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: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: IR-induced down-regulation of Mcl-1 and apoptosis induction are impaired in resistant Jurkat subclone. Sensitive (IR-sensitive) and resistant (IR-resistant) Jurkat cells were irradiated with 10 Gy. ΔΨm dissipation (A, C), DNA degradation (B, D), and survival (E) were analyzed by flow cytometry at indicated time points after irradiation. (A) Representative histograms show cells with intact (ΔΨm-H for high ΔΨm) and dissipated (ΔΨm-L for low ΔΨm) mitochondrial membrane potential 24 hours after irradiation. (B) Representative histograms display cells with degraded DNA visualized in the sub-G1 fraction 48 hours after irradiation. Results of ΔΨm dissipation and DNA fragmentation are shown in C and D, respectively (n = 6, mean ± SD). Radiation-induced ΔΨm dissipation and DNA fragmentation were significantly reduced in IR-resistant cells. (E) Cell death and survival were quantified by flow cytometry employing a propidium iodide exclusion dye (n = 8, mean ± SD). (F) Long-term survival was analyzed in a clonogenic assay using limiting dilutions of the IR-resistant and IR-sensitive cells. The SF was calculated for cells irradiated with 0, 2, 4, 6, and 8 Gy (normalized to respective nonirradiated cells). Error bars of SF are within the symbols. (G) Western blot analysis shows cleavage of caspase-9, caspase-3, and the caspase-3 substrate PARP in sensitive cells in response to IR, which is abrogated in resistant cells. Down-regulation of Mcl-1 and Puma levels are detected in IR-sensitive cells only. Levels of other Bcl-2 proteins (Bcl-xL, Bcl-2, Bak, Bim, and Noxa) were not affected by irradiation. GAPDH was used as loading control. ***P < .001.

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Irradiation, Flow Cytometry, Membrane, Clonogenic Assay, Western Blot, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: Mcl-1 prevents mitochondrial permeabilization in IR-sensitive and IR-resistant cells. Mcl-1 siRNA (mcl1; 500 nM) or nontargeting siRNA (nt) was electroporated into IR-sensitive and IR-resistant Jurkat cells. (A) Three and six hours after electroporation, lysates were made, and down-regulation of Mcl-1 was analyzed by Western blot analysis. GAPDH was used as loading control. (B) Six hours after electroporation, dissipation of mitochondrial membrane potential (ΔΨm low) was analyzed by flow cytometry. In both IR-sensitive and IR-resistant cells, the expression of Mcl-1 is essential for maintaining mitochondrial homeostasis. ***P < .001.

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Electroporation, Western Blot, Control, Membrane, Flow Cytometry, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: GSK-3β does not regulate Mcl-1 stability in irradiated Jurkat cells. (A) IR-sensitive Jurkat cells were irradiated with 10 Gy. Cells were lysed 24 hours after irradiation. Western blot analysis shows that phopsho-GSK-3β levels were not changed by IR, whereas the levels of GSK-3β substrates Mcl-1 and β-catenin were clearly reduced. (B–D) IR-sensitive Jurkat cells were irradiated with 10 Gy in the presence of 30 nM GSK-3 inhibitor XV. (B) Twenty-four hours later, cells were lysed and analyzed by Western blot. Treatment of IR-sensitive Jurkat cells with 30 nM GSK-3 inhibitor XV for 24 hours resulted in an accumulation of β-catenin without having an effect on Mcl-1 levels. GSK-3 inhibitor XV did not alter a radiation-induced Mcl-1 decline. Dissipation of mitochondrial membrane potential (ΔΨm) was analyzed by flow cytometry 24 hours after irradiation. (D) DNA fragmentation was analyzed by flow cytometry 48 hours after irradiation. GSK-3 inhibitor XV had no effect on radiation-induced ΔΨm dissipation or DNA degradation. Flow cytometric data show means ± SD (n = 6); NS indicates no significance (P > .05).

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Irradiation, Western Blot, Membrane, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: Bim, Puma, and Noxa are dispensable for radiation-induced apoptosis. (A) IR-sensitive Jurkat cells were treated with 30 µM caspase inhibitor zVAD or the solvent only and irradiated with 10 Gy. Twenty-four hours later, cell lysates were made and analyzed by Western blot analysis. Radiation-induced decline of Puma but not of Mcl-1 could be blocked by the caspase inhibitor, indicating that down-regulation of Mcl-1 occurs upstream of caspase activation, whereas Puma is downregulated after caspase activation. (B) Bim siRNA (500 nM), (C) Puma siRNA (1 µM), (D) Noxa siRNA (1 µM), or respective concentrations of nontargeting (nt) siRNA were electroporated into IR-sensitive Jurkat cells. Forty-eight hours later, cells were irradiated with 10 Gy. ΔΨm dissipation was examined 24 hours after irradiation by flow cytometry (B–D, middle panels), whereas DNA fragmentation was analyzed 48 hours after irradiation (B.D, lower panels). Data show means ± SD (n = 3); NS indicates no significance (P > .05). Down-regulation of Bim, Puma, and Noxa and Mcl-1 levels were verified by Western blot analysis 72 hours after electroporation (B and C, upper panels). The knockdown of the respective proteins was quantified by densitometry. Abrogation of Bim, Puma, or Noxa expression did not affect radiation-induced apoptosis in Jurkat cells.

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Solvent, Irradiation, Western Blot, Activation Assay, Flow Cytometry, Electroporation, Knockdown, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: Irradiation enhances interaction of Mcl-1 with USP9x in resistant cells and prevents Mcl-1 degradation. (A) IR-sensitive and IR-resistant Jurkat cells were irradiated with 10 Gy. Lysates were made 4, 8, 12, and 24 hours later and analyzed by Western blot. USP9x levels did not change in response to irradiation. The levels of potential USP9x substrates Mcl-1 and β-catenin decreased only in IR-sensitive cells upon irradiation. Densitometric quantification of USP9x and Mcl-1 is shown below the Western blots of the respective cells. (B) IR-sensitive and IR-resistant cells were irradiated with 10 Gy. Twenty-four hours later, Mcl-1 was precipitated from irradiated and nonirradiated cells. More USP9x coprecipitated in irradiated IR-resistant than in IR-sensitive cells. The whole-cell lysate (WCL) shows the overall Mcl-1 and USP9x protein levels before precipitation. (C–E) Twenty-four hours after irradiation with 10 Gy, cells were treated with 1 µM cycloheximide (CHX) for 0 to 3 hours. Cells were lysed at respective time points. (C) Mcl-1 levels were analyzed by Western blot. (D) Mcl-1 levels were quantified by densitometry. The values were fitted and the half-life of Mcl-1 was calculated. (E) Graph shows the mean values ± SD of Mcl-1 half-life time (n = 7). *P < .05.

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Irradiation, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Deubiquitinase USP9x Confers Radioresistance through Stabilization of Mcl-1 1 2

doi:

Figure Lengend Snippet: Ionizing radiation induces activation of USP9x and deubiquitylation of Mcl-1 in IR-resistant cells. (A) IR-sensitive and IR-resistant Jurkat cells were irradiated with 10 Gy. Twenty-four hours after irradiation, USP9x was precipitated from the lysates. (A) The activity of USP9x was determined by adding a fluorogenic substrate to the precipitated USP9x. Increased USP9x activity could be detected only in resistant cells 24 hours after irradiation. Data show mean values ± SD (n = 6); ***P < .001; NS indicates no significance (P > .05). (B) IR-resistant Jurkat cells were irradiated with 10 Gy. Twenty-four hours after irradiation, USP9x was precipitated from the lysates of irradiated and nonirradiated control cells. In parallel, IR-resistant cells were treated with 5 µM proteasome inhibitor MG132 for 3 hours to accumulate ubiquitylated Mcl-1. To start the deubiquitylation reaction, we added ubiquitylated Mcl-1 precipitated from MG132-treated lysates to the USP9x precipitated from irradiated lysates. Mcl-1 deubiquitylation was analyzed by Western blot using an antibody against ubiquitin and Mcl-1. Where indicated by (i), USP9x activity was inhibited by adding 5 µM WP1130 before the incubation with precipitated Mcl-1. Less ubiquitylated Mcl-1 and more p40 Mcl-1 were detected after incubation with USP9x precipitated from irradiated cells. (C) Twenty-four hours after irradiation with 10 Gy, Mcl-1 was precipitated from the lysates of irradiated or nonirradiated control cells. The ubiquitylation state of Mcl-1 was detected by Western blot using antibodies against ubiquitin and Mcl-1. IR-induced decrease of high molecular, ubiquitylated Mcl-1 was detected only in IR-resistant cells 24 hours after irradiation. (D) IR-sensitive and IR-resistant Jurkat cells were either irradiated with 10 Gy (left panel) or treated with 5 µM MG132 (right panel). Cell lysates were analyzed by Western blot. No change of ubiquitylated proteins in WCLs was observed 24 hours after irradiation. In contrast, treatment with MG132 for 3 hours resulted in an accumulation of ubiquitylated proteins as well as USP9x and Mcl-1.

Article Snippet: Jurkat E6 T lymphoma cells ( Jurkat-sensitive) and K562 chronic myelogenous leukemia cells were from ATCC (Bethesda, MD).

Techniques: Activation Assay, Irradiation, Activity Assay, Control, Western Blot, Ubiquitin Proteomics, Incubation

Journal: RSC Advances

Article Title: Stick, stretch, and scan imaging method for DNA and filaments †

doi: 10.1039/d1ra07067c

Figure Lengend Snippet: Rosette stretching under light microscope. Jurkat cells were bound to microbeads (A). Filopodia was stretched by the beads in the magnetic field for 2 h (B and C). The beads were aligned along the magnetic field.

Article Snippet: Jurkat T-cells were purchased from the China Center for Type Culture Collection and cultured with 5% fetal bovine serum (Gibco Thermo Fisher, USA) in RPMI 1640 (Gibco) under 5% CO 2 in an incubator (Thermo Scientific).

Techniques: Light Microscopy

Journal: RSC Advances

Article Title: Stick, stretch, and scan imaging method for DNA and filaments †

doi: 10.1039/d1ra07067c

Figure Lengend Snippet: Fine structures observed by SEM. (A–D) Elongated filopodia were observed when the GRGDNP-coated bead was stretched by magnetic force for 5 h. The extension and thickness of the stretched filopodia varied. (E and F) A Jurkat cell was bound to beads coated with GRGDNP, without magnetic force, as a control. (G) A Jurkat cell was bound to the glass cover, without magnetic beads, as a control.

Article Snippet: Jurkat T-cells were purchased from the China Center for Type Culture Collection and cultured with 5% fetal bovine serum (Gibco Thermo Fisher, USA) in RPMI 1640 (Gibco) under 5% CO 2 in an incubator (Thermo Scientific).

Techniques: Control, Magnetic Beads