Journal: Nature Communications

Article Title: Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma

doi: 10.1038/s41467-023-40987-3

Figure Lengend Snippet: a Three-dimensional visualization of α-tubulin and Fn in E109 cells. The cells were infected with Fn (MOI of 1:10) for 48 h. Scale bar: 50 μm (left). b , c Intracellular bacterial proliferation was assessed by a gentamycin protection assay. E109, Kyse150 and AKR cells were lysed at the indicated time points after Fn (MOI of 1:10) infection, and the numbers of total viable bacteria ( b ) and viable bacteria per ESCC cell ( c ) were determined by the serial dilution method. P means vs . the 24 h group. d , e FACS of PD-L1 + membrane expression in E109, Kyse150 and AKR cells after infection with Fn (MOI of 1:10) for 48 h and quantification. f , g Dichromatic IF staining of PD-L1 and Fn in E109, Kyse150 and AKR cells. The cells were infected with Fn (MOI of 1:10) for 48 h. f Representative images. Scale bar: 20 μm. g Quantification. h qRT‒PCR analysis of PD-L1 in E109, Kyse150 and AKR cells after infection with Fn (MOI of 1:1, 1:10, 1:100) for 48 h or infection with Fn (MOI of 1:10) for 24, 48 or 72 h. P means vs . the Con group. i , j Immunoblotting analysis of PD-L1 in E109, Kyse150 and AKR cells after infection with Fn ( i ) and quantification ( j ). P means vs . the Con group. k Immunoblotting analysis and quantification of PD-L1 in tumors from C57BL/6 xenografts (mean ± SD; the experiment was done once; n = 3 mice per group). l IF staining of PD-L1 or Fn in tumor tissues from C57BL/6 xenografts. Scale bar: 50 μm. Images in a and l were representative results of n = 3 independent experiments with similar results. Results in b , c , e , g , h and j were presented as n = 3 biological replicates, mean ± SD, ns means not significant. The statistical significance of results in b , c , h , j and k were determined by one-way ANOVA analysis. e and g were determined by a two-tailed unpaired t -test.

Article Snippet: The human ESCC cell line Eca109 (E109) and Kyse150 (a kind gift from professor Musheng Zeng, SYSUCC, Guangzhou, China), Jurkat cell line (ATCC TIB-152), embryonic kidney 293 T cells (ATCC CRL-3216), and mouse ESCC cell line AKR (C945, Wuhan Sunncell Biotechnology, China) were cultured in RPMI 1640 or DMEM medium (Gibco, CA, USA), respectively.

Techniques: Infection, Bacteria, Serial Dilution, Membrane, Expressing, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma

doi: 10.1038/s41467-023-40987-3

Figure Lengend Snippet: a – c Dichromatic IF staining of Fn and Fn-Dps ( a ), scale bar: 40 μm (top). Immunoblotting ( b ) and FACS ( c ) analysis of PD-L1. Cells were infected with Fn (MOI of 1:10) or treated with Fn-Dps (1 μM) for 48 h. d – f Venn diagram showing the unique and overlapping predicted transcription factors in different groups. The blue dots represent differentially expressed transcription factor genes (POU6F1, CEBPB, NR4A2 and MAFG). qRT‒PCR analysis ( f ). g Analysis of the relevance of the CD274 (PD-L1) and ATF3 genes in ESCA tissues (TCGA database, n = 173). h – j ATF3 and PD-L1 expression was analyzed by western blotting. ESCC cells were transfected with ATF3 overexpression (OE) or negative control (CTRL) vectors for 48 h ( h ). Co-IP assay ( i ). Cells were treated with Fn-Dps (1 μM) for 48 h after transfection with siRNA against ATF3 for 48 h ( j ). k , l Representative images of IHC staining ( k ) and IF staining quantification ( l ) of ATF3 or PD-L1 expression in tumors from C57BL/6 xenografts. Scale bar: 10 μm. m The interaction of Fn-Dps with ATF3 in ESCC cells after treated with Fn-Dps (1 μM) for 48 h was detected by Co-IP assay. n Analysis of CD274 WT or mutant promoter activity in 293 T cells transfected with ATF3-Flag and treated with Fn-Dps (1 μM) for 48 h. o ChIP‒qPCR analysis of the relative enrichment of ATF3 at the CD274 gene promoter in Kyse150 cells. p , q Dichromatic IF staining of ATF3 and Fn-Dps (p), scale bar: 50 μm (top). Immunoblotting of nuclear (N) and cytoplasmic (C) ( q ). Cells were treated with Fn-Dps (1 μM) for 48 h. Images in a , h , j , m , p and q were representative results of n = 3 independent experiments with similar results. Results in b , c , n , and o were presented as n = 3 biological replicates, mean ± SD. Statistical significance in b , c was determined by a two-tailed unpaired t -test. l , n and o were determined by two-way ANOVA multiple comparisons. g was determined by two-tailed nonparametric Spearman correlation analysis.

Article Snippet: The human ESCC cell line Eca109 (E109) and Kyse150 (a kind gift from professor Musheng Zeng, SYSUCC, Guangzhou, China), Jurkat cell line (ATCC TIB-152), embryonic kidney 293 T cells (ATCC CRL-3216), and mouse ESCC cell line AKR (C945, Wuhan Sunncell Biotechnology, China) were cultured in RPMI 1640 or DMEM medium (Gibco, CA, USA), respectively.

Techniques: Staining, Western Blot, Infection, Expressing, Transfection, Over Expression, Negative Control, Co-Immunoprecipitation Assay, Immunohistochemistry, Mutagenesis, Activity Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Haemophilus parasuis Encodes Two Functional Cytolethal Distending Toxins: CdtC Contains an Atypical Cholesterol Recognition/Interaction Region

doi: 10.1371/journal.pone.0032580

Figure Lengend Snippet: The cells were analyzed for cell cycle distribution by treated with Cdt proteins alone or in various combinations (as indicated) following staining with propidium iodide. (A), PAM cell; (B), PK-15 cell; (C) Jurkat cell or (D) PAM cells were exposed to 200 µl CdtB alone (50 µg/ml) or in the presence of CdtA (50 µg/ml) or CdtC (50 µg/ml). The cells were analyzed for cell cycle distribution by flow cytometry based upon propidium iodide fluorescence. The data represent the mean ± SEM of three experiments; at least 20,000 cells were analyzed per sample.

Article Snippet: The porcine alveolar macrophage cell line PAM and the human T cell leukemia cell line Jurkat E6-1 were obtained from the ATCC and cultured in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 10 mM HEPES.

Techniques: Staining, Flow Cytometry, Fluorescence

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 4 HL-60 (a), Raji (b), and Jurkat cells (c) were treated with 150 nm flavopiridol7500 nm MG-132, after which the percentage of cells exhibiting apoptotic morphology and reduced DCm were determined, respectively. Values represent the means7s.d. for three separate experiments performed in triplicate. Alternatively, cells were lysed and subjected to Western blot using PARP antibody (d). CF ¼ cleavage fragment. Two additional studies yielded equivalent results

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Western Blot

Journal: Oncogene

Article Title: Proteasome inhibitors potentiate leukemic cell apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol through a SAPK/JNK- and NF-kappaB-dependent process.

doi: 10.1038/sj.onc.1206863

Figure Lengend Snippet: Figure 5 U937 cells were treated with 100 nm flavopiridol7300 nm MG-132 for 24 h, after which expression of phosphorylated MAP kinases (e.g. SAPK/JNK, ERK, and p38) (a), cell cycle regulators (e.g. p34cdc2, p21CIP/WAF1, p27KIP1), and apoptotic regulators (e.g. Bcl- 2, Bcl-xL, Bax, XIAP, and Mcl-1) (b) were monitored by Western blot. Alternatively, cell lysates were subjected to SAPK/JNK kinase assay. SAPK/JNK activity was reflected by phosphorylated GST-c-Jun (a, bottom panel). Similarly, U937 cells were treated with 100 nm flavopiridol7500 nm lactacystin for 24 h (c), after which expression of indicated protein was analysed by Western blot. Phosphorylation of SAPK/JNK was also monitored in HL- 60, Jurkat, and Raji cells after treatment with 150 nm flavopir- idol þ 500 nm MG-132 for 24 h (d). Each lane was loaded with 25 mg of protein; blots were stripped and reprobed with antibodies to tubulin or actin to ensure equal loading and transfer. Results are representative of three separate experiments

Article Snippet: U937 human histiocytic leukemia, HL-60 acute promyelocytic leukemia, Jurkat acute T-cell leukemia, and Raji Burkitt’s lymphoma cells were obtained from ATCC and maintained in 10% FBS-RPMI 1640 medium as previously reported (Dai et al., 2001).

Techniques: Expressing, Western Blot, Kinase Assay, Activity Assay, Phospho-proteomics

Journal: eLife

Article Title: Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes

doi: 10.7554/eLife.81608

Figure Lengend Snippet: ( A ) Dose-dependent effects of didemnin (orange) and ternatin-4 (red) on protein synthesis in HCT116 cells under continuous treatment (4 hr). Protein synthesis was quantified by homopropargylglycine pulse (1 hr) followed by fixation and copper-mediated conjugation to CF405M azide fluorophore and analyzed by FACS. ( B ) HCT116 cells were treated with didemnin (100 nM, orange) or ternatin-4 (500 nM, red) for 4 hr, followed by washout. Protein synthesis was quantified as in ( A ) at 1, 2-, 4-, 8-, or 24 hr post-washout. ( C ) Histograms corresponding to panel ( B ) for didemnin ( left ) and ternatin-4 ( right ). ( D ) Jurkat cells were treated with didemnin (100 nM, left ) or ternatin-4 (500 nM, right ) or for 1, 2, 4, 8, or 24 hr, stained with Annexin V-FITC (AV+, apoptotic) and propidium iodide (PI+, green, dead), and analyzed by FACS. ( E ) Jurkat cells were treated with didemnin (100 nM, orange) or ternatin-4 (500 nM, red) for 2 hr followed by washout and 22 hr incubation in drug-free media or for 24 hr and analyzed as in ( D ). Bar graphs show the ratio of live cells. See also .

Article Snippet: Cell line ( Homo sapiens ) , Jurkat cells , ATCC , Cat#TIB-152 , .

Techniques: Conjugation Assay, Staining, Incubation

Journal: eLife

Article Title: Didemnin B and ternatin-4 differentially inhibit conformational changes in eEF1A required for aminoacyl-tRNA accommodation into mammalian ribosomes

doi: 10.7554/eLife.81608

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , Jurkat cells , ATCC , Cat#TIB-152 , .

Techniques: Expressing, Generated, Plasmid Preparation, Recombinant, Purification, Sequencing, Protease Inhibitor, Magnetic Beads, Software

Journal: Immunity

Article Title: Targeting the aminopeptidase ERAP enhances antitumor immunity by disrupting the NKG2A-HLA-E inhibitory checkpoint.

doi: 10.1016/j.immuni.2024.10.013

Figure Lengend Snippet: Figure 4. Loss of Erap1 changes MHC class I peptide presentation in mouse tumor models (A) Flow cytometry histogram showing classical MHC class I (H-2K, H-2D) and non-classical MHC class I (Qa-1b) surface expression in control and Erap1- deficient tumor cells with or without IFN-g stimulation. Representative results from Renca, CT26, KPC, and B16 are shown. (B) Schematic TMT-labeled immunopeptidomics experiment design. (C and D) Pie charts showing the peptide length distribution of differential Qa-1b (C), H-2Dd (D, left), and H-2Kd (D, right) peptides in control versus Erap1-deficient conditions. (E) Bar plots of normalized intensities of the 10 most abundant Qa-1b peptides in both control and Erap1 KO cells. Data represented as mean ± SD. Statistical significance determined by Student’s t test. (F) Volcano plot showing differential Qa-1b peptides in control versus Erap1 KO cells. Significantly depleted (blue) and enriched (red) (adjusted p < 0.05 and log2 fold change > 0.5 or < 0.5) peptides in Erap1 KO cells are shown, and top five are labeled; FL9 (Fam49b) highlighted as positive control. (G) Schematic of NKG2A/CD94 CAR reporter system. (H) Activation of NKG2A/CD94 CAR+ reporter Jurkat cells by the Erap1 KO, Qa-1b KO, and B2m KO cells, where the y axis shows the mean fluorescence intensity (MFI ± SD) of CD69. Statistical significance determined by Student’s t test.

Article Snippet: The Jurkat T-lymphoblast cell line used to generate the human NKG2A/CD94 CAR reporter cell line was purchased from DSMZ (DSMZ, ACC 282); the line used to generate the mouse NKG2A/CD94 CAR reporter cell line was purchased from ATCC (ATCC, TIB-152).

Techniques: Flow Cytometry, Expressing, Control, Labeling, Immunopeptidomics, Positive Control, Activation Assay

Journal: Immunity

Article Title: Targeting the aminopeptidase ERAP enhances antitumor immunity by disrupting the NKG2A-HLA-E inhibitory checkpoint.

doi: 10.1016/j.immuni.2024.10.013

Figure Lengend Snippet: Figure 5. ERAP KO alters MHC class I peptide presentation in human tumor models (A–C) Cell surface expression of classical MHC class I (A), HLA-E (B), and VL9/HLA-E (C) in IFN-g-stimulated human tumor models after indicated gene deletions. Left, representative flow plot from HT-29; right, summary of relative expression in 7 human models. Relative expression determined by normalizing MFI of each condition to the MFI of control cells. Data represented as mean ± SD. (D) Schematic of human NKG2A/CD94 CAR+ reporter Jurkat cell assay. (E) Flow cytometry showing activation of human NKG2A/CD94 CAR+ reporter Jurkat cells by the indicated gene-deleted human tumor cell lines. Left, repre- sentative flow plot of reporter Jurkat cells activated by HT-29 cells; right, summarized analysis of 5 models. Relative CD69 expression was determined by normalizing the MFI of each condition to the control MFI for each model. Data represented as mean ± SD. (F) HLA-E surface expression of unstimulated control, ERAP1/2 KO, HLA-E KO, and B2M KO HT-29 cells overexpressing (OE) HLA-E. Data represented as mean ± SEM. (G) VL9/HLA-E-specific antibody staining of HLA-E OE cells in (F). Data represented as mean ± SEM. (H) NKG2A/CD94 CAR+ reporter Jurkat cell activation (CD69 MFI) by unstimulated HLA-E OE HT-29 tumor cells. Data represented as mean ± SEM. (I) Schematic of the HLA-E label-free immunopeptidomics experiment in HT-29 cells. (J) Pie charts of the proportion of highest HLA-E peptide intensities in the control and ERAP1/2 KO conditions. (K) Quantification of total VL9 peptides presented by HLA-E in ERAP1 KO, ERAP2 KO, ERAP1/2 KO, HLA-E KO, and B2M KO conditions from SCaBER, HCT116, and NCI-H1650 human tumor models. VL9 peptide concentration determined by IP-MS with isotope-labeled VL9 peptide spike-in. Data represented as mean ± SD. Statistical significance determined by Student’s t test.

Article Snippet: The Jurkat T-lymphoblast cell line used to generate the human NKG2A/CD94 CAR reporter cell line was purchased from DSMZ (DSMZ, ACC 282); the line used to generate the mouse NKG2A/CD94 CAR reporter cell line was purchased from ATCC (ATCC, TIB-152).

Techniques: Expressing, Control, Flow Cytometry, Activation Assay, Staining, Immunopeptidomics, Concentration Assay, Protein-Protein interactions, Labeling