thp1 human acute monocytic leukemia cells  (ATCC)

Journal: The Journal of Clinical Investigation

Article Title: LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9 + macrophages and enhances radiation-induced antitumor immunity

doi: 10.1172/JCI195279

Figure Lengend Snippet: ( A ) The coculture system. Human or mouse tumor cells were pretreated with IFN-β for 48 hours and cocultured with THP1-derived macrophages (THP1-MΦ) or RAW264.7 cells for another 48 hours. The macrophages were collected for analysis. ( B ) Heatmap showing the ISG15 , IFI44 , CXCL9 , CXCL10 , and SPP1 mRNA expression in cocultured THP1-MΦ. ( C ) Relative Cxcl9 and Cxcl10 expression in cocultured RAW264.7 cells. ( D ) CD86 and HLA-DR expression on cocultured THP1-MΦ. ( E ) CD40 expression on RAW264.7 cells cocultured with Usp5-deficient CT26 cells. ( F ) p-STAT2 and p-STAT1 in large extracellular vesicles (LEVs) from the culture medium of USP5-deficient SUNE1 and HK1 cells after IFN-β treatment. ( G ) p-STAT1 and p-STAT2 levels in THP1-MΦ after coculture with SUNE1 cells. ( H – J ) THP1-MΦ were treated with LEVs collected from the culture medium of STAT2-EGFP–transfected control and USP5-deficient SUNE1 cells after IFN-β treatment. The proportion of EGFP + macrophages ( H ), CD86 and HLA-DR expression ( I ), and relative CXCL9 and CXCL1 expression ( J ) in EGFP + and EGFP – macrophages were measured. ( K – M ) Indicated B16 tumors were subjected to irradiation on day 10 after inoculation. The proportions of CXCL9 + CD11b + macrophages measured by multiplex immunofluorescent staining ( K , n = 6 per group), the iNOS + CD11b + macrophages and IFN-γ + TNF-α + CD8 + T cells measured by flow cytometry ( L , n = 5 per group), and tumor growth ( M , n = 6 per group) are reported. The results are representative of 3 independent experiments ( B – J ). Data are presented as mean ± SD. Comparisons were performed using 1-way ANOVA ( H ) or 2-way ANOVA with Bonferroni’s test ( C – E and I – M ) for multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human monocyte leukemia cells THP1, mouse monocyte-macrophage leukemia cells RAW264.7, B16 murine melanoma cells, and MC38 and CT26 murine colon adenocarcinoma cells were obtained from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Transfection, Control, Irradiation, Multiplex Assay, Staining, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9 + macrophages and enhances radiation-induced antitumor immunity

doi: 10.1172/JCI195279

Figure Lengend Snippet: ( A ) The cytosolic and nuclear p-STAT1 and p-STAT2 levels in WT and USP5-deficient SUNE1 cells. ( B ) Representative confocal microscopy images and line scan analysis showing the different localization of p-STAT2 in WT or USP5-deficient SUNE1 cells. Scale bar: 10 μm. ( C ) Bubble diagram shows the enriched biological process of anti-STAT2 immunoprecipitated proteins in USP5-deficient SUNE1 cells after IFN-β treatment. ( D ) Volcano plot shows 1,353 upregulated and 566 downregulated proteins (FC ≥ 1.5 and P < 0.05) in anti-STAT2 immunoprecipitates from USP5-deficient compared with control SUNE1 cells. ( E and F ) Co-IP shows the interaction between STAT2 and LC3B-II in USP5-deficient SUNE1 cells ( E ) and HK1 cells ( F ) after IFN-β treatment. ( G and H ) Representative immunofluorescence assay images ( G ) showing the colocalization of p-STAT2 and LC3B in USP5-deficient SUNE1 cells after IFN-β treatment. Scale bar: 10 μm. ( H ) The Manders’ colocalization coefficients are reported. ( I ) The decreased p-STAT2 and p-STAT1 in large extracellular vesicles (LEVs) from the culture medium of USP5-deficient SUNE1 after LC3B knockdown. ( J ) LC3B knockdown in STAT2-EGFP SUNE1 cells decreased the EGFP + population in coculture with THP1-MΦ. ( K and L ) Control and USP5-deficient SUNE1 cells were transfected with si-LC3B and treated with IFN-β for 48 hours. LEVs were collected from the culture medium and used to treat the THP1-MΦ. The relative CXCL 9 and CXCL10 expression in macrophages was measured by RT-qPCR ( K ); the CD86 and HLA-DR expression on macrophages was measured by flow cytometry ( L ). The results are representative of 3 independent experiments ( A , B , and E – L ). The data are presented as mean ± SD ( H and J – L ). Comparisons were performed using 1-way ANOVA ( H ) or 2-way ANOVA ( J – L ) with Bonferroni’s test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human monocyte leukemia cells THP1, mouse monocyte-macrophage leukemia cells RAW264.7, B16 murine melanoma cells, and MC38 and CT26 murine colon adenocarcinoma cells were obtained from American Type Culture Collection (ATCC).

Techniques: Confocal Microscopy, Immunoprecipitation, Control, Co-Immunoprecipitation Assay, Immunofluorescence, Knockdown, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9 + macrophages and enhances radiation-induced antitumor immunity

doi: 10.1172/JCI195279

Figure Lengend Snippet: ( A ) Ubiquitination of STAT2 in control and USP5-depleted cells after IFN-β treatment. ( B ) The binding of STAT2 and LC3B-II in USP5-deficient SUNE1 cells after WT or C335A-mutated USP5 restoration and IFN-β treatment. ( C ) PRY41 treatment decreased p-STAT1 and p-STAT2 levels in LEVs from USP5-deficient SUNE1 cells. ( D ) Knockout of USP5 increased K63O-linked ubiquitination of STAT2 in IFN-β–treated SUNE1 cells. ( E ) Mass spectrometry analysis of STAT2 ubiquitination sites. ( F ) Ubiquitination of FLAG-tagged WT and K167R, K197R, and K239R-STAT2 mutants in USP5-deficient SUNE1 cells. ( G ) Representative confocal microscopy images showing the different localization of phosphorylated WT or K167R-mutated STAT2 in WT or USP5-deficient SUNE1 cells. Scale bar: 10 μm. ( H and I ) The colocalization relationship and Manders’ colocalization coefficients between p-STAT2 and LC3B in STAT2- and USP5-deficient SUNE1 cells with WT STAT2 or K167R-mutant reconstruction and IFN-β treatment. Scale bar: 10 μm. ( J ) The binding between LC3B with WT or K167R-STAT2 in SUNE1 cells after IFN-β treatment. ( K ) The p-STAT1 and p-STAT2 levels in LEVs collected from the culture medium of STAT2-deficient SUNE1 cells transfected with WT or K167R-mutated STAT2 after IFN-β treatment. ( L and M ) Control and USP5-deficient SUNE1 cells were transfected with WT STAT2 or K167R-mutant and treated with IFN-β for 48 hours. LEVs were collected from culture medium and treated for THP1-MΦ. The CXCL 9 and SPP1 expression in macrophages was measured by RT-qPCR ( L ), and the CD86 expression on macrophages was measured by flow cytometry ( M ). The results are representative of 3 independent experiments ( A – D and F – M ). The data are presented as mean ± SD. Comparisons were performed using 2-way ANOVA with Bonferroni’s test ( I , L , and M ) for multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The human monocyte leukemia cells THP1, mouse monocyte-macrophage leukemia cells RAW264.7, B16 murine melanoma cells, and MC38 and CT26 murine colon adenocarcinoma cells were obtained from American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Control, Binding Assay, Knock-Out, Mass Spectrometry, Confocal Microscopy, Mutagenesis, Transfection, Expressing, Quantitative RT-PCR, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: LC3-dependent intercellular transfer of phosphorylated STAT1/2 elicits CXCL9 + macrophages and enhances radiation-induced antitumor immunity

doi: 10.1172/JCI195279

Figure Lengend Snippet: ( A ) USP5-IN-1 increased STAT2 ubiquitination in SUNE1 and HK1 cells after IFN-β treatment. ( B ) Co-IP shows that the USP5-IN-1 treatment promotes the interaction between STAT2 and LC3B-II. ( C ) Immunofluorescence assay and representative images show the colocalization of p-STAT2 and LC3B in USP5-IN-1– and IFN-β–treated SUNE1 and HK1 cells. Scale bar: 10 μm. The Manders’ colocalization coefficients are reported. ( D ) Western blot showing the p-STAT1 and p-STAT2 levels in large extracellular vesicles (LEVs) from SUNE1 and HK1 cells treated with USP5-IN-1 and IFN-β. ( E and F ) Relative mRNA expression of ISG15 , IFI44 , CXCL9 , CXCL10 , and SPP1 ( E ), and HLA-DR and CD86 expression ( F ) in THP1-MΦ after coculture with SUNE1 cells pretreated with DMSO or USP5-IN-1 and IFN-β. The results are representative of 3 independent experiments ( A – F ). The data are presented as mean ± SD. Comparisons were performed using 2-way ANOVA with Bonferroni’s test ( C and F ) for multiple comparisons. * P < 0.05, **** P < 0.0001.

Article Snippet: The human monocyte leukemia cells THP1, mouse monocyte-macrophage leukemia cells RAW264.7, B16 murine melanoma cells, and MC38 and CT26 murine colon adenocarcinoma cells were obtained from American Type Culture Collection (ATCC).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Immunofluorescence, Western Blot, Expressing

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: GATA2 Mediates Macrophage Proliferation During Atherosclerosis

doi: 10.1161/JAHA.125.043646

Figure Lengend Snippet: A , Cytoscape network analysis identified enrichment of a proliferative network in the transcriptome of GATA2+ macrophages compared with monocytes from healthy controls. B , Overexpression of GATA2 in THP1‐derived macrophages increases the expression of MYB and decreases the expression of JUN compared with wild‐type THP1 macrophages. C and D , Quantification of JUN ( C ) and MYB ( D ) promoter activity using firefly luciferase reporters containing the MYB or JUN promoters, in WT vs GATA2‐overexpressing (GATA2) THP1‐derived macrophages. Cells are either untransfected (Mock), transfected with a promoterless vector (Empty) or transfected with a MYB‐ or JUN‐promoter containing vector. E , MYB expression measured by RT‐PCR in WT, GATA2‐overexpressing (GATA2), and GATA2‐shRNA expression (shGATA2) THP1 monocytes and THP1‐derived macrophages. F , Impact of GATA2 on the proliferation rate of GATA2‐overexpressing (GATA2) vs WT THP1 monocytes as they differentiate into macrophages. G , GATA2 expression in THP1‐macrophages following exposure to the indicated concentrations of oxLDL for 48 hours. P values are in comparison to 0 mmol/L. H , Impact of GATA2 and MYB siRNA knockdown, vs a scrambled siRNA control (Scrm), on the proliferation of THP1‐macrophages that are untreated (UT) or treated with oxLDL. I , THP1 proliferation during monocyte–macrophage differentiation and during macrophage‐foam cell differentiation. THP1 cells are either WT or GATA2 overexpressing (GATA2)±treatment with oxLDL during monocyte‐to‐macrophage differentiation (oxLDL), or were first differentiated fully into macrophages before loading with proliferation dye, and proliferation quantified during FC differentiation. Data are presented as the integrated cell proliferation dye intensity of >250 cells. Proliferation is identified as a decrease in the intensity of the cell proliferation dye compared with the fixed control sample (Fixed). * P <0.0001, † P >0.9999 between indicated groups. J , RT‐PCR quantification of GATA2 and MYB expression during the polarization of (HC) vs Pt monocytes into Mφ and FC. K , THP1‐derived macrophage apoptosis under control conditions vs following stimulation with 2.5 mmol/L oxLDL, or 10 μmol/L Tg. Macrophages are either derived from WT THP1 cells (green), GATA2‐overexpressing THP1 cells (magenta), or THP1 cells expressing a GATA2‐depleating shRNA (shGATA2, orange). n=3 ( B , E , I ), 5 ( C , D , H , K ), 6 ( F ), or 4 ( G , J ) biological replicates, Mann–Whitney U test ( B through D , H ) or Kruskal–Wallis test with Dunn correction ( G , I , J ). FC indicates foam cell; HC, healthy control; Mφ, macrophage; oxLDL, oxidized low‐density lipoprotein; Pt, Patient; RT‐PCR, reverse transcription polymerase chain reaction; Tg, thapsigargin; and WT, wild‐type.

Article Snippet: Lympholyte poly and THP1 human monocytic cells (ATCC TIB 202) were purchased from Cedarlane Labs (Burlington, Canada).

Techniques: Over Expression, Derivative Assay, Expressing, Activity Assay, Luciferase, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, shRNA, Comparison, Knockdown, Control, Cell Differentiation, MANN-WHITNEY, Reverse Transcription, Polymerase Chain Reaction

Journal: Nature Communications

Article Title: AR + TREM2 + macrophage induced pathogenic immunosuppression promotes prostate cancer progression

doi: 10.1038/s41467-025-62381-x

Figure Lengend Snippet: a Representative immunofluorescence staining images of AR and F4/80 in WT BMDMs and TREM2 KO BMDMs. Scale bar: 5 μm. b Representative flow plots and quantification of AR expression in WT BMDMs and TREM2 KO BMDMs ( n = 5 biologically independent samples). c Representative immunofluorescence staining images of AR in WT BMDMs treated with RM1 CM, RM1 CM + DMSO or RM1 CM + ENZA, respectively. Scale bar: 10 μm. d , e Protein expression levels of AR ( d ) and TREM2 ( e ) in WT BMDMs treated with DMSO or 10 μM ENZA for 48 h were quantified by flow cytometry ( n = 5 biologically independent samples). f , g Quantification of AR ( f ) and TREM2 ( g ) expression in WT BMDMs treated with RM1 CM for 48 h ( n = 5 biologically independent samples). h , i Relative protein expression of AR ( h ) and TREM2 ( i ) in THP1 cells treated with 22Rv1 CM for 48 h ( n = 5 biologically independent samples). j , k Protein expression levels of AR ( j ) and TREM2 ( k ) in THP1 cells treated with Lncap CM for 48 h ( n = 5 biologically independent samples). l , m Representative flow plots and quantification of TREM2 ( l ) and AR ( m ) in TAMs of Pten PC-/- TREM2 f/f mice and Pten PC-/- TREM2 f/f -Lyz2-cre mice ( n = 5 biologically independent samples). n The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways following ASC-J9 treatment in WT BMDMs. o The bubble chart of KEGG pathway enrichment analysis illustrated the significantly downregulated signalling pathways in RM1 CM treated TREM2 KO BMDMs compared to WT BMDMs. p Representative immunoblot analysis of AR, TREM2, p-STAT3, ROR-γ, p-Src, p-Syk, and p-JAK1 in WT BMDMs, TREM2 KO BMDMs and DAP12 KO BMDMs treated with RM1 CM for 48 h. Experiment was repeated three times independently with similar results. q Representative immunoblot analysis of AR and ROR-γ in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 5 μM SR2211 (ROR-γ inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. r Representative immunoblot analysis of AR and p-STAT3 in WT BMDMs and TREM2 KO BMDMs treated with DMSO plus RM1 CM or 10 μM C188-9 (p-STAT3 inhibitor) plus RM1 CM for 48 h. Experiment was repeated three times independently with similar results. For a and c, experiments were repeated three times independently with similar results. All the data are presented as mean ± SD. The P- values were determined by two-sided Mann-Whitney U test for ( b , d − m ); and by Hypergeometric Test for ( n , o ). Source data are provided as a Source Data file.

Article Snippet: Mouse prostate cancer cells RM1 (Cat# CRL-3310; RRID: CVCL_B459), human prostate cancer cells 22Rv1 (Cat# CRL-2505; RRID: CVCL_1045) and LnCap (Cat# CRL-1740; RRID: CVCL_0395), human monocytic-leukemia cells THP1 (Cat# TIB-202; RRID: CVCL_0006), and mouse fibroblasts L929 (Cat# CCL-1; RRID: CVCL_AR58) were purchased from ATCC and cultured in accordance with the instructions provided by the manufacturer.

Techniques: Immunofluorescence, Staining, Expressing, Flow Cytometry, Western Blot, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Interrogation of macrophage-related prognostic signatures reveals a potential immune-mediated therapy strategy by histone deacetylase inhibition in glioma

doi: 10.3389/fonc.2025.1554845

Figure Lengend Snippet: Prediction and validation of the potential anticancer drugs. (A) Correlation between CD163 expression and risk score in the TCGA database (p <0.0001). (B) Differences in mRNA expression of various prognostic genes and CD163 in the indirect co-culture model (*p <0.05; * *p <0.01; * * *p <0.001; * * * *p <0.0001). (C) Expression of CD163 and risk score in the indirect co-culture model. (D) Potential anticancer drugs were predicted using signature genes and a risk score. (E) The U87MG (RP) was cocultured with the PMA-induced adherent THP1 (GP), and the treatment groups were then treated with Vorinostat. (F) Analysis of growth of U87MG and U87MG cocultured with PMA-induced adherent THP1 before and after Vorinostat treatment. (G) Expression of macrophage-related immune checkpoints of THP1 before and after the treatment with Vorinostat in the U87MG-THP1 indirect co-culture model (*p <0.05; * *p <0.01; * * *p <0.001; * * * *p <0.0001). (H) The expression of Cd163 in RAW264.7 cells at the protein level, with GL261 conditioned medium and Vorinostat as variables.

Article Snippet: Human glioma cell lines A172 and U87MG, human monocytic leukemia cell line THP1, mouse macrophages cell line RAW264.7 were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).

Techniques: Biomarker Discovery, Expressing, Co-Culture Assay