depicts the quantifications of the ubiquitin charging status of each E2 enzyme based on immunoblot signal of ubiquitin-charged over total E2, normalized to UBA1 WT . Some E2 enzymes are known to be charged independent of UBA1 (UBA1-independent), while the majority of other E2s show varying degrees of sensitivity to loss of cytoplasmic UBA1 activity (UBE2Ds = UBE2D1/2/3/4). ( D ) Confirmation of the specific E2 charging defect of ERAD-associated UBE2J1 and UBE2G2 in patient cells. CD14 + monocytes from control or VEXAS patients were isolated and subjected to immunoblotting with indicated antibodies. ( E ) Quantification of the ubiquitin charging status for each E2 (E2∼Ub/ total E2) depicted in panel e. n ≥ 4 biological replicates, error bar = s.e.m, ** = p < 0.01, *** = p < 0.001, student’s t-test. ( F ) ERAD inhibition leads to activation of the unfolded protein response (UPR), as shown by immunoblotting for UPR and integrated stress response (ISR) components upon treatment of parental THP1 cells with 5 μM ERAD inhibitor CP26. ( G ) ERAD inhibition leads to cytoplasmic vacuole formation, as assessed via Wright-Giemsa staining after treatment of THP1 cells with the ERAD inhibitors EerI (2.5 μM) and CP26 (5 μM). Vacuoles were quantified via a machine learning algorithm (see methods). n = 3 biologic replicates, **** = p < 0.001, student’s t-test. ( H ) Loss of cytoplasmic UBA1 activity in THP1 cells causes abnormal ER morphology with dilated, vacuole-sized, and proteinaceous cisterna. UBA1 WT and UBA1 M41V THP1 cells were treated with doxycycline for 10 days and analyzed by transmission electron microscopy (TEM, upper panel) or focused ion beam scanning electron microscopy (FIB-SEM, lower panel). For TEM, Representative images of 2 biologic replicates are shown (for quantifications, see Fig. S10). For FIB-SEM, 3D reconstructions depict a representative UBA1 WT and UBA1 M41V THP1 cell, demonstrating dilated ER coursing through the UBA1 M41V THP1 cell. ( I ) VEXAS-associated vacuoles originate from ER, as demonstrated by overlay of brightfield (BF) and confocal anti-calnexin immunofluorescence images of UBA1 WT and UBA1 M41V THP1 cells treated with doxycycline for 10 days in both single plane and maximal intensity projection (max IP). Scale bar = 10 μm

Article Snippet: Human THP1 cells (ATCC, TIB-202) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 Medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/mL Penicillin-Streptomycin (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate.

Techniques: Expressing, Western Blot, Activity Assay, Ubiquitin Proteomics, Incubation, Control, Isolation, Inhibition, Activation Assay, Staining, Transmission Assay, Electron Microscopy, Immunofluorescence

Journal: bioRxiv

Article Title: Cell autonomous inflammation in VEXAS is mediated by cGAS-STING

doi: 10.64898/2026.05.26.727520

Figure Lengend Snippet: ( A ) STING is the sole immune response-related ERAD/ HRD1 substrate that accumulates in VEXAS CD14 + cells. The volcano plot depicts differentially expressed proteins (DEPs) of healthy donor and VEXAS CD14 + monocytes. DEPs colored in black highlight previously identified HRD1 substrates and DEPs in blue highlight HRD1 substrates significantly upregulated in VEXAS CD14 + cells. ( B ) Innate immune adaptor STING accumulates in VEXAS-patient derived CD14 + cells, UBA1 M41V THP1 monocytes, UBA1 M41L 32D cells, and upon ERAD inhibition (CP26, 1 μM or 2 μM for 24h), but not upon autophagy inhibition (BafA, 1 μM or 10 μM for 24h) or ESCRT-mediated degradation pathway inhibition (NSC69, 3 μM and 4 μM for 24h) in THP1 parental cells, as quantified from anti-STING immunoblots. n ≥ 3 biologic replicates as indicated, error bar = s.e.m, * = p < 0.05, ** = p < 0.01, student’s t-test. ( C ) The STING pathway is active in CD14 + cells from VEXAS patients, as shown via immunoblotting of cell lysates with indicated antibodies. (D ) The STING pathway is active in UBA1 M41V THP1 monocytes as shown via immunoblotting of cell lysates with indicated antibodies. (E) STING is activated in VEXAS model cells, as evidenced by increased STING trafficking to the Golgi in UBA1 M41V THP1 monocytes observed by anti-STING and anti-GM130 immunofluorescence. Scale bar = 10 μm. ( F ) Quantification of the percentage of Golgi-localized STING of the experiment shown in panel e. n ≥ 150 cells across 10 fields, error bar = s.e.m, **** = p < 0.0001, student’s t-test. ( G ) STING inhibition reverses VEXAS-associated inflammatory signaling in THP1 model cells. UBA1 WT and UBA1 M41V THP1 macrophages (Mϕ) were treated with STING inhibitors H-151 (1 μM) and SN-011 (10 μM) for 24h and subjected to immunoblot analysis using indicated antibodies. ( H ) Schematic representation of experimental design for STING or cGAS knockout (KO) and assessment of inflammation. ( I ) STING or cGAS KO inhibit VEXAS-associated inflammatory signaling in THP1 model cells. UBA1 WT and UBA1 M41V THP1 macrophages (Mϕ) were electroporated with indicated gRNAs and subjected to immunoblot analysis using indicated antibodies. ( J ) Graphs depict quantifications of p-STAT1/STAT1 ratio normalized to control gRNA-electroporated UBA1 WT THP1 macrophages. n = 3 biological replicates, error bar = s.e.m, ** = p < 0.01, student’s t-test. ( K ) STING and cGAS KO inhibit VEXAS-associated inflammatory signaling in THP1 model cells. Same experiments as in panel I, but cell lysates were either subjected to qPCR analysis to determine mRNA levels of different type I interferon stimulated genes ( left graph , IFN score), or cell supernatants were subjected to ELISA to measure secreted protein concentrations of TNFα ( middle graph ) or IL-6 ( right graph ). ( L ) Scheme depicting molecular events triggered by canonical VEXAS mutations in myeloid cells. Loss of cytoplasmic UBA1 activity causes ERAD impairment, which induces UPR/ISR signaling, vacuoles, and accumulation of the innate immune adaptor STING, which is activated in a cGAS-dependent manner.

Article Snippet: Human THP1 cells (ATCC, TIB-202) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 Medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/mL Penicillin-Streptomycin (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate.

Techniques: Derivative Assay, Inhibition, Western Blot, Immunofluorescence, Knock-Out, Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: bioRxiv

Article Title: Cell autonomous inflammation in VEXAS is mediated by cGAS-STING

doi: 10.64898/2026.05.26.727520

Figure Lengend Snippet: ( A ) Vacuole-containing VEXAS THP1 cells contain less mitochondria, as demonstrated by brightfield (BF) and single-plane confocal anti-TOM20 immunofluorescence images of UBA1 WT and UBA1 M41V THP1 monocytes treated with doxycycline for 10 d. Representative images of 2 biological replicates are shown. For more examples, see Fig. S16. Scale bar = 10 μm. ( B ) UBA1 M41V THP1 monocytes exhibit mitochondrial morphology abnormalities, indicative of impaired function. UBA1 WT and UBA1 M41V THP1 cells were treated with dox for 10 d and analyzed by transmission electron microscopy. Scale bar = 1 μm. Red arrowheads point to dilated cristae often seen in UBA1 M41V mitochondria (see also Movie S2) ( C ) UBA1 M41V THP1 monocytes exhibit a higher percentage of mitochondria with dilated cristae per cell as compared to UBA1 WT control cells. Graphs depict quantifications of mitochondrial morphology on TEM images of the experiment shown in panel b. n > 20 cells, error bar = s.e.m, * = p < 0.05, ** = p < 0.01, student’s t-test. ( D ) VEXAS THP1 cells exhibit elevated levels of cytoplasmic mitochondrial DNA, as revealed by qPCR analysis of whole cell or cytoplasmic fractions of UBA1 WT and UBA1 M41V THP1 monocytes. n = 3 biological replicates with 2 technical replicates for two different mitochondrial DNA probes (see methods), error bar = s.e.m, ** = p < 0.01, student’s t-test. ( E ) Schematic representation of experimental design to test whether mtDNA release into the cytosol activates cGAS-STING-based VEXAS inflammation. ( F ) Blocking mtDNA release into the cytosol using either a scavenger of reactive oxygen species (MitoTEMPO), an inhibitor of VDAC oligomerization (VBIT4), or an inhibitor of the mitochondrial permeability transition pore (CsA) dampens STING signaling and attenuates VEXAS inflammation in THP1 UBA1 M41V model cells. UBA1 WT and UBA1 M41V THP1 monocytes were treated with indicated inhibitors (10 μM MitoTEMPO, 10 μM VDAC, and 1 μM CsA) for 24h and subjected to immunoblot analysis using indicated antibodies. Graphs depict quantifications of p-STING/STING, p-STAT1/STAT1, or p-STAT3/STAT3 ratio normalized to DMSO-treated UBA1 WT THP1 monocytes. n = 3 biological replicates, error bar = s.e.m, * = p < 0.05, ** = p < 0.01, student’s t-test. ( G ) Blocking mtDNA release into the cytosol restores STING localization by preventing its trafficking to the Golgi, as evidenced by anti-STING and anti-GM130 immunofluorescence analysis of THP1 model monocytes treated as described in panel F. ( H ) Quantification of the percentage of Golgi-localized STING of the experiment depicted in panel g. n ≥ 150 cells across 10 fields, error bar = s.e.m, **** = p < 0.0001, one-way ANOVA. ( I ) Verification that treatment of THP1 VEXAS model cells with indicated inhibitors blocks mtDNA release into the cytosol, as revealed by qPCR analysis of cytoplasmic fractions of UBA1 WT and UBA1 M41V THP1 monocytes. n = 3 biological replicates, 2 technical replicates for two different mitochondrial DNA probes (see methods), error bar = s.e.m, **** = p < 0.0001, one-way ANOVA. (J) Scheme depicting the molecular mechanisms triggering STING activation in VEXAS myeloid cells. Mitochondria in VEXAS myeloid cells are defective and release mtDNA into the cytosol, causing activation of cGAS-STING signaling, which drives multi-cytokine inflammation.

Article Snippet: Human THP1 cells (ATCC, TIB-202) were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI 1640 Medium (Gibco) supplemented with 10% heat-inactivated FBS, 100 U/mL Penicillin-Streptomycin (Gibco), 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate.

Techniques: Immunofluorescence, Transmission Assay, Electron Microscopy, Control, Blocking Assay, Permeability, Western Blot, Activation Assay

Journal: Cell Death & Disease

Article Title: Insulin enables acquisition of the IL7R + memory phenotype in PD1 + T cells in RA tissues

doi: 10.1038/s41419-026-08916-6

Figure Lengend Snippet: A Confocal microscopy images of THP1 cells stained with antibodies to H3K27ac (red) in nucleus (blue) after stimulation with insulin (top), and HDAC-inhibitor (bottom) for 24 h. Images are acquired with 40× magnification with additional digital magnification 1.5×. B Scatter plot of nuclear H3K27ac staining intensity after stimulation with insulin (top) and HDAC-inhibitor (bottom). P -values are calculated by Wilcoxon unpaired test. Asterisks indicate * < 0.05, ** < 0.01, *** <0.001, **** <0.0001. C Histogram of phosphorylated serine 473 (p)AKT1 mean fluorescence intensity in THP1 cells stimulated with 10 nM insulin, by flow cytometry. D Histogram of H3K27ac mean fluorescence intensity in THP1 cells stimulated with increasing concentrations of insulin for 24 h, by flow cytometry. E Histogram of pAKT1 mean fluorescence intensity in human lymphocytes stimulated with increasing concentrations of insulin for 30 min, by flow cytometry.

Article Snippet: Human monocytic cell line THP1 (TIB-202, ATCC, Manassas, VA, USA) were propagated in RPMI medium (Gibco, Waltham, Massachusetts, USA) containing 50 μM β-mercaptoethanol (Gibco), Glutamax 2 mM (Gibco), sodium pyruvate 1 mM (Gibco), HEPES 10 mM (Gibco), gentamicin 50 μg/mL (Sanofi-Aventis) and 10% fetal bovine serum (Sigma-Aldrich) at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Confocal Microscopy, Staining, Fluorescence, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: PS ASO innate immunogenicity varies with cell system, treatment duration, and concentration. ( A ) Model PS-ASOs used for this study. “o”indicates a phosphodiester linkage; all other linkages are phosphonothioate. Constrained Ethyl (cEt) and MOE 2’ modifications are indicated for each PS-ASO. Fast-acting or slow-acting TLR9 agonism is listed as defined by Pollak et al. 2022 (, ). ( B ) Pulse treatment in the BJAB cell line (left) and the THP1-TLR9 cell line (right). Relative qRT-PCR levels of CCL22 mRNA following a 1, 2, 3, or 4 h incubation with 1.6 µM of the indicated PS ASOs in serum-free RPMI media. RNA lysates were collected 24 h following treatment. ( C ) Continuous treatment in the BJAB cell line (left) and the THP1-TLR9 cell line (right). Relative qRT-PCR levels of CCL22 following a 2-, 4-, 8-, or 24-h incubation with 1.6 µM of indicated PS ASOs in serum-free RPMI media. RNA lysates were collected immediately following treatment. ( D ) Dose response in BJAB cell line (left) or THP1-TLR9 cell line (right). Cells were treated for 2 h with varying concentrations (0.064, 0.32, 1.6, or 8 µM) of PS ASOs. RNA lysates were collected 24 h after treatment. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Immunopeptidomics, Concentration Assay, Quantitative RT-PCR, Incubation

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Differential kinetics of cytokine secretion following PS ASO stimulation in BJAB and THP1-TLR9 cells. THP1-TLR9 (left) and BJAB cells (right) were treated with 1.6 μM of the indicated PS ASOs for 2 h, before supernatants were collected for cytokine analysis of TNF-α ( A ) and IL-10 ( B ). THP1-TLR9 cells were also assessed for levels of secreted IL-1β ( C ) and IL-6 ( D ). Values were interpolated to a standard curve of known protein concentration, and are expressed as mean at each time point. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Protein Concentration

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Trp degradation enzymes, IDO1 and IL4I1 , are upregulated in immune cells stimulated with PS ASOs. ( A ) Correlation of tryptophan oxidation enzymes and TLR9 activation in BJAB cells (top) and THP1-TLR9 cells (bottom) after treatment with 1.6 µM of the indicated PS ASOs for 2 h. Relative qRT-PCR levels of CCL22 and IL4I1/IDO1 mRNA were measured at 8, 24, 48, and 72 h post-treatment. Spearman’s rank correlation was used to assess the association between CCL22 mRNA and IL4I1/IDO1 mRNA. ( B ) Maximum values observed in each experiment with the indicated PS ASOs for BJAB cells (top) and THP1-TLR9 cells (bottom). ( C ) Kinetics of IL4I1 and CCL22 qRT-PCR levels in BJAB cells, and ( D ) THP1-TLR9 cells after dosing with the indicated PS ASOs. All data are presented as a percentage of UTC control (mRNA expression/Ribogreen/UTC) and expressed as mean ± S.E.M. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Activation Assay, Quantitative RT-PCR, Control, Expressing

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Immunogenic PS ASOs stimulate Kyn production via the tryptophan catabolism pathway, and genetic manipulation of tryptophan catabolizing enzymes alters TLR9-mediated PS ASO activation. Total extracellular ( A ) tryptophan and ( B ) kynurenine metabolite levels from THP1-TLR9 cells 24- or 48-h post-treatment with 0.32, 1.6, or 8 μM of indicated PS ASOs. ( C ) Relative CCL22 and IDO1 mRNA expression levels in IDO1 siRNA-treated THP1-TLR9 stimulated with CpG ASO. ( D ) Validation of IDO1 siRNA knockdown and representative western blot measuring IDO1 protein expression. THP1-TLR9 cells were electroporated with 1 μM of siRNA 24 h prior to treatment with 1.6 μM CpG ASO for 2 h. Protein lysates were collected 24 h following treatment. ( E ) Relative CCL22 and IL4I1 mRNA expression levels in IL4I1 overexpression plasmid-treated BJAB cells stimulated with CpG ASO. All bar graph data are expressed as mean ± S.E.M.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Knockdown, Western Blot, Over Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Directed metabolomics of tryptophan metabolites after PS ASO treatment in THP1-TLR9 cells. Cells were treated with 8 μM PS-ASOs for 2 h, recovered for 48 h, and the collected cellular supernatant was subjected to directed metabolomic analysis of tryptophan metabolites to quantify specific changes. Biological replicates ( n = 3) were submitted for metabolomic analysis. Significant differences in means of metabolite quantities (ng/mL) were determined via one-way ANOVA with Dunnett post-hoc tests (statistical significance is denoted at P < 0.05*, P < 0.005**, and P < 0.0005***).

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Metabolomic

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Exogenous kynurenine and indole metabolites attenuate PS-ASO-induced innate immune responses. ( A ) Relative CCL22 mRNA expression levels in BJAB and THP1-TLR9 cells and ( B ) Relative IRF reporter activity and NF-kB reporter activity in THP1-TLR9 cells following treatment with 100 µM of the indicated metabolite and 0.8 µM CpG ASO. Relative TLR9 activation of ( C ) THP1-TLR9 cells and ( D ) BJAB cells pretreated with various doses (1.5 mM–0.08 µM) of kynurenine, kynurenic acid, 3-hydroxy anthranilic acid, and indole-3-pyruvate for 2 h, and subsequently treated with 1.6 µM of ASO-20 or ASO-95 for 24 h. TLR9 activation was measured as CCL22 mRNA expression. Metabolite IC50s derived from the top five doses are shown for each ASO, calculated using linear regression with normalized response and variable slope. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet:

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: RNA sequencing, In Vitro, Ex Vivo

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet: A) Genetically predicted lower LACTB expression—instrumented using macrophage cis-eQTLs at the LACTB locus—is associated with lower AD risk (left panel). Genetically predicted lower LACTB expression is also associated with higher succinylcarnitine levels in the CSF (middle panel). Furthermore, genetically predicted higher succinylcarnitine levels are associated with lower AD risk (right panel). B) LACTB is expressed higher in the immune cluster compared to other brain cells, in contrast to another mitochondrial protein (SDHA), data from Brain Atlas . C, D) LACTB mRNA (C) and protein levels (D) are increased upon differentiation (THP1 macrophages vs monocytes and WTC11 iMGLs vs iPSC). E) Succinylcarnitine levels are increased in the lysate of LACTB KD/KO myeloid cells compared to SCR/WT. F) Succinylcarnitine is increased in the cell culture media of LACTB KD/KO myeloid cells compared to SCR/ WT. G) Higher succinylcarnitine levels in mice with lower LACTB expression (C57B6 x SLJ background, 2 months old). H) Higher succinylcarnitine levels in the liver, brain, isolated microglia and astrocytes of LACTB enzymatically-dead (ED) mice compared to WT (n=2 mice per genotype, 3 months old). I) LACTB protein levels increase with age in WT mice (12 vs 2 months old). J) Succinylcarnitine levels decrease with age in WT mice (12 vs 2 months old). Graphs display individual data points (left) alongside estimated marginal means with 95% confidence intervals (right). For the raw data, dot shapes represent independent macrophage differentiations, and dot colors indicate distinct microglia clones. Statistical details are provided in .

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: Expressing, Cell Culture, Isolation, Clone Assay

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet: A) Representative curves of succinyl-D-Asp substrate cleavage over time, showing strong reduction/abolishment in LACTB KD/KO myeloid cells compared to SCR/WT (a1: n=3 THP1 macrophages differentiations, a2: n=3 clones per genotype WTC11 iMGLs, a3: mouse BMDMs n=6 mice per genotype). Area under the curve (AUC) was calculated and used as outcome variable in statistical analyses. B) 13 C 3 -carnitine generation after substrate ( 13 C 7 -succinylcarnitine) incubation with LACTB recombinant protein in the absence or presence of LACTB inhibitor (Ac-IEPD-CHO) in a cell-free assay. C) Reduced label incorporation into carnitine in LACTB KO iMGLs after incubation with 13 C 3 -succinylcarnitine (n=3 clones per genotype) compared to WT WTC11 iMGLs. D) Representative heatmap showing label incorporation from 13 C 7 -succinylcarnitine into the indicated metabolites in WT iMGLs. E-J) LACTB is succinylated after treatment with diethyl-succinate (5 μM for 4 hours) in HEK cells (n=2 transfections, 4-10 well replicates each) (E) and THP1 macrophages (n=2 differentiations, 4 well replicates each) (H). Succinylation of LACTB is associated with a reduced rate of succinyl-D-Asp cleavage in HEK cells (n=2 transfections, 3 well-replicates each) (F) and THP1 macrophages (n=2 differentiations, 3 well-replicates each). AUC was calculated and used as outcome variable in statistical analyses. (I) LACTB succinylation is associated with higher succinylcarnitine levels in HEK cells (n=3) (G) and THP1 macrophages (n=3) (J). Graphs display individual data points (left) alongside estimated marginal means with 95% confidence intervals (right). For the raw data, dot shapes represent independent macrophage differentiations, and dot colors indicate distinct microglia clones. Statistical details are provided in .

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: Clone Assay, Incubation, Recombinant, Cell-Free Assay, Transfection

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet: A) Pathways enriched in GSEA analysis of bulk RNAseq data from LACTB KD THP1 macrophages compared to SCR (n=5 independent macrophages differentiations), NES = Normalized Enrichment Score. B) Pathways enriched in GSEA analysis of bulk RNAseq data from LACTB KO iMGLs compared to WT (3 clones per genotype, 2 differentiations per clone). C) Heatmap of Pearson correlation coefficients between LACTB expression and genes encoding pro-inflammatory cytokines across microglial states in publicly available human single-cell microglia datasets [ – ]. D) Single-cell clusters and corresponding annotations of WT and LACTB KO iMGLs (3 clones per genotype, 1-2 differentiations per clone). E) LACTB expression across clusters in WT iMGLs. F) Cluster proportions in WT and LACTB KO iMGLs. Cluster proportions were estimated using crumblr and compared by genotype using a generalized linear model with batch as a covariate (see Methods for further details). G) Pathways enriched in GSEA analysis of bulk RNAseq data from mouse microglia isolated from the brains of LACTB ED mice compared to controls (3 months old, n=3). Complete lists of genes and pathways are provided in and .

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: RNA sequencing, Clone Assay, Expressing, Single Cell, Isolation

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet: LACTB KD/KO in myeloid cells increases oxidative phosphorylation and reduces protein synthesis, cholesteryl esters, and triacylglycerides . A) Increased in OXPHOS (measured by mitostress seahorse assays) in LACTB KD THP1 macrophages (n=10 differentiations, 3-6 technical replicates each), and WTC11 LACTB KO iMGLs (3 clones per genotype, 3 differentiations per clone, 3-6 technical replicates each) compared to controls. B) Reduction in nascent protein synthesis (measured with a methionine analog) in LACTB KD/KO myeloid cells compared to SCR/WT. Graphs display individual data points (left) alongside estimated marginal means with 95% confidence intervals (right). For the raw data, dot shapes represent independent macrophage differentiations, and dot colors indicate distinct microglia clones. Full statistical details are provided in . C) Lipidomics alterations in LACTB KD vs SCR THP1 macrophages (n=3) and WTC11 LACTB KO vs WT iMGLs (n=3 clones, 1-2 differentiations each), showing a consistent reduction in CE and TG across the two myeloid cell types. Statistical details are provided in .

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: Phospho-proteomics, Clone Assay

Journal: bioRxiv

Article Title: Reduced LACTB expression in myeloid cells is associated with elevated succinylcarnitine levels and reduced Alzheimer’s disease risk

doi: 10.64898/2026.03.24.711053

Figure Lengend Snippet: A) LACTB mRNA expression increases after stimulation with IFN-β, IFN-γ, or TNF-α in THP1 macrophages (6 hours treatment) or WTC11 iMGLs (24 hours treatment). B) Succinylcarnitine levels decrease after stimulation with IFN-β, IFN-γ, or TNF-α in THP1 macrophages (6 hours treatment) or WTC11 iMGLs (24 hours treatment). C) Efferocytosis-related assays (myelin phagocytosis and lysosomal acidification, mass and proteolytic capacity) in LACTB KD/KO myeloid cells compared to SCR/WT. Graphs display individual data points (left) alongside estimated marginal means with 95% confidence intervals (right). For the raw data, dot shapes represent independent differentiations, and dot colors indicate distinct microglia clones. Statistical details are provided in .

Article Snippet: We used the monocytic human immortalized cell line THP1 (ATCC, TIB-202 RRID:CVCL_0006), which we treated with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml) for 72 hours to differentiate into macrophages.

Techniques: Expressing, Clone Assay