Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet: MtbCM affects the PKCε-IRAK3-MKP-1 signaling cascades in macrophages RAW 264.7 macrophages were either treated with medium alone (cells) or infected with either Msmeg-pVV or Msmeg-MtbCM at MOI of 1:10. After 2 h of infection cells were washed to remove extracellular bacilli and incubated further for 3 h. Cells were lysed and the levels of MKP-1 were examined by Western blotting using anti-MKP-1 Ab (A). GAPDH was used as loading control and densitometric quantification of Western blots was performed using ImageJ software (B). Results shown are Mean ± SEM of 3 independent experiments. In another experiment, RAW 264.7 macrophages were infected with either Msmeg-pVV or Msmeg-MtbCM at 1:10 MOI. After 2 h of infection, cells were washed to remove extracellular bacilli and further incubated for another 3 h. Next cells were washed, fixed with 4% paraformaldehyde followed by permeabilization using 0.1% Triton X-100 and stained with anti-IRAK3 Ab at a dilution of 1:1000. Next, cells were washed and incubated with Alexa Flour-488 conjugated goat anti-rabbit secondary Ab at a dilution of 1:1000. After mounting with DAPI containing mounting medium, cells were observed under confocal microscope (C), (Scale bar: 5 μm). Results shown are representative of 3 different experiments. Also, lysates in Figure 3 A were used to check the levels of phospho-PKCε and total PKCε by Western blotting using anti-phospho-PKCε Ab and total PKCε Ab respectively (D) and densitometric quantification of Western blot was performed using ImageJ software (E). Results shown are Mean ± SEM of 3 independent experiments. Unpaired t -test was used to calculate the p values for Figures 3 B and 3E. In another experiment, RAW 264.7 macrophages were either treated with medium alone (cells) or infected with either Msmeg-pVV or Msmeg-MtbCM with MOI of 1:10 in the absence or presence of DCP-LA (100 nM). After 2 h of infection, cells were washed with 1X PBS to remove extracellular bacilli and incubated for another 2 h. Cells were harvested and protein lysates were prepared and analyzed for phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK (F) as well as MKP-1 (G) protein levels by Western blotting using specific antibodies. GAPDH was used as a loading control. Results shown are representative of 3 different experiments.

Article Snippet: Rabbit anti-IRAK3 Ab , Cell Signaling Technology, USA , Cat# 4369.

Techniques: Infection, Incubation, Western Blot, Control, Software, Staining, Microscopy

Journal: iScience

Article Title: The SH3-binding domain of chorismate mutase protein of Mycobacterium tuberculosis contributes to mycobacterial virulence

doi: 10.1016/j.isci.2024.111044

Figure Lengend Snippet:

Article Snippet: Rabbit anti-IRAK3 Ab , Cell Signaling Technology, USA , Cat# 4369.

Techniques: Recombinant, Adjuvant, Protease Inhibitor, Transfection, Control, Clone Assay, Software, Microscopy

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: IRAK3 expression and methylation levels in different types of human cancers. (A) Increased or decreased IRAK3 in datasets of various cancers compared with normal tissues by the Oncomine database. (B) IRAK3 expression levels in different tumors of TCGA data are obtained from the TIMER database (*, P<0.05; **, P<0.01; ***, P<0.001). (C) IRAK3 mRNA expression in LUAD tissues and normal tissues (N=59; T=522) (****, P<0.0001). (D) Methylation status of IRAK3 in different types of cancers and normal tissues from TCGA data (*, P<0.05; ****, P<0.0001). (E) Pearson correlation coefficient between IRAK3 mRNA expression and each CpG site methylation status in LUAD. (F) Scatter plots showing the methylation status of two CpG sites in promoter region of IRAK3 (cg20395892, cg26279550), which are correlated with reduced IRAK3 expression in 476 cases of LUAD tissue samples. TPM, transcripts per million; 5'UTR, 5' untranslated region; 3'UTR, 3' untranslated region; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing, Methylation

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: IRAK3 is commonly down-regulated in LUAD tissues and lack of IRAK3 predicts poor prognosis. (A) IHC staining reveals that IRAK3 is often down-regulated in LUAD relative to adjacent non-tumor tissues (****, P<0.0001). (B) Representative images from IHC staining of IRAK3 in LUAD tumor tissues and non-tumor tissues. (C) Overall survival analysis based on the expression level of IRAK3 measured by IHC in 222 LUAD patients from National Cancer Center. Survival rates were determined by the Kaplan-Meier survival analysis. (D) OS and PFS survival curves of lung cancer (n=2,166, n=1,252) in the Kaplan-Meier plotter database. (E) OS and PFS survival curves of LUAD (n=1,161, n=906) and LUSC (n=780, n=220) respectively. HR, hazard ratio; CI, confidence interval; OS, overall survival; PFS, progression-free survival; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; IHC, immunohistochemical.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Immunohistochemistry, Expressing, Immunohistochemical staining

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: Univariate and multivariate analysis of overall survival among patients with LUAD

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques:

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: Correlation between IRAK3 expression and clinicopathological characteristics of LUAD patients

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: IRAK3 expression predicts the clinical outcomes of LUAD patients with lymphatic metastasis. Kaplan-Meier survival plots (OS) comparing high and low expression of IRAK3 in stage N0 (A) and N1+2 (B) LUAD patients from the NCC cohort. Kaplan-Meier survival curves of OS comparing high- IRAK3 and low- IRAK3 group in stage N0 (C) and N1 (D) patients from LUAD cohort of the Kaplan-Meier plotter database. NCC, National Cancer Center; LUAD, lung adenocarcinoma; OS, overall survival; HR, hazard ratio; CI, confidence interval.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: The coexpression genes with IRAK3 and signaling pathways altered by IRAK3 in LUAD. (A) Every significantly associated gene with IRAK3 distinguished by Pearson test in the TCGA-LUAD cohort from the LinkedOmics database. (B,C) Top 50 genes positively and top 50 genes negatively related to IRAK3 in LUAD are showed by heatmap respectively. (D) Survival maps of the top 50 genes positively and negatively associated with IRAK3 in LUAD. (E) GSEA of multiple KEGG pathways in the LUAD cohort. (F) GO term annotations enrichment analysis of IRAK3 in the LUAD cohort. LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques:

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: The correlation of IRAK3 expression with immune infiltration level in LUAD. (A) Different infiltration levels of 20 immune cells in IRAK3 high and low tumors analyzed using CIBERSORT algorithm (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001). (B) The correlation between the infiltration of immune cells and the expression of IRAK3 . (C,D) IRAK3 expression significantly positively correlates with infiltrating levels of macrophage cells and CD4 + memory resting T cells (****, P<0.0001). (E) The correlation of IRAK3 expression with immune score, stromal score and ESTIMATE score in LUAD. NK, natural killer; LUAD, lung adenocarcinoma.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: Expression level of IRAK3 at the single-cell stage. (A) UMAP graphs indicate cellular clusters and the expression level of IRAK3 in diverse cellular types of LUAD datasets. (B) Single-cell IRAK3 expression profile of different cellular types in LUAD. (C) The heatmap of IRAK3 expression level in diverse cellular types of all NSCLC cohorts from TISCH database. (D) The correlation between IRAK3 expression and myeloid cell lineage-specific genes in TCGA-LUAD cohort. LUAD, lung adenocarcinoma; DC, dendritic cell; NK, natural killer; TPM, transcripts per million; UMAP, uniform manifold approximation and projection; NSCLC, non-small cell lung cancer; TCGA, The Cancer Genome Atlas.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: The correlation of IRAK3 expression with immunotherapy response. (A) IRAK3 expression is higher in ICB-resistant LUAD patients (n=220) than in ICB-responsive LUAD patients (n=285) (*, P<0.05). (B) TMB in high- IRAK3 and low- IRAK3 patients (***, P<0.001). (C) TIDE, T-cell dysfunction, T-cell exclusion scores and MSI comparison between the different IRAK3 subgroups using the Wilcoxon test (***, P<0.001; ns, P>0.05). (D) The relationship between IRAK3 and CTL dysfunction in TCGA-LUAD cohort. (E) Violin plots showing differential expression of immune checkpoint markers in different IRAK3 subgroups (**, P<0.01; ****, P<0.0001). (F) The correlation of IRAK3 and known immune checkpoint markers in LUAD (**, P<0.01; ***, P<0.001). (G) The lollipop plot for GSEA enrichment results of hallmark gene sets for LUAD samples. TPM, transcripts per million; TMB, tumor mutation burden; TIDE, tumor immune dysfunction and exclusion; CTL, cytotoxic T lymphocyte; MSI, microsatellite instability; NES, normalized enrichment score; ICB, immune checkpoint blockade; LUAD, lung adenocarcinoma; GSEA, gene set enrichment analysis.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing, Comparison, Mutagenesis

Journal: Translational Lung Cancer Research

Article Title: IL-1 receptor-associated kinase 3 (IRAK3) in lung adenocarcinoma predicts prognosis and immunotherapy resistance: involvement of multiple inflammation-related pathways

doi: 10.21037/tlcr-24-391

Figure Lengend Snippet: Assessment of chemotherapy responses related to IRAK3 expression level. (A) The correlations of IRAK3 expression and responses of six common chemotherapeutic and molecular targeted agents. (B) The responses of patients to the same six drugs in the high- and low- IRAK3 groups (***, P<0.001). IC 50 , half maximal inhibitory concentration.

Article Snippet: Slides were incubated with anti-IRAK3 (HPA043097, Sigma-Aldrich, Darmstadt, Germany) antibody at 4 ℃ overnight, followed by incubation with the secondary antibody.

Techniques: Expressing, Concentration Assay