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: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: Effect of lipid emulsions on the deformation and size (projected area) of non-activated Jurkat cells. The cells were incubated for 48 h in SMOFlipid ( A , C ) or Omegaven ( B , D ). Each data point on the graph represents the mode of measurements of at least 900 cells analyzed from a single experiment. Bar plots show the mean ± SEM of these values, and bar colors indicate lipid emulsion concentrations. Statistical significance was assessed using mixed effects analysis followed by Dunnett’s post hoc test, comparing all samples to the control. ( E ) A typical example of density plots showing differences in cell size (projected area) and deformation for control cells and cells treated for 48 h with 0.01 mg/mL Omegaven or 0.1 mg/mL SMOFlipid. The grey lines represent theoretical isoelasticity curves calculated by CytoPlot analysis (calculation model: LE-2D-FEM-19).

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Incubation, Emulsion, Control

Journal: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: Machine-learning-based classification of images. ( A ) Examples of distinct image classes observed in samples of Jurkat cells (from top to bottom): debris, dead cells, anomalous cells, cell aggregates, and intact cells. Note that the distinction between intact and anomalous cells is not always clear, even to the human eye. Additionally, some cell aggregates appear smaller than intact cells. ( B ) Confusion matrix, illustrating the performance of the supervised machine-learning classification model based on a deep neural network. ( C )–( E ) Typical examples of a deformation–area scatter plots of automatically classified cells: ( C ) control cells (N = 21,391), ( D ) cells incubated in Omegaven (0.01 mg/mL, N = 12,020) and ( E ) cells incubated with SMOFlipid (0.1 mg/mL; N = 15,841). These concentrations were the highest at which cell viability after 48 h of incubation remained sufficient for further analysis with deformability cytometry. The fraction of events classified as dead cells or debris is clearly higher in the Omegaven- and SMOFlipid-treated samples compared to the control.

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Control, Incubation, Cytometry

Journal: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: Effect of lipid emulsions on the viability of non-activated Jurkat cells. The cells were incubated for 48 h in SMOFlipid ( A , C ) or Omegaven ( B , D ). ( A ) and ( B ): relative viability measured by MTS assay, calculated as the absorbance at 490 nm relative to the control. Each data point represents the average of 4 to 5 technical replicates. Bar plots show the mean ± SEM, and bar colors indicate lipid emulsion concentrations. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s post hoc test, comparing all samples to the control. ( C ) and ( D ): relative abundance of five image classes—intact cells, anomalous cells, cell aggregates, dead cells, and debris—based on image-based classification from deformability cytometry, illustrating the overall cell state after incubation. Results represent the average cell class abundances from 5 to 6 replicates, each containing at least 3,000 events.

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Incubation, MTS Assay, Control, Emulsion, Cytometry

Journal: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: Lipid droplet accumulation in non-activated Jurkat cells incubated for 48h with lipid emulsions. ( A ) A brightfield image from deformability cytometry highlighting a Jurkat cell with visible lipid droplets. ( B ) Representative brightfield and LipidTOX-stained fluorescence microscopy images of Jurkat cells after incubation with lipid emulsions, showing intracellular lipid droplets B.1—epifluorescence, B.2—brightfield and B.3—maximum intensity projection confocal microscopy image. ( C ) and ( D ): flow cytometry analysis showing mean LipidTOX fluorescence intensity of single cells positive for LipidTOX in response to increasing concentrations of lipid emulsions SMOFlipid ( C ) and Omegaven ( D ). Each data point on the graph represents the average measurement of 30,000 cells. Bar plots show the mean ± SEM of these values, and bar colors indicate lipid emulsion concentrations. Statistical significance was assessed using repeated measures one-way ANOVA followed by Dunnett’s post hoc test, comparing all samples to the control. ( E ) and ( F ): label-free quantification of average number of lipid droplets per cell using deformability cytometry across different concentrations of SMOFlipid ( E ) and Omegaven ( F ). Each data point on the graph represents the average number of lipid droplets measured in at least 900 cells from a single experiment. While the number of lipid droplets per cell is always an integer, the displayed values are averages across many cells and are therefore expressed as non-integer numbers. Bar plots show the mean ± SEM of these values, and bar colors indicate lipid emulsion concentrations. Statistical significance was assessed using mixed effects analysis followed by Dunnett’s post hoc test, comparing all samples to the control.

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Incubation, Cytometry, Staining, Fluorescence, Microscopy, Confocal Microscopy, Flow Cytometry, Emulsion, Control, Quantitative Proteomics

Journal: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: The effect of lipid emulsions on energy metabolism of non-activated Jurkat cells. The cells were incubated for 48h at 0.1 mg/mL lipid concentration for SMOFlipid and 0.01 mg/mL for Omegaven, after which the rate of oxygen consumption (OCR) and extracellular acidification rate (ECAR) were determined using a modified Seahorse Mito Stress Assay with a prior etomoxir injection. The assay is used to measure the contribution of beta oxidation, the primary metabolic pathway for fatty acid catabolism. No significant altered baseline or maximal oxygen consumption rate (OCR), baseline extracellular acidification rate (ECAR) or the ratio between the two (OCR/ECAR) were observed (B–E). ( A ) A representative OCR graph for one experiment. After 3 measurement cycles, 5 µM etomoxir or medium was injected, after which baseline OCR ( B ) and ECAR ( C ) were measured and the OCR to ECAR ratio ( D ) was calculated. 1.5 µM oligomycin, 1.5 µM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), and 0.5 µM rotenone + antimycin A (AA) were then sequentially injected to determine the ATP-synthase independent, maximal ( E ) and non-mitochondrial ( F ) OCR, respectively. Data represent mean ± SEM of two independent experiments. No statistically significant differences were found by two-way ( B – E ) or one-way (F) ANOVA with Dunnett’s post-hoc test.

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Incubation, Concentration Assay, Modification, Injection

Journal: Scientific Reports

Article Title: Effects of intravenous lipid emulsions on Jurkat cells assessed using label-free deformability cytometry

doi: 10.1038/s41598-025-33582-7

Figure Lengend Snippet: Impact of lipid emulsions on activated versus non-activated Jurkat cells. The cells were incubated for 48 h in lipid emulsions SMOFlipid and Omegaven. ( A ) Cell viability was assessed using an MTS assay with increasing concentrations of SMOFlipid and Omegaven for both non-activated and activated Jurkat cells. Relative viability was calculated by comparing the absorbance value at 490 nm for incubated cells with that for their respective controls. Each data point on the graph represents an average of 4 technical replicates. Bar plots show the mean ± SEM of these values. ( B ) Comparison of cell deformability of non-activated and activated Jurkat cells measured by deformability cytometry. Statistical significance was assessed using mixed effects analysis followed by Šidak’s multiple comparison test. ( C ) Quantification of lipid droplet formation using deformability cytometry image analysis. Statistical significance was assessed using two-way ANOVA with multiple comparisons. Each data point on the graph represents the mode ( B ) or mean ( C ) of at least 1,000 measured cells in one experiment. Bar plots show the mean ± SEM of these values.

Article Snippet: Jurkat cells (clone E6-1, ATCC, lot number: 70053340) were cultured in RPMI-1640 medium (Gibco, Life Technologies Limited, UK) supplemented with 10% fetal bovine serum (Gibco, Life Technologies Limited, UK) and 1% penicillin–streptomycin (Gibco, Life Technologies Limited, UK).

Techniques: Incubation, MTS Assay, Comparison, Cytometry