Journal: Experimental & Molecular Medicine

Article Title: Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

doi: 10.1038/s12276-022-00886-x

Figure Lengend Snippet: a Volcano plots of differentially expressed genes in THP1 cells after treatment with the indicated PM types for 24 h. b Multidimensional scaling (MDS) plot of RNA-seq expression profiles in two dimensions. c Heatmap of 452 differentially expressed human cytokine and growth factor genes between THP1-Control cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. d Venn diagram of upregulated cytokines and growth factors. e Venn diagram of downregulated cytokines and growth factors.

Article Snippet: The human lung cancer cell line A549 and human monocytic leukemia cell line THP1 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: RNA Sequencing Assay, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

doi: 10.1038/s12276-022-00886-x

Figure Lengend Snippet: a Wound healing assay of A549 cells incubated with CM from PM-treated THP1 cells; images were acquired at 0 and 36 h. Scale bar, 250 μm. b Transwell migration (top) and invasion (bottom) assays of A549 cells incubated with CM from PM-treated THP1 cells. Scale bar, 200 μm. c Phospho-RTK array assay of A549 CM-Control (Con) and A549 CM-PM cells. The dots inside the rectangle represent EGFR (left). Relative pixel intensities indicating phospho-EGFR levels in the phospho-RTK array (right). d Gene set enrichment analysis plot of EGFR-induced genes in A549 CM-PM cells. NES normalized enrichment score, FWER familywise error rate, FDR false discovery rate. e Immunoblot analysis of EGFR phosphorylation in A549 cells after incubation with CM from PM-treated THP1 cells for the indicated time. β-Actin was used as the loading control. f Immunoblot analysis of EGFR phosphorylation in A549 cells after incubation with the indicated concentrations of CM from PM-treated THP1 cells at for 20 min. * P ≤ 0.05, *** P ≤ 0.001.

Article Snippet: The human lung cancer cell line A549 and human monocytic leukemia cell line THP1 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Wound Healing Assay, Incubation, Migration, Western Blot

Journal: Experimental & Molecular Medicine

Article Title: Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

doi: 10.1038/s12276-022-00886-x

Figure Lengend Snippet: a Heatmap showing the differential expression of the indicated EGFR ligands between THP1-Control (Con) cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. b qRT‒PCR analysis of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. c ELISA of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. AD Arizona dust. d Immunoblot analysis of THP1 cells treated with the indicated concentrations of different types of PM for 24 h. e qRT‒PCR analysis of human cord blood-derived macrophages treated with 5 μg/cm 2 PM for 24 h. f ELISA of human cord blood-derived macrophages treated with 5 μg/cm 2 PM for 24 h. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: The human lung cancer cell line A549 and human monocytic leukemia cell line THP1 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay

Journal: Experimental & Molecular Medicine

Article Title: Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

doi: 10.1038/s12276-022-00886-x

Figure Lengend Snippet: a Heatmap of differential gene expression between A549 CM-Control (Con) and A549 CM-PM cells identified by RNA-seq. b Gene set enrichment analysis plot for the “Epithelial-to-mesenchymal transition” gene set from the HALLMARK gene collection. NES normalized enrichment score; FWER familywise error rate; FDR false discovery rate. c Immunoblot analysis of A549 cells incubated for 24 h with the indicated concentration of CM from PM-treated THP1 cells. β-Actin was used as the loading control. d Immunoblot analysis of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. e qRT‒PCR analysis of A549 cells incubated for 24 h with the indicated concentration of CM from PM-treated THP1 cells. f qRT‒PCR analysis of A549 cells treated with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. g Immunofluorescence analysis of A549 cells cultured with CM-PM immunoprecipitated using control IgG or an anti-HBEGF antibody for 24 h. Scale bar, 20 μm. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; ns not significant.

Article Snippet: The human lung cancer cell line A549 and human monocytic leukemia cell line THP1 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Incubation, Concentration Assay, Immunoprecipitation, Immunofluorescence, Cell Culture

Journal: Experimental & Molecular Medicine

Article Title: Particulate matter promotes cancer metastasis through increased HBEGF expression in macrophages

doi: 10.1038/s12276-022-00886-x

Figure Lengend Snippet: a Heatmap of the differential expression of AhR-related genes between THP1-Control (Con) cells and THP1-PM2.5, THP1-PM10, and THP1-KRPM cells identified by RNA-seq. b qRT‒PCR analysis of THP1 cells treated with different types of PM (25 μg/cm 2 ) for 24 h. c Immunoblot analysis of PM2.5 (25 μg/cm 2 )-stimulated AhR translocation. β-Actin and Lamin A were used as the cytoplasmic fraction and nuclear fraction loading controls, respectively. d Immunoblot analysis of THP1 cells treated with the indicated concentrations of PM2.5 for 24 h. e , f qRT-PCR ( e ) and ELISA ( f ) of THP1 cells transfected with 50 nM siAhR and treated with 25 μg/cm 2 PM2.5 for 24 h. g , h qRT‒PCR ( g ) and ELISA ( h ) of THP1 cells pretreated with 10 nM CH-223191 for 1 h and treated with 25 μg/cm 2 PM2.5 for an additional 24 h. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001; ns not significant.

Article Snippet: The human lung cancer cell line A549 and human monocytic leukemia cell line THP1 were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Translocation Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Intracellular pathogen Leishmania intervenes in iron loading into ferritin by cleaving chaperones in host macrophages as an iron acquisition strategy

doi: 10.1016/j.jbc.2022.102646

Figure Lengend Snippet: Leishmania infection affects iron chaperon PCBP1 and PCBP2 protein expression in host macrophages. A , splenocytes were isolated from mouse (n = 3) and subjected to Leishmania donovani (LD) infection for 2 h (MOI = 1:10) or kept uninfected. Cell lysates were immunoblotted using PCBP1, PCBP2, and actin antibody. B , J774 macrophages were infected with LD (MOI = 1:10) for 0 to 2 h. Cell lysates were immunoblotted using PCBP1, PCBP2, hnRNP K, and actin antibody. C , murine macrophage cell lines (J774 and RAW264.1) and human monocytic cell lines (THP1 and U937) were infected with LD for 2 h (MOI = 1:10) or kept uninfected and subjected to immunoblot analyses using PCBP1, PCBP2, and actin antibodies. D , J774 cells were infected with Leishmania major (LM) and Leishmania tarentolae (LT) (MOI = 1:10) for 4 h, and immunoblot was performed using PCBP1, PCBP2, and actin antibody. Results are representatives of one of the three independent experiments. Right panels show quantifications of intact PCBP1 or PCBP2 representing ±SD from three independent experiments. E , mice splenocytes were isolated and infected with LD (up to 120 min). PCBP1 and PCBP2 mRNA expressions were determined from total RNA using quantitative RT–PCR. β-Actin was used as endogenous control. Data represent mean ± SD from three independent experiments performed in triplicates. hnRNP, heterogeneous nuclear ribonucleoprotein; J774, J774A.1; MOI, multiplicity of infection; PCBP, poly(rC)-binding protein.

Article Snippet: Murine macrophage cell J774A.1, RAW264.1, and human monocytic cell lines THP1 and U937 were procured from American Type Culture Collection and maintained in RPMI1640 medium (Sigma–Aldrich) supplemented with 10% (v/v) FBS and 10,000 units/ml penicillin, and 10,000 μg/ml streptomycin in a humidified atmosphere containing 5% CO 2 at 37 °C in a CO 2 incubator ( , , ).

Techniques: Infection, Expressing, Isolation, Western Blot, Quantitative RT-PCR, Binding Assay

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis infection elevates SLIT2 expression to modulate oxidative stress responses in macrophages

doi: 10.1101/2022.10.13.512188

Figure Lengend Snippet: (A) THP1 macrophages were infected with Mtb H37Rv for the indicated time points. Whole cell lysates were assessed for SLIT2 protein expression by immunoblotting. (B) Mouse peritoneal macrophages were infected with Mtb H37Rv for 24 h and assessed for the transcript levels of ROBO receptors. (C) Mouse peritoneal macrophages were infected with Mtb H37Rv for the indicated time points and ROBO2 levels were assessed by immunoblotting. All immunoblotting data are representative of three independent experiments. β-ACTIN was utilized as loading control. qRT-PCR data represents mean±S.E.M. from three independent experiments.

Article Snippet: RAW 264.7 mouse monocyte-like cell line and human monocytic cell line THP1 cells were obtained from American Type Culture Collection (ATCC), USA.

Techniques: Infection, Expressing, Western Blot, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis infection elevates SLIT2 expression to modulate oxidative stress responses in macrophages

doi: 10.1101/2022.10.13.512188

Figure Lengend Snippet: (A) Schematic representing the probable mechanism leading to Slit2 upregulation during Mtb infection. (B) THP1 macrophages were infected with Mtb H37Rv for 24 h and assessed for the recruitment of H3K27me3 and p-Histone H3 (Ser28) over the Slit2 promoter by ChIP assay. (C) Mouse peritoneal macrophages were treated with the indicated inhibitors for 1 h, followed by 24 h infection with Mtb H37Rv and assessed for the expression of Slit ligands transcripts by qRT-PCR (D) Mouse peritoneal macrophages were treated with the indicated inhibitors for 1 h, followed by 24 h infection with Mtb H37Rv and assessed for the expression of indicated proteins by immunoblotting (E, F) Mouse peritoneal macrophages were treated with the indicated inhibitors for 1 h. Cells were infected with Mtb H37Rv for 24 h and assessed for the accumulation of ROS by fluorescence microscopy (CellROX Deep Red); (E) representative image and (F) respective quantification. . All immunoblotting and immunofluorescence data are representative of three independent experiments. β-ACTIN was utilized as loading control. qRT-PCR data represents mean±S.E.M. from three independent experiments. ROS, reactive oxygen species; h, hours; ChIP, Chromatin immunoprecipitation; d, days; kda, kilodalton; CTCF, corrected total cell fluorescence. *, p<0.05; ****, p < 0.0001 (Student’s t-test in B, One-way ANOVA in C,F; GraphPad Prism 6.0 and 9.0). Scale bar, 5 μm. SB203580: p38 inhibitor, SP600125: JNK1/2 inhibitor.

Article Snippet: RAW 264.7 mouse monocyte-like cell line and human monocytic cell line THP1 cells were obtained from American Type Culture Collection (ATCC), USA.

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Fluorescence, Microscopy, Immunofluorescence, Chromatin Immunoprecipitation

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis infection elevates SLIT2 expression to modulate oxidative stress responses in macrophages

doi: 10.1101/2022.10.13.512188

Figure Lengend Snippet: (A) THP1 macrophages were infected with Mtb H37Rv for 24 h and assessed for the recruitment of H3K27me3 over the Slit2 promoter by ChIP assay. (B) Mouse peritoneal macrophages were infected with Mtb H37Rv for the indicated time points and assessed for the levels of p-Histone H3 (Ser28) by immunoblotting. (C) THP1 macrophages were infected with Mtb H37Rv for the indicated time points and assessed for the levels of p-Histone H3 (Ser28) by immunoblotting. (D) THP1 macrophages were treated with the indicated inhibitors for 1 h, followed by 24 h infection with Mtb H37Rv and assessed for the expression of SLIT2 by immunoblotting. All immunoblotting data are representative of three independent experiments. β-ACTIN was utilized as loading control.

Article Snippet: RAW 264.7 mouse monocyte-like cell line and human monocytic cell line THP1 cells were obtained from American Type Culture Collection (ATCC), USA.

Techniques: Infection, Western Blot, Expressing

Journal: bioRxiv

Article Title: Mycobacterium tuberculosis infection elevates SLIT2 expression to modulate oxidative stress responses in macrophages

doi: 10.1101/2022.10.13.512188

Figure Lengend Snippet: (A) THP1 macrophages were infected with Mtb H37Rv for 24 h and assessed for the levels of VNN1 by immunoblotting. (B) Mouse peritoneal macrophages were treated with rSLIT2 at the indicated concentrations for 12 h and assessed for the levels of VNN1 by immunoblotting. (C) Mouse peritoneal macrophages were transfected with NT or Vnn1 siRNAs. Transfected cells were infected with Mtb H37Rv for 24 h and assessed for the accumulation of ROS by fluorescence microscopy (CellROX Deep Red). All immunoblotting and immunofluorescence data are representative of three independent experiments. β-ACTIN was utilized as loading control. Scale bar, 5 μm.

Article Snippet: RAW 264.7 mouse monocyte-like cell line and human monocytic cell line THP1 cells were obtained from American Type Culture Collection (ATCC), USA.

Techniques: Infection, Western Blot, Transfection, Fluorescence, Microscopy, Immunofluorescence

Journal: Scientific Reports

Article Title: Proteomic networks associated with tumor-educated macrophage polarization and cytotoxicity potentiated by heat-killed tuberculosis

doi: 10.1038/s41598-022-10463-x

Figure Lengend Snippet: Flow cytometric analysis of macrophage phenotypes. Phenotypes of ( A ) primary peripheral blood macrophages from healthy subjects and lung cancer patients and ( B ) THP1-derived classically activated macrophage (M) and tumor-educated macrophage (TEM) models were determined before and after 3 days of heat-killed tuberculosis (HKTB) stimulation. Data with ≤ 50 cells within the gate were omitted. ns, not significant.

Article Snippet: An in vitro study was developed utilizing the THP1 human monocytic cell line (ATCC® TIB-202™, ATCC, Manassas, VA, USA) and the A549 lung adenocarcinoma cell line (ATCC® CCL-185™) for macrophage polarization, cytokine, and cell viability assays.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Mutation in wbaP cps Gene Cluster Selected by Phage-Borne Depolymerase Abolishes Capsule Production and Diminishes the Virulence of Klebsiella pneumoniae

doi: 10.3390/ijms222111562

Figure Lengend Snippet: Comparison of virulence of capsule-deficient Kp7De mutant and its parental Kp486 strain. Adhesion ( A ) and time-dependent internalization ( B ) by the A549 lung epithelial cells expressed in CFU per well and shown as the mean ± SEM ( n = 4). ( C ) Phagocytosis by the human monocyte cell line THP1 determined by flow cytometry. UV-killed FITC-labeled bacterial cells were mixed with monocytes at a 100:1 ratio. Quantification of cellular uptake of bacterial cells by phagocytes is expressed as the geometric mean fluorescence intensity (gMFI) from the gated sample with phagocytosing cells ± SEM ( n = 3). ( D ) Time-dependent bactericidal effect of 50% NHS or heat-inactivated NHS for 3 h at 37 °C. The percentage of survival was determined as the number of bacteria that survived relatively to the initial bacterial loading ( n = 3). Asterisks indicate a statistically significant difference ( p < 0.05).

Article Snippet: Briefly, Human monocyte/macrophage cell line THP1 (ATCC, TIB-202) was maintained in RPMI-1640 medium (Lonza, Verviers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (HIFBS; GIBCO, Life Technologies, Grand Island, NY, USA), 1× glutaMAX (GIBCO, Life Technologies, Grand Island, NY, USA), and 1× antibiotic–antimycotic solution (GIBCO, Life Technologies, Grand Island, NY, USA) at 37 °C in a 5% CO 2 atmosphere. (i) Fluorescence labeling of Klebsiella strains Washed bacteria (ca.

Techniques: Mutagenesis, Flow Cytometry, Labeling, Fluorescence