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resiquimod (MedChemExpress)

Name: resiquimod
Brand: MedChemExpress
Rating: 94 (27 reviews)

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MedChemExpress resiquimod
Resiquimod, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/resiquimod/product/MedChemExpress
Average 94 stars, based on 27 article reviews

resiquimod - by Bioz Stars, 2026-03

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(A) Schematic of IRF7 activation, nuclear localization, and DNA binding downstream of multiple pattern recognition receptor pathways. (B) Overview of experimental approach employed in HEK Blue hTLR7 cells. C) Representative Western blot image of HEK Blue hTLR7 nuclear lysates following stimulation with R848 for 3-hours. β-actin and total H3 are included as loading controls. (D) Densitometry analysis of band intensities for risk and protective haplotype bands based on the quantitative Western blot presented in Figure S2. IRF7 bands were normalized to a total protein stain to calculate normalized signal as described in Methods. Welch’s t-test was used to determine significance. (E) Normalized ISRE luciferase activity (ratio of ISRE firefly to Renilla luciferase relative light units (RLU)) following 24-hours of R848 stimulation. Welch’s t-test was used to determine significance. (F) IFN-α measured in cell-free supernatants from cells stimulated with R848 for 3– and 24-hours. Data are mean ± SEM of triplicate samples (unless otherwise indicated) and are representative of at least three independent experiments. Significance was assessed with two-way ANOVA with a Holm-Šidak’s multiple comparisons correction. *P<0.05; ***P<0.005; ns indicates non-significant (P>0.05). ISRE: Interferon stimulated response element; EV: Empty Vector; R848: Resiquimod.

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: (A) Schematic of IRF7 activation, nuclear localization, and DNA binding downstream of multiple pattern recognition receptor pathways. (B) Overview of experimental approach employed in HEK Blue hTLR7 cells. C) Representative Western blot image of HEK Blue hTLR7 nuclear lysates following stimulation with R848 for 3-hours. β-actin and total H3 are included as loading controls. (D) Densitometry analysis of band intensities for risk and protective haplotype bands based on the quantitative Western blot presented in Figure S2. IRF7 bands were normalized to a total protein stain to calculate normalized signal as described in Methods. Welch’s t-test was used to determine significance. (E) Normalized ISRE luciferase activity (ratio of ISRE firefly to Renilla luciferase relative light units (RLU)) following 24-hours of R848 stimulation. Welch’s t-test was used to determine significance. (F) IFN-α measured in cell-free supernatants from cells stimulated with R848 for 3– and 24-hours. Data are mean ± SEM of triplicate samples (unless otherwise indicated) and are representative of at least three independent experiments. Significance was assessed with two-way ANOVA with a Holm-Šidak’s multiple comparisons correction. *P<0.05; ***P<0.005; ns indicates non-significant (P>0.05). ISRE: Interferon stimulated response element; EV: Empty Vector; R848: Resiquimod.

Article Snippet: Ligands used in cell stimulations include Resiquimod (R848) (Invivogen: 1ug/mL), polydA:dT-Lyovec (Invivogen: 2.5ug/mL), and Poly(I:C) LMW (Invivogen: 2.5ug/mL).

Techniques: Activation Assay, Binding Assay, Western Blot, Staining, Luciferase, Activity Assay, Plasmid Preparation

Using CRISPR targeting and homologous recombination, we mutated C/G to T/A at position 141,263,661 on chromosome 7 (Genome Reference Consortium Mouse Build 38), resulting in an R334Q substitution in mouse IRF7 that is analogous to the rs1131665 SLE risk-variant R412Q substitution in human IRF7. (A) 100 micrograms of resiquimod (R848) was epicutaneously applied three times per week to the ears of genome edited C57BL/6 mice with the lupus risk or non-risk IRF7 genotype for a total of eight weeks. (B) Sera was collected and used to quantify anti-double-stranded DNA (ds-DNA) antibodies by ELISA. (C) Eightweek-old male mice with the lupus risk or non-risk IRF7 genotype were intranasally infected with 1 million plaque-forming units of VSV-NJ. (D) After 24 hours, virus was quantified in lung homogenates using viral plaque assays. (E) Model for the lupus risk genotype in IRF7 enhancing both viral responses (with the lupus risk genotype enhancing viral clearance) and autoimmunity (with the lupus risk genotype enhancing the production of autoantibodies).

Journal: medRxiv

Article Title: A highly prevalent lupus risk haplotype increases IRF7-dependent induction of IFN-α, enhancing antiviral defense and exacerbating autoimmunity

doi: 10.64898/2026.01.21.26344474

Figure Lengend Snippet: Using CRISPR targeting and homologous recombination, we mutated C/G to T/A at position 141,263,661 on chromosome 7 (Genome Reference Consortium Mouse Build 38), resulting in an R334Q substitution in mouse IRF7 that is analogous to the rs1131665 SLE risk-variant R412Q substitution in human IRF7. (A) 100 micrograms of resiquimod (R848) was epicutaneously applied three times per week to the ears of genome edited C57BL/6 mice with the lupus risk or non-risk IRF7 genotype for a total of eight weeks. (B) Sera was collected and used to quantify anti-double-stranded DNA (ds-DNA) antibodies by ELISA. (C) Eightweek-old male mice with the lupus risk or non-risk IRF7 genotype were intranasally infected with 1 million plaque-forming units of VSV-NJ. (D) After 24 hours, virus was quantified in lung homogenates using viral plaque assays. (E) Model for the lupus risk genotype in IRF7 enhancing both viral responses (with the lupus risk genotype enhancing viral clearance) and autoimmunity (with the lupus risk genotype enhancing the production of autoantibodies).

Article Snippet: Ligands used in cell stimulations include Resiquimod (R848) (Invivogen: 1ug/mL), polydA:dT-Lyovec (Invivogen: 2.5ug/mL), and Poly(I:C) LMW (Invivogen: 2.5ug/mL).

Techniques: CRISPR, Homologous Recombination, Variant Assay, Enzyme-linked Immunosorbent Assay, Infection, Virus

The concentration of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 in the mRNA-1273 group PBMC supernatants after stimulation with LPS, R848, Curdlan, or Pam3Csk4 assessed via ELISA. Data is presented as mean ± SD, with dots representing individual participants. Statistical significance was assessed using either one-way ANOVA followed by Tukey’s post-hoc test or Kruskal–Wallis test followed by Dunn’s post-hoc test, as appropriate

Journal: Journal of Clinical Immunology

Article Title: Signatures of Trained Immunity Following mRNA Vaccination: Differences Between mRNA-1273 and BNT162b2

doi: 10.1007/s10875-025-01977-w

Figure Lengend Snippet: The concentration of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 in the mRNA-1273 group PBMC supernatants after stimulation with LPS, R848, Curdlan, or Pam3Csk4 assessed via ELISA. Data is presented as mean ± SD, with dots representing individual participants. Statistical significance was assessed using either one-way ANOVA followed by Tukey’s post-hoc test or Kruskal–Wallis test followed by Dunn’s post-hoc test, as appropriate

Article Snippet: The following day, cells were stimulated with one of the following ligands: lipopolysaccharide (LPS, InvivoGen, cat# tlrl-eklps, 5 ng/mL, TLR-4), Resiquimod (R848, InvivoGen, cat# tlrl-r848, 5 μg/mL, TLR-7/8), Curdlan (InvivoGen, cat# tlrl-curd, 10 μg/mL, TLR-4/2), or Pam3CSK4 (InvivoGen, cat# tlrl-pms, 10 μg/mL, TLR-1/2).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

The concentration of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 in the BNT162b2 group PBMC supernatants after stimulation with LPS, R848, Curdlan, or Pam3Csk4 assessed via ELISA. Data is presented as mean ± SD, with dots representing individual participants. Statistical significance was assessed using either one-way ANOVA followed by Tukey’s post-hoc test or Kruskal–Wallis test followed by Dunn’s post-hoc test, as appropriate

Journal: Journal of Clinical Immunology

Article Title: Signatures of Trained Immunity Following mRNA Vaccination: Differences Between mRNA-1273 and BNT162b2

doi: 10.1007/s10875-025-01977-w

Figure Lengend Snippet: The concentration of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 in the BNT162b2 group PBMC supernatants after stimulation with LPS, R848, Curdlan, or Pam3Csk4 assessed via ELISA. Data is presented as mean ± SD, with dots representing individual participants. Statistical significance was assessed using either one-way ANOVA followed by Tukey’s post-hoc test or Kruskal–Wallis test followed by Dunn’s post-hoc test, as appropriate

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

The comparison between mRNA-1273 and BNT162b2 group in production of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 after stimulation with LPS, R848,Curdlan, or Pam3Csk4 and measured by ELISA. In the box plots, the middle line indicates the median, the box represents the interquartile range (Q1–Q3), dots indicate individual participants, and the error bars denote the minimum and maximum values. Statistical differences were determined using two-way ANOVA followed by Tukey’s post-hoc test

Journal: Journal of Clinical Immunology

Article Title: Signatures of Trained Immunity Following mRNA Vaccination: Differences Between mRNA-1273 and BNT162b2

doi: 10.1007/s10875-025-01977-w

Figure Lengend Snippet: The comparison between mRNA-1273 and BNT162b2 group in production of ( A ) IL-6, ( B ) IL-10, ( C ) TNFα, ( D ) IFNγ, ( E ) CCL2, ( F ) CXCL1 after stimulation with LPS, R848,Curdlan, or Pam3Csk4 and measured by ELISA. In the box plots, the middle line indicates the median, the box represents the interquartile range (Q1–Q3), dots indicate individual participants, and the error bars denote the minimum and maximum values. Statistical differences were determined using two-way ANOVA followed by Tukey’s post-hoc test

Techniques: Comparison, Enzyme-linked Immunosorbent Assay

(A) Priming schematic for Hoxb8 macrophages. ( B) Intracellular TNF staining of primary/secondary TLR stimulations. Cells were primed with TLR ligands: LPS 100ng/ml (TLR4), Pam3CSK4 200ng/ml (TLR2), R848 100ng/ml (TLR7) and CpG-B 100nM (TLR9). Priming ligand was washed out after 24h and cells were re-stimulated with secondary TLR agonists: LPS 100ng/ml (TLR4), Pam3CSK 100ng/ml (TLR2), R848 10ng/ml (TLR7) and CpG-B 50nM (TLR9). Colors indicate observed effect: red = enhanced response upon secondary stimulation (training), blue = reduced response (tolerance). Black = unchanged. Representative experiment out of N=3. ( C) Legendplex assay of TNF and IL-6 (N=2) of cells stimulated as in (B). (D) Western blot analysis of TLR7 protein levels after LPS priming (N=3). E: Western blot analysis of TLR9 protein levels after LPS priming (N=3). CL: cleaved. P values were calculated with ratio paired t-test. ns: not significant.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A) Priming schematic for Hoxb8 macrophages. ( B) Intracellular TNF staining of primary/secondary TLR stimulations. Cells were primed with TLR ligands: LPS 100ng/ml (TLR4), Pam3CSK4 200ng/ml (TLR2), R848 100ng/ml (TLR7) and CpG-B 100nM (TLR9). Priming ligand was washed out after 24h and cells were re-stimulated with secondary TLR agonists: LPS 100ng/ml (TLR4), Pam3CSK 100ng/ml (TLR2), R848 10ng/ml (TLR7) and CpG-B 50nM (TLR9). Colors indicate observed effect: red = enhanced response upon secondary stimulation (training), blue = reduced response (tolerance). Black = unchanged. Representative experiment out of N=3. ( C) Legendplex assay of TNF and IL-6 (N=2) of cells stimulated as in (B). (D) Western blot analysis of TLR7 protein levels after LPS priming (N=3). E: Western blot analysis of TLR9 protein levels after LPS priming (N=3). CL: cleaved. P values were calculated with ratio paired t-test. ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Staining, Western Blot

(A) LPS primes TLR7-dependent IL-6 production in murine ex vivo alveolar macrophages (N=4). ( B-C) TLR7 or TLR9-HA staining in Hoxb8 macrophages upon priming with LPS 100ng/ml (N=3). (D) Hoxb8 macrophages were primed with LPS 100ng/ml or left unprimed for 24h; cells were then fed with CpG-B-Cy5 for 0-180 minutes and uptake was quantified with flow cytometry (N=3). P values were calculated with ratio paired t-test at t=180min. (E) Intracellular TNF staining of Hoxb8 macrophages primed with LPS 100ng/ml and restimulated with increasing concentrations of R848: 5, 10, or 20ng/ml (N=4). P-values were calculated with two-way ANOVA and Tukey’s multiple comparison test (F) Intracellular TNF staining of Hoxb8 macrophages primed with decreasing concentrations of LPS: 100, 50, 10 or 1ng/ml. After 24h, cells were restimulated with 10ng/ml R848 (N=4). P-values were calculated with one-way ANOVA with Tukey’s post-test and each compared to the unprimed+R848 group. ns: not significant G) Hoxb8 macrophages were primed as in E) and then restimulated with 100ng/ml LPS. Flow histograms show a representative experiment out of N=4. Graphs show pooled repeats (N=4). P-values were calculated with one-way ANOVA with Tukey’s post-test. ns: not significant.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A) LPS primes TLR7-dependent IL-6 production in murine ex vivo alveolar macrophages (N=4). ( B-C) TLR7 or TLR9-HA staining in Hoxb8 macrophages upon priming with LPS 100ng/ml (N=3). (D) Hoxb8 macrophages were primed with LPS 100ng/ml or left unprimed for 24h; cells were then fed with CpG-B-Cy5 for 0-180 minutes and uptake was quantified with flow cytometry (N=3). P values were calculated with ratio paired t-test at t=180min. (E) Intracellular TNF staining of Hoxb8 macrophages primed with LPS 100ng/ml and restimulated with increasing concentrations of R848: 5, 10, or 20ng/ml (N=4). P-values were calculated with two-way ANOVA and Tukey’s multiple comparison test (F) Intracellular TNF staining of Hoxb8 macrophages primed with decreasing concentrations of LPS: 100, 50, 10 or 1ng/ml. After 24h, cells were restimulated with 10ng/ml R848 (N=4). P-values were calculated with one-way ANOVA with Tukey’s post-test and each compared to the unprimed+R848 group. ns: not significant G) Hoxb8 macrophages were primed as in E) and then restimulated with 100ng/ml LPS. Flow histograms show a representative experiment out of N=4. Graphs show pooled repeats (N=4). P-values were calculated with one-way ANOVA with Tukey’s post-test. ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Ex Vivo, Staining, Flow Cytometry, Comparison

(A) Schematic of TLR4-dependent signaling pathways and respective KOs and inhibitors used. (B) Functional validation of CRISPR-Cas9 knockouts of IFNAR, TRIF or IRF3 in Hoxb8 macrophages. Cells were stimulated with LPS 100ng/ml or IFN-β 800U/ml, and expression of the interferon stimulated gene Viperin was measured by intracellular staining and flow cytometry (each dot represents an independent KO line) (C) Intracellular TNF staining of knockout lines after 100ng/ml LPS stimulation for 6 hours (N=2). (D) Characterization of IRAK4 inhibitor Zimlovisertib: WT Hoxb8 macrophages were treated with Zimlovisertib (20µM) or vehicle and stimulated with TLR ligands: R848 20ng/ml, CpG-B 100nM, LPS 100ng/ml. Viperin and TNF production was measured by intracellular staining (N=1). (E) Intracellular TNF staining and viability control for Polymyxin-B: Hoxb8 WT macrophages were stimulated with LPS 100ng/ml for 6 hours after preincubation with increasing doses of Polymyxin-B (0, 10, 25, 50µg/ml) (N=1). (F) IL6-ELISA of WT Hoxb8 macrophages primed for 24 hours with LPS 100ng/ml in presence or absence of IRAK4 inhibitor Zimlovisertib (20µM) and re-stimulated with R848 10ng/ml or CpG-B 50nM (N=3) after washout of the inhibitor. (G) IL-6 ELISA of Hoxb8 macrophages primed with Poly:IC 10µg/ml for 24 hours and re-stimulated with R848 10ngml for 6 hours (N=4). (H) TLR7 staining of Hoxb8 macrophages left unprimed or primed with Poly:IC 10µg/ml for 24 hours. Representative histogram out of N=3. Graph shows pooled repeats. P-values were calculated with ratio paired t-test, ns: not significant.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A) Schematic of TLR4-dependent signaling pathways and respective KOs and inhibitors used. (B) Functional validation of CRISPR-Cas9 knockouts of IFNAR, TRIF or IRF3 in Hoxb8 macrophages. Cells were stimulated with LPS 100ng/ml or IFN-β 800U/ml, and expression of the interferon stimulated gene Viperin was measured by intracellular staining and flow cytometry (each dot represents an independent KO line) (C) Intracellular TNF staining of knockout lines after 100ng/ml LPS stimulation for 6 hours (N=2). (D) Characterization of IRAK4 inhibitor Zimlovisertib: WT Hoxb8 macrophages were treated with Zimlovisertib (20µM) or vehicle and stimulated with TLR ligands: R848 20ng/ml, CpG-B 100nM, LPS 100ng/ml. Viperin and TNF production was measured by intracellular staining (N=1). (E) Intracellular TNF staining and viability control for Polymyxin-B: Hoxb8 WT macrophages were stimulated with LPS 100ng/ml for 6 hours after preincubation with increasing doses of Polymyxin-B (0, 10, 25, 50µg/ml) (N=1). (F) IL6-ELISA of WT Hoxb8 macrophages primed for 24 hours with LPS 100ng/ml in presence or absence of IRAK4 inhibitor Zimlovisertib (20µM) and re-stimulated with R848 10ng/ml or CpG-B 50nM (N=3) after washout of the inhibitor. (G) IL-6 ELISA of Hoxb8 macrophages primed with Poly:IC 10µg/ml for 24 hours and re-stimulated with R848 10ngml for 6 hours (N=4). (H) TLR7 staining of Hoxb8 macrophages left unprimed or primed with Poly:IC 10µg/ml for 24 hours. Representative histogram out of N=3. Graph shows pooled repeats. P-values were calculated with ratio paired t-test, ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Protein-Protein interactions, Functional Assay, Biomarker Discovery, CRISPR, Expressing, Staining, Flow Cytometry, Knock-Out, Control, Enzyme-linked Immunosorbent Assay

(A-C) IL-6 ELISA of IFNAR -/- , TRIF -/- and IRF3 -/- macrophages. Cells were primed with LPS 100ng/ml and restimulated with R848 10ng/ml (N=3). (D) IL-6 ELISA of WT and IRF3 -/- macrophages primed with LPS 100ng/ml and IFN-b 800U/ml (N=3). (E) IL-6 ELISA of WT macrophages primed with IFN-b 800U/ml (N=3). (F) IL-6 ELISA of supernatant (SN) transfer: WT Hoxb8 macrophages were primed with LPS 100ng/ml for 24h; supernatant was then transferred to naive cells to prime subsequent TLR7 activation. Neutralization of residual LPS in the supernatant with 50µg/ml Polymyxin B prevented priming of naive cells. Graph shows pooled independent repeats (N=3). (A-D): P-values were calculated with paired t-test. (E-F): P-values were calculated with one-way ANOVA with Tukey’s post-test. ns: not significant.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A-C) IL-6 ELISA of IFNAR -/- , TRIF -/- and IRF3 -/- macrophages. Cells were primed with LPS 100ng/ml and restimulated with R848 10ng/ml (N=3). (D) IL-6 ELISA of WT and IRF3 -/- macrophages primed with LPS 100ng/ml and IFN-b 800U/ml (N=3). (E) IL-6 ELISA of WT macrophages primed with IFN-b 800U/ml (N=3). (F) IL-6 ELISA of supernatant (SN) transfer: WT Hoxb8 macrophages were primed with LPS 100ng/ml for 24h; supernatant was then transferred to naive cells to prime subsequent TLR7 activation. Neutralization of residual LPS in the supernatant with 50µg/ml Polymyxin B prevented priming of naive cells. Graph shows pooled independent repeats (N=3). (A-D): P-values were calculated with paired t-test. (E-F): P-values were calculated with one-way ANOVA with Tukey’s post-test. ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Neutralization

(A-C) MAP kinase immunoblots of ERK and p38 phosphorylation upon TLR7, TLR9 or TLR4 stimulation in LPS-primed or naive cells. Representative blots out of N=3. Quantification performed on 3 pooled repeats. (D) Bioassay measuring IFN-b production by naïve or primed Hoxb8 macrophages upon TLR7/9 restimulation with increasing concentrations (N=3, shown is SEM). (E) Legendplex assay of IL-10 secretion: Hoxb8 macrophages were primed with LPS and re-stimulated with R848 10ng/ml. (N=2).

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A-C) MAP kinase immunoblots of ERK and p38 phosphorylation upon TLR7, TLR9 or TLR4 stimulation in LPS-primed or naive cells. Representative blots out of N=3. Quantification performed on 3 pooled repeats. (D) Bioassay measuring IFN-b production by naïve or primed Hoxb8 macrophages upon TLR7/9 restimulation with increasing concentrations (N=3, shown is SEM). (E) Legendplex assay of IL-10 secretion: Hoxb8 macrophages were primed with LPS and re-stimulated with R848 10ng/ml. (N=2).

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Western Blot, Phospho-proteomics, Bioassay

( A) Schematic of MyD88-mNG knock-in mouse and Myddosome assembly downstream of activated TLR2/4/7/9. (B) Intracellular TNF staining of WT and MyD88-mNG Hoxb8 macrophages in response to TLR stimulation for 6h: MyD88-dependent TLR4 (LPS 100ng/ml), TLR2 (Pam3CSK4 200ng/ml), TLR7 (R848 500ng/ml), TLR9 (CpG-B 1µM), and MyD88-independent TLR3 (Poly:IC 10µg/ml). Representative experiment out of N=4. (C) Pooled repeats of (B). P-values were calculated with a paired t-test. (D) IL-6 ELISA of WT and MyD88-mNG macrophages primed for 24h with 100ng/ml LPS and re-stimulated with R848 500ng/ml (N=4). P-values were calculated with two-way ANOVA with Tukey’s multiple comparisons test. (E) Widefield imaging of Myddosome formation in MyD88-mNG macrophages stimulated for 1 hour with the TLR ligands and concentrations as used in (B). (F) Fixed SIM microscopy of MyD88-mNG macrophages stimulated with LPS 100ng/ml for 1 hour or primed with LPS for 24h and then stimulated with R848 500ng/ml for 1 hour, co-stained with Lamp1 and EEA1. White arrows point to Myddosomes. ns: not significant.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: ( A) Schematic of MyD88-mNG knock-in mouse and Myddosome assembly downstream of activated TLR2/4/7/9. (B) Intracellular TNF staining of WT and MyD88-mNG Hoxb8 macrophages in response to TLR stimulation for 6h: MyD88-dependent TLR4 (LPS 100ng/ml), TLR2 (Pam3CSK4 200ng/ml), TLR7 (R848 500ng/ml), TLR9 (CpG-B 1µM), and MyD88-independent TLR3 (Poly:IC 10µg/ml). Representative experiment out of N=4. (C) Pooled repeats of (B). P-values were calculated with a paired t-test. (D) IL-6 ELISA of WT and MyD88-mNG macrophages primed for 24h with 100ng/ml LPS and re-stimulated with R848 500ng/ml (N=4). P-values were calculated with two-way ANOVA with Tukey’s multiple comparisons test. (E) Widefield imaging of Myddosome formation in MyD88-mNG macrophages stimulated for 1 hour with the TLR ligands and concentrations as used in (B). (F) Fixed SIM microscopy of MyD88-mNG macrophages stimulated with LPS 100ng/ml for 1 hour or primed with LPS for 24h and then stimulated with R848 500ng/ml for 1 hour, co-stained with Lamp1 and EEA1. White arrows point to Myddosomes. ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Knock-In, Staining, Enzyme-linked Immunosorbent Assay, Imaging, Microscopy

(A) Intracellular TNF staining of WT and MyD88-mNG bone marrow-derived macrophages in response to TLR stimulation for 6h: MyD88-dependent TLR4 (LPS 100ng/ml), TLR2 (Pam3CSK4 200ng/ml), TLR7 (R848 500ng/ml), TLR9 (CpG-B 1µM), and MyD88-independent TLR3 (Poly:IC 10µg/ml). Representative histograms out of N=4. (B) Pooled repeats of (A). P-values were calculated with paired t-test. (C) Representative fixed SIM image of MyD88-mNG Hoxb8 macrophages stably expressing IRAK2-mScarlet and stimulated with R848 (500ng/ml) for 1 hour. (D) Mass spectrometry analysis of naïve and LPS primed whole cell lysates. Volcano blot shows log2 fold change over p-value of differentially expressed proteins (N=4). (E) Heat map shows log2fold change of mass spec. hits of relevant pathway proteins. Color code: Red = upregulated, blue = downregulated. (F) Free-floating Myddosomes: Fixed SIM microscopy of MyD88-mNG macrophages primed with LPS for 24h and then stimulated with R848 500ng/ml for 1 hour, co-stained with Lamp1 and EEA1. White arrows point to Myddosomes.

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A) Intracellular TNF staining of WT and MyD88-mNG bone marrow-derived macrophages in response to TLR stimulation for 6h: MyD88-dependent TLR4 (LPS 100ng/ml), TLR2 (Pam3CSK4 200ng/ml), TLR7 (R848 500ng/ml), TLR9 (CpG-B 1µM), and MyD88-independent TLR3 (Poly:IC 10µg/ml). Representative histograms out of N=4. (B) Pooled repeats of (A). P-values were calculated with paired t-test. (C) Representative fixed SIM image of MyD88-mNG Hoxb8 macrophages stably expressing IRAK2-mScarlet and stimulated with R848 (500ng/ml) for 1 hour. (D) Mass spectrometry analysis of naïve and LPS primed whole cell lysates. Volcano blot shows log2 fold change over p-value of differentially expressed proteins (N=4). (E) Heat map shows log2fold change of mass spec. hits of relevant pathway proteins. Color code: Red = upregulated, blue = downregulated. (F) Free-floating Myddosomes: Fixed SIM microscopy of MyD88-mNG macrophages primed with LPS for 24h and then stimulated with R848 500ng/ml for 1 hour, co-stained with Lamp1 and EEA1. White arrows point to Myddosomes.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Staining, Derivative Assay, Stable Transfection, Expressing, Mass Spectrometry, Microscopy

$(A) Representative images of widefield microscopy of TLR7-dependent Myddosome formations in MyD88-mNG macrophages with or without prior LPS priming. (B) Quantification of MyD88 puncta per cell (only cells that contained at least 3 Myddosomes were included). Graph shows all individual cells from 3 independent repeats and corresponding means (N=3). Total number of analyzed cells: unstimulated=652, R848 only=620, LPS+R848=550, LPS acute=565, LPS no secondary stim.=504, LPS+LPS restim.=523. (C) Quantification of the cell fraction containing at least 3 Myddosomes: data is pooled from three independent experiments (N=3). Total number of analyzed cells: unstimulated=99, R848 only=262, LPS+R848=369, LPS acute=437, LPS no secondary stim.=121, LPS+LPS restim.=303. P-values for (B) and (C) were calculated by unpaired t-test. (D) Western blot analysis of WT MyD88 and MyD88-mNeonGreen in naïve and LPS-primed macrophages. Representative blot out of N=3. E: Quantification of (D). P-values were calculated by unpaired t-test, ns: not significant.$

Journal: bioRxiv

Article Title: Microbial Priming Enhances TLR7-Driven Myddosome Assembly Dynamics

doi: 10.64898/2025.12.16.693659

Figure Lengend Snippet: (A) Representative images of widefield microscopy of TLR7-dependent Myddosome formations in MyD88-mNG macrophages with or without prior LPS priming. (B) Quantification of MyD88 puncta per cell (only cells that contained at least 3 Myddosomes were included). Graph shows all individual cells from 3 independent repeats and corresponding means (N=3). Total number of analyzed cells: unstimulated=652, R848 only=620, LPS+R848=550, LPS acute=565, LPS no secondary stim.=504, LPS+LPS restim.=523. (C) Quantification of the cell fraction containing at least 3 Myddosomes: data is pooled from three independent experiments (N=3). Total number of analyzed cells: unstimulated=99, R848 only=262, LPS+R848=369, LPS acute=437, LPS no secondary stim.=121, LPS+LPS restim.=303. P-values for (B) and (C) were calculated by unpaired t-test. (D) Western blot analysis of WT MyD88 and MyD88-mNeonGreen in naïve and LPS-primed macrophages. Representative blot out of N=3. E: Quantification of (D). P-values were calculated by unpaired t-test, ns: not significant.

Article Snippet: TLR7 was stimulated with R848 (Resiquimod) (Invivogen, #tlrl-r848-1).

Techniques: Microscopy, Western Blot

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