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InvivoGen thp1 dualtm cells thpd nfis

a, immune cell types in the TME identified through scRNAseq of CD45 + cells isolated from orthotopically implanted M3-9-M OVA cells; b, number of immune cell types detected in M3-9-M OVA tumours through scRNAseq experiment (n = 2 pooled samples per experimental groups); c, number of significantly impacted genes obtained from pairwise comparisons (Wilcoxon rank sum test); d, circos plot of all significantly impacted genes, obtained from pairwise comparisons of all cell types measured, that overlap with each other through identical or shared pathways; e, bar graph of transcriptional regulators of 3,2-HPP impacted genes; f-m, NF-κB and IRF induction in mouse RAW-Dual™ cells and human <t>THP1-Dual™</t> cells with different pathogen recognition receptor agonists in the presence of HPP metabolites (HI = heat inactivated, three combined experiments, error bars represent standard error, one-way ANOVA test).

Thp1 Dualtm Cells Thpd Nfis, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 515 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells"

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

Journal: bioRxiv

doi: 10.64898/2026.04.23.720410

Figure Legend Snippet: a, immune cell types in the TME identified through scRNAseq of CD45 + cells isolated from orthotopically implanted M3-9-M OVA cells; b, number of immune cell types detected in M3-9-M OVA tumours through scRNAseq experiment (n = 2 pooled samples per experimental groups); c, number of significantly impacted genes obtained from pairwise comparisons (Wilcoxon rank sum test); d, circos plot of all significantly impacted genes, obtained from pairwise comparisons of all cell types measured, that overlap with each other through identical or shared pathways; e, bar graph of transcriptional regulators of 3,2-HPP impacted genes; f-m, NF-κB and IRF induction in mouse RAW-Dual™ cells and human THP1-Dual™ cells with different pathogen recognition receptor agonists in the presence of HPP metabolites (HI = heat inactivated, three combined experiments, error bars represent standard error, one-way ANOVA test).

Techniques Used: Isolation

a, NF-κB activity in THP1-Dual™ cells treated with LPS in the presence of vehicle or HPP metabolites for 0-24 hours (two combined experiments, error bars represent standard error); b-e, NF-κB or IRF activity in THP1-Dual™ cells treated with different inducers in the presence or absence of HPP isomers for 16 hours (three combined experiments, error bars represent standard error, one-way ANOVA test).

Figure Legend Snippet: a, NF-κB activity in THP1-Dual™ cells treated with LPS in the presence of vehicle or HPP metabolites for 0-24 hours (two combined experiments, error bars represent standard error); b-e, NF-κB or IRF activity in THP1-Dual™ cells treated with different inducers in the presence or absence of HPP isomers for 16 hours (three combined experiments, error bars represent standard error, one-way ANOVA test).

Techniques Used: Activity Assay

a, representation of the proteins with denaturing curves significantly altered (red) or unaltered (black) by 3,2-HPP treatment of THP1-Dual™ cells, using thermal proteome profiling (two combined experiments, proteomic coverage: 4301, NPARC test); b, protein-protein interaction network of proteins with denaturation curves altered by 3,2-HPP treatment (red) and the transcriptional regulators of 3, 2-HPP impacted genes in M3-9-M tumours (yellow); c, impact of 3,2-HPP treatment of THP1-Dual™ cells on the denaturation curve of GSDMD (two-combined experiments, NPARC test); d, NF-κB induction in THP1-Dual™ reporter cells treated with conditioned media harvested from WT or Gsdmd- KO THP1 cells treated with LPS ± HPP metabolites ± IL-1α and −1β neutralizing antibody (three combined experiments, one-way ANOVA with Bonferroni multiple comparison test); e-h, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT or Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); i-l, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT vs. Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS and NG ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); m, western blot of THP-1 cells treated with LPS ± NG ± HPPs (representative of two experiments).

Figure Legend Snippet: a, representation of the proteins with denaturing curves significantly altered (red) or unaltered (black) by 3,2-HPP treatment of THP1-Dual™ cells, using thermal proteome profiling (two combined experiments, proteomic coverage: 4301, NPARC test); b, protein-protein interaction network of proteins with denaturation curves altered by 3,2-HPP treatment (red) and the transcriptional regulators of 3, 2-HPP impacted genes in M3-9-M tumours (yellow); c, impact of 3,2-HPP treatment of THP1-Dual™ cells on the denaturation curve of GSDMD (two-combined experiments, NPARC test); d, NF-κB induction in THP1-Dual™ reporter cells treated with conditioned media harvested from WT or Gsdmd- KO THP1 cells treated with LPS ± HPP metabolites ± IL-1α and −1β neutralizing antibody (three combined experiments, one-way ANOVA with Bonferroni multiple comparison test); e-h, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT or Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); i-l, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT vs. Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS and NG ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); m, western blot of THP-1 cells treated with LPS ± NG ± HPPs (representative of two experiments).

Techniques Used: Comparison, Cell Culture, Western Blot

a, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells treated with LPS±HPPs for 16 hours, measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); b, LDH release from THP1-WT vs. – GSDMD -KO cells treated with LPS±HPP for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test); c-d, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells © and human PBMCs (d) treated with LPS and NG±HPPs for 16 hours. measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); e-f, LDH release from THP1-WT vs. – GSDMD -KO cells € and human PBMCs (f) treated with LPS and NG±HPPs for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test).

Figure Legend Snippet: a, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells treated with LPS±HPPs for 16 hours, measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); b, LDH release from THP1-WT vs. – GSDMD -KO cells treated with LPS±HPP for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test); c-d, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells © and human PBMCs (d) treated with LPS and NG±HPPs for 16 hours. measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); e-f, LDH release from THP1-WT vs. – GSDMD -KO cells € and human PBMCs (f) treated with LPS and NG±HPPs for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test).

Techniques Used: Activity Assay

Image Search Results

Journal: bioRxiv

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

doi: 10.64898/2026.04.23.720410

Figure Lengend Snippet: a, immune cell types in the TME identified through scRNAseq of CD45 + cells isolated from orthotopically implanted M3-9-M OVA cells; b, number of immune cell types detected in M3-9-M OVA tumours through scRNAseq experiment (n = 2 pooled samples per experimental groups); c, number of significantly impacted genes obtained from pairwise comparisons (Wilcoxon rank sum test); d, circos plot of all significantly impacted genes, obtained from pairwise comparisons of all cell types measured, that overlap with each other through identical or shared pathways; e, bar graph of transcriptional regulators of 3,2-HPP impacted genes; f-m, NF-κB and IRF induction in mouse RAW-Dual™ cells and human THP1-Dual™ cells with different pathogen recognition receptor agonists in the presence of HPP metabolites (HI = heat inactivated, three combined experiments, error bars represent standard error, one-way ANOVA test).

Article Snippet: RAW-DualTM cells (rawd-ismip), THP1-DualTM cells (thpd-nfis), HEK-Blue TM IL-1β cells (hkb-il1bv2; InvivoGen), THP1-Null2 Cells (thp-nullz) and THP1-KO-GSDMD cells (thp-kogsdmdz) were obtained from InvivoGen (San Diego, CA, USA).

Techniques: Isolation

Journal: bioRxiv

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

doi: 10.64898/2026.04.23.720410

Figure Lengend Snippet: a, NF-κB activity in THP1-Dual™ cells treated with LPS in the presence of vehicle or HPP metabolites for 0-24 hours (two combined experiments, error bars represent standard error); b-e, NF-κB or IRF activity in THP1-Dual™ cells treated with different inducers in the presence or absence of HPP isomers for 16 hours (three combined experiments, error bars represent standard error, one-way ANOVA test).

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

doi: 10.64898/2026.04.23.720410

Figure Lengend Snippet: a, representation of the proteins with denaturing curves significantly altered (red) or unaltered (black) by 3,2-HPP treatment of THP1-Dual™ cells, using thermal proteome profiling (two combined experiments, proteomic coverage: 4301, NPARC test); b, protein-protein interaction network of proteins with denaturation curves altered by 3,2-HPP treatment (red) and the transcriptional regulators of 3, 2-HPP impacted genes in M3-9-M tumours (yellow); c, impact of 3,2-HPP treatment of THP1-Dual™ cells on the denaturation curve of GSDMD (two-combined experiments, NPARC test); d, NF-κB induction in THP1-Dual™ reporter cells treated with conditioned media harvested from WT or Gsdmd- KO THP1 cells treated with LPS ± HPP metabolites ± IL-1α and −1β neutralizing antibody (three combined experiments, one-way ANOVA with Bonferroni multiple comparison test); e-h, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT or Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); i-l, [secreted IL-1α and −1β] in conditioned media harvested from THP-1 WT vs. Gsdmd -KO cells, or human PBMCs, cultured for 16 hours in LPS and NG ± HPPs (two combined experiments, one-way ANOVA with Bonferroni multiple comparison test); m, western blot of THP-1 cells treated with LPS ± NG ± HPPs (representative of two experiments).

Techniques: Comparison, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Microbiome-derived hydroxyphenyl propanoates enhance antitumour immunity by potentiating gasdermin D activity in tumour-associated myeloid cells

doi: 10.64898/2026.04.23.720410

Figure Lengend Snippet: a, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells treated with LPS±HPPs for 16 hours, measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); b, LDH release from THP1-WT vs. – GSDMD -KO cells treated with LPS±HPP for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test); c-d, IL-1β activity in the supernatant of THP1-WT vs. - GSDMD -KO cells © and human PBMCs (d) treated with LPS and NG±HPPs for 16 hours. measured using HEK-Blue™ IL-1β reporter cells (three combined experiments, error bars represent standard error, one-way ANOVA with Bonferroni test); e-f, LDH release from THP1-WT vs. – GSDMD -KO cells € and human PBMCs (f) treated with LPS and NG±HPPs for 16 hours (three combined experiments, one-way ANOVA with Bonferroni test).

Techniques: Activity Assay

Anti-cancer potency of PL-MNP-p BIRC5 /As- BIRC5 and PL-MNP-p BIRC5 /dN-BIRC5 in BIRC5-Expressing cells. ( A ) Western blot analysis of BIRC5 expression in MIA PaCa-2 and NTUB1 cells. ACTA1 served as a loading control to verify equal protein loading. (B) MIA PaCa-2 cells were co-treated with lysosomotropic agent 3 nM BAF and IC 50 concentrations of PL-MNP-p BIRC5 /As- BIRC5 or PL-MNP-p BIRC5 /dN-BIRC5 for 96 h. Cell viability was determined using the MTT assay. (C) MIA PaCa-2 cells were treated with PL-MNP-p BIRC5 /Emp or PL-MNP-p BIRC5 /dN-BIRC5 at their respective IC 50 concentrations for 48 h. BIRC5-CASP3 protein–protein interactions were detected by in situ PLA using anti-BIRC5 and anti-CASP3 antibodies. Scale bars: 10 µm. (D) γ-H2AX expression in MIA PaCa-2 cells treated with IC 50 concentrations of PL-MNP-p BIRC5 /As- BIRC5 or PL-MNP-p BIRC5 /dN-BIRC5 for 48 h, assessed by Western blotting. ACTA1 served as a loading control. Data represent at least three independent experiments. The bar graph represents the quantified band intensities from independent experiments, with γ-H2AX expression levels normalized to the corresponding ACTA1 band intensity for each sample. (E) Western blot analysis of ABCB1 expression in NTUB1 and NTU0.017 cells. ACTA1 served as a loading control to verify equal protein loading. (F) Cell viability of MIA PaCa-2, NTUB1, NTU0.017, SK-OV-3 (with or without 25 ng/mL IFNγ), MCF10A, HMEC-1, WI-38 VA13 subline 2RA, HaCaT, and THP1 cells treated with PL-MNP-p BIRC5 /Emp, PL-MNP-p BIRC5 /As- BIRC5 , or PL-MNP-p BIRC5 /dN-BIRC5 for 96 h, measured by the MTT assay and WST assay (for THP-1 cells). Data represent at least three independent experiments. Bar and curve graphs indicate the mean ± SD. Statistical analyses were performed using one-way ANOVA for panels ( B – D ), and two-way ANOVA for panel ( F ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. PL-MNP-p BIRC5 /Emp or non-BAF-treated group; n.s., not significant. Statistical symbols are color-coded to match the corresponding experimental groups in the figure.

Journal: International Journal of Nanomedicine

Article Title: BIRC5 Promoter-Driven Nanodrugs Suppress BIRC5-Positive Cancers Independent of ABCB1 Status and IDO1 Expression

doi: 10.2147/IJN.S588571

Figure Lengend Snippet: Anti-cancer potency of PL-MNP-p BIRC5 /As- BIRC5 and PL-MNP-p BIRC5 /dN-BIRC5 in BIRC5-Expressing cells. ( A ) Western blot analysis of BIRC5 expression in MIA PaCa-2 and NTUB1 cells. ACTA1 served as a loading control to verify equal protein loading. (B) MIA PaCa-2 cells were co-treated with lysosomotropic agent 3 nM BAF and IC 50 concentrations of PL-MNP-p BIRC5 /As- BIRC5 or PL-MNP-p BIRC5 /dN-BIRC5 for 96 h. Cell viability was determined using the MTT assay. (C) MIA PaCa-2 cells were treated with PL-MNP-p BIRC5 /Emp or PL-MNP-p BIRC5 /dN-BIRC5 at their respective IC 50 concentrations for 48 h. BIRC5-CASP3 protein–protein interactions were detected by in situ PLA using anti-BIRC5 and anti-CASP3 antibodies. Scale bars: 10 µm. (D) γ-H2AX expression in MIA PaCa-2 cells treated with IC 50 concentrations of PL-MNP-p BIRC5 /As- BIRC5 or PL-MNP-p BIRC5 /dN-BIRC5 for 48 h, assessed by Western blotting. ACTA1 served as a loading control. Data represent at least three independent experiments. The bar graph represents the quantified band intensities from independent experiments, with γ-H2AX expression levels normalized to the corresponding ACTA1 band intensity for each sample. (E) Western blot analysis of ABCB1 expression in NTUB1 and NTU0.017 cells. ACTA1 served as a loading control to verify equal protein loading. (F) Cell viability of MIA PaCa-2, NTUB1, NTU0.017, SK-OV-3 (with or without 25 ng/mL IFNγ), MCF10A, HMEC-1, WI-38 VA13 subline 2RA, HaCaT, and THP1 cells treated with PL-MNP-p BIRC5 /Emp, PL-MNP-p BIRC5 /As- BIRC5 , or PL-MNP-p BIRC5 /dN-BIRC5 for 96 h, measured by the MTT assay and WST assay (for THP-1 cells). Data represent at least three independent experiments. Bar and curve graphs indicate the mean ± SD. Statistical analyses were performed using one-way ANOVA for panels ( B – D ), and two-way ANOVA for panel ( F ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 vs. PL-MNP-p BIRC5 /Emp or non-BAF-treated group; n.s., not significant. Statistical symbols are color-coded to match the corresponding experimental groups in the figure.

Article Snippet: THP1 cells were kindly provided by Dr. Tzeng-Horng Leu (Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan) and cultured in RPMI containing 10% FBS and PSG.

Techniques: Expressing, Western Blot, Control, MTT Assay, Protein-Protein interactions, In Situ, WST Assay

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.

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

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.

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

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.

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

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.

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

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***).

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

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.

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

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