Journal: RSC Advances

Article Title: Development of substituted 2-(4-(sulfonyl)piperazin-1-yl)quinazoline molecular hybrids as a new class of antimalarials

doi: 10.1039/d5ra08515b

Figure Lengend Snippet: Effect of the test compounds on the growth of the activated macrophages from THP1 monocytes (the results were the average of the five independent experiments).

Article Snippet: Lung cancer A549 epithelial cells and THP1 monocytes were obtained from ATCC.

Techniques:

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