Journal: iScience

Article Title: Atypical B cells consist of subsets with distinct functional profiles

doi: 10.1016/j.isci.2023.108496

Figure Lengend Snippet:

Article Snippet: One day prior to sorting B cells, wells of a 96-well plate were each seeded with 30,000 adherent, CD40L-expressing 3T3 cells (kind gift from Dr. Mark Connors, NIH) in 100 μl IMDM/Glutamax/ 10% FBS containing 2× MycoZap Plus-PR (Lonza #VZA-2021), 100 ng/ml human IL-2 (GoldBio #1110-02-50), and 100 ng/ml human IL-21 (GoldBio #1110-21-10) to promote expansion and differentiation of B cells into antibody-secreting cells.

Techniques: Staining, Recombinant, Cell Isolation, Multiplex Assay, Sequencing, Plasmid Preparation, Software

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: MDSCs identification. A, the indicated myeloid cell subsets were tested for suppressive activity against CFSE-labeled autologous T cells stimulated with beads coated with anti-CD3/anti-CD28 antibodies. Data normalized on the control (no MDSC) are cumulative of five independent experiments using PBMCs from 5 patients. P value for the ANOVA test (Pa) and the Tukey post hoc test are reported. B, example of multicolor FACS analysis for MDSC phenotype CD33+IL4Rα+ cells are highlighted in blue. C, intratumoral CD33+ IL4Rα+ cells were retrospectively evaluated in the tumor specimen of recurrent or nonrecurrent OSCC patients by immunofluorescence microscopy. P value for t test is reported.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Activity Assay, Labeling, Control, Immunofluorescence, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: An intermediate tadalafil dose modulates most effectively tumor immunity. The ratio between the MDSCs (A) or the log2-ratio of the CD8 proliferation (B) after (t2) and before (t1) pharmacologic treatment was plotted against the weight-normalized tadalafil dose. Best-fitting quadratic curve and confidence interval (gray area) are reported. cGMP (C) and cAMP (D) were measured by ELISA in the following FACS-sorted cell population from patients (n = 3) treated with intermediate or high dosage of tadalafil: CD33+IL4Rα+ (MDSCs), HLADRhigh (APC), or CD3+ (T cells). Pt, paired t test; BDL, below detection limit.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: Tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy. Pa, P ANOVA test.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Concentration Assay, Fluorescence, Microscopy

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: IL-33 and ST2L are overexpressed in human GC tissues and predict poor prognosis. a QRT-PCR analysis of IL-33 mRNA expression in GC and corresponding normal tissues ( n = 18). b Histogram displaying the relative mRNA expression of IL-33 in 18 GC tissues. Data are shown as −ΔΔCt and 2 −ΔCt . c IHC staining of α-SMA, IL-33 and ST2L in GC tissues (200×; scale bar = 100 μm). d Histogram displaying the number of α-SMA, IL-33 and ST2L positive cells/field in GC tissues. e Histogram displaying the correlation between IL-33 expression and ST2L expression determined by IHC ( r = 0.6503). f , g Survival of patients with low or high levels of IL-33 and ST2L protein expression. h – j Immunofluorescence staining of IL-33, α-SMA, FAP, ST2L and Cytokeratin in GC tissues (200×; scale bar = 400 μm). k – m Histogram displaying the percentage of IL-33 + /α-SMA + , IL-33 + /FAP + and ST2L + /Cytokeratin ++ cells in GC tissues. Data are represented as the mean ± SD; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry, Immunohistochemistry-Resin, Immunofluorescence, Staining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Relationship between IL-33 and ST2L expression levels and clinicopathologic parameters in 134 GC tissues

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Expressing, Immunostaining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: CAFs-derived IL-33 promotes the migration, invasion and EMT of GC cells. a – d The migration and invasion ability of SGC7901 and MKN45 cells were analyzed after culture in medium alone (blank) or treatment with: exogenous IL-33 (300 ng/ml); co-culture with CAFs supplemented with IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). Histograms show the average cell number/field (100×; scale bar = 100 μm). e – h The migration and invasion ability of SGC7901 and MKN45 cells was detected after culture in medium alone (blank), co-culture with CAFs transfected with IL-33/siRNA or nc/siRNA. Histograms display the average cell number/field (100×; scale bar = 100 μm). i – l The migration and invasion ability of SGC7901 and MKN45 cells was analyzed after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) plus IgG isotype antibody or ST2L neutralizing antibody (3 μg/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). Histograms show the average cell number/field. (100×; scale bar = 100 μm). m , n QRT-PCR of the genes for EMT in SGC7901 and MKN45 cells after culture in medium alone (blank); or activation with exogenous IL-33 (300 ng/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). o – r Western blot of EMT markers in SGC7901 and MKN45 cells cultured in medium alone (blank); or stimulated with exogenous IL-33 (300 ng/ml) or co-culture with CAFs or co-cultured with CAFs in the presence of DMSO, U0126 (20 μM), IL-33 neutralizing antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). p and r Densitometric analysis shows the expression level of EMT markers in GC cells stimulated by the above factors. Data are represented as the mean ± SD of three independent experiments; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Migration, Co-Culture Assay, Transfection, Quantitative RT-PCR, Activation Assay, Western Blot, Cell Culture, Expressing

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: The metastatic potential of GC cells induced by CAFs-derived IL-33 is mediated by the activation of ERK1/2-SP1-ZEB2 pathway via ST2L. a – d Western blot analysis showing the phosphorylation of ERK1/2 in SGC7901 and MKN45 cells treated with medium alone or stimulated as follows: exogenous IL-33 (300 ng/ml), U0126 (20 μM), CAFs supernatants (CAFsu) supplemented with IgG isotype control antibody (3 μg/ml), DMSO; co-cultured with CAFs transfected with IL-33/siRNA or NC/siRNA; CAFsu supplemented with IL-33 neutralizing antibody (3 μg/ml). e Western blot analysis of pERK1/2 in SGC7901 and MKN45 cells incubated with medium alone; exogenous IL-33 (300 ng/ml); U0126 (20 μM); co-culture with CAFs; IL-33 neutralizing antibody (3 μg/ml); and/or ST2L neutralizing antibody (3 μg/ml). f , g The migration and invasion ability of SGC7901 and MKN45 cells after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) and/or U0126 (20 μM); co-culture with CAFs and/or U0126 (20 μM). Histograms show the average cell number per field. (100×; scale bar = 100 μm). h , i QRT-PCR analysis of SP1 mRNA expression in SGC7901 and MKN45 cells incubated in medium alone or stimulation with exogenous IL-33 (300 ng/ml) or CAFs in the presence of IgG isotype control antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). j , k Western blot analysis of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells incubated in medium alone or stimulated by co-culture with CAFs in the presence of exogenous IL-33 (300 ng/ml) or U0126 (20 μM). l Western blot analysis showing the protein expression of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells cultured in medium alone (Mock); or transfected with control plasmid (nc) or SP1-expressing plasmid. m Dual luciferase reporters containing four different lengths of the ZEB2 promoter were co-transfected with SP1-expressing plasmid into 293T cells. Firefly luciferase activity was assessed relative to Renilla luciferase activity. Data are representative of three independent experiments. Densitometry shows relative protein expression. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Activation Assay, Western Blot, Phospho-proteomics, Control, Cell Culture, Transfection, Incubation, Co-Culture Assay, Migration, Quantitative RT-PCR, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Knockdown of IL-33 expression in CAFs or ST2L expression in GC cells inhibits peritoneal dissemination of GC cells in nude mice. a , c In vivo tumor peritoneal dissemination was examined by co-injection of SGC7901 or MKN45 cells and CAFs transfected with control siRNA (NC) or IL-33 siRNA into nude mice. Metastatic nodules are indicated by the red arrows. b , d Histograms displaying the number of peritoneal nodules in nude mice. e – h In vivo tumor peritoneal dissemination was examined by co-injection of CAFs and GC cells with ST2L knockdown (KD) into nude mice. Metastatic nodules are indicated by the red arrows. f , h Histograms displaying the number of peritoneal nodules in nude mice. Data are representative of three independent experiments. * P <0.05, * P <0.05, ** P <0.01

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Knockdown, Expressing, In Vivo, Injection, Transfection, Control

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Model for the epithelial–stromal interactions in the tumor microenvironment of GC. a IL-33 released from CAFs promotes the migration, invasion and EMT of GC cells via the ST2L-ERK1/2-SP1-ZEB2 pathway. b GC cell-derived TNF-α upregulates IL-33 expression in CAFs via the TNFR2-NF-κB-IRF-1 pathway

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Migration, Derivative Assay, Expressing

Journal: BMC Immunology

Article Title: Increased circulating Tfh to Tfr ratio in chronic renal allograft dysfunction: a pilot study

doi: 10.1186/s12865-019-0308-x

Figure Lengend Snippet: The frequency of cTfh and cTfr cells and the level of associated factors between CAD group and stable group . Squares refer to chronic allograft dysfunction (CAD) group, cycles refer to stable renal function group; a cTfh: CXCR5 + Foxp3 − on CD4 + cells; b cTfr: CXCR5 + Foxp3 + on CD4 + cells; c cTfh to cTfr ratio; d Tregs: CD25 + Foxp3 + on CD4 + cells; e CXCR5 + PD-1 + on CD4 + cells; f PD-1 + on CXCR5 + CD4 + cells; g CXCR5 + ICOS + on CD4 + cells; h ICOS + on CXCR5 + CD4 + ; i CXCR5 + STAT3 + on CD4 + cells; j CXCR5 + STAT5 + on CD4 + cells; k STAT3 + on CXCR5 + CD4 + cells; l STAT5 + on CXCR5 + CD4 + cells; m CXCR5 + STAT4 + on CD4 + cells; n STAT4 + on CXCR5 + CD4 + cells; o CXCR5 + IL-21 + on CD4 + cells; p IL-21 + on CXCR5 + CD4 + cells; q The serum level of CXCL13; r The serum level of TGF-β

Article Snippet: Then samples were stained with IL-21-PE (BD Bioscience, New Jersey, US).

Techniques:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I interacted with TRIM21’s PRY-SPRY domain. (A) The Co-IP result indicated the interaction of Tg ROP18 I and TRIM21. (B,C) The Co-IP result indicated that ROP18-KD still bound with TRIM21. (D) Sketch map of TRIM21 WT and truncation mutants. (E) The Co-IP results showed the PRY-SPRY domain of TRIM21 was indispensable for Tg ROP18 I -TRIM21 interaction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001; IP, immunoprecipitation).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted the phosphorylation of TRIM21. (A,B) HEK293T cells were transfected with different amount of pcDNA3.1-ROP18 I -FLAG as indicated, and the cell lysates were subjected to TRIM21 IP and Western blotting. The more plasmids were transfected to HEK293T cells, the higher levels of phosphorylated TRIM21 were observed, and the differences between groups were significant. (C,D) HFF cells were infected with CEP or CEP- rop18 I , and then the total protein was extracted and subjected to TRIM21 IP and Western blotting. The results showed that more phosphorylated TRIM21 was detected in the CEP- rop18 I infected cells than which detected in the CEP infected cells and uninfected cells, while the phosphorylation levels of TRIM21 were not significantly different between the CEP infected and uninfected cells. The experiments were repeated three times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Phospho-proteomics, Transfection, Western Blot, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I promoted TRIM21 degradation through lysosomal pathway. (A) HEK293T cells were transfected with the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. (B) HEK293T cells were co-transfected with a stable amount of pcDNA3.1-TRIM21-HA and the increased amounts of pcDNA3.1-ROP18 I -FLAG as indicated. The endogenous or overexpressed TRIM21 level was decreased with the increased ROP18 level. (C) HEK293T cells were co-transfected with pcDNA3.1-TRIM21-HA and pcDNA3.1-ROP18 I -FLAG or pcDNA3.1-ROP18 I -KD-FLAG as indicated. The results of Western blotting detection with the cell lysates indicated that much more TRIM21 was detected in the ROP18-KD overexpression group than in the ROP18 overexpression group. (D) Lysates of HFFs infected with RH or RH-△ rop18 was detected by Western blotting, and more TRIM21 was detected in the RH-△ rop18 infection group than in the RH infection group. (E) HEK293T cells were co-transfected with 1mg of pcDNA3.1-TRIM21-HA and increased amounts of pcDNA3.1-ROP18 I -FLAG. The cells were treated with MG132 or Leupeptin, or left untreated. Cell lysates were subjected to Western blotting, and the results showed that TRIM21’s level was decreased with the increased amount of Tg ROP18 I in the cells treated with MG132 or DMSO. However, TRIM21’s level was kept stable in the Leupeptin treated group. All the experiments were repeated three times. IB, immunoblot.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Transfection, Western Blot, Over Expression, Infection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 transcription and translation levels were upregulated following T. gondii infection. HFF cells infected with RH or CEP were harvested for detection of TRIM21 transcription and translation level. (A) Comparison of the TRIM21 transcription levels between the indicated groups with qRT-PCR. The CEP infection resulted in the highest TRIM21 transcription level, followed by RH infection, and uninfection at 1 h post infection, the differences between groups were significant. At 24 h post infection, the RH infection resulted in a significant higher TRIM21 transcription level than in CEP infection or uninfection groups between which no significant difference was observed in TRIM21 transcription level. (B,C) Comparison of the translation level of TRIM21 in the HFF cells infected with RH or CEP for the indicated time with Western blot (up panels). The densitometrical analysis for the intensity of TRIM21 bands normalized to its corresponding GAPDH intensity showed that, both CEP and RH infection resulted in significant higher transcription levels of TRIM21 than in uninfected cells (down panels). All the experiments were repeated four times. The values were analyzed using the one-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Infection, Comparison, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 is involved in the IFN-γ induced inhibition of CEP proliferation (but not RH proliferation) in HFFs. The proliferation of RH and CEP tachyzoies in the HFFs was evaluated after stimulation with IFN-γ for 24 h (A–C) , or transfection with pcDNA3.1-TRIM21-HA for 24 h (E–F) ; and proliferation of CEP-WT was evaluated after transfection with si-TRIM21 for 48 h in HFFs followed by IFN-γ treatment for 24 h (H–I) . TRIM21 protein levels were measured by Western blotting (A,D,G) . The average number of tachyzoites in 100 parasitophorous vacuoles (PVs) was counted (B,E,H) , and the percentage of the PVs containing 1, 2, 4, or 8 parasites was determined by immune fluorescence assay (C,F,I) . (A) The TRIM21 protein level was significantly up-regulated under IFN-γ stimulation in a dose-dependent manner, with the response concentration ranged from 50 to 500 U/ml. (B,C) The proliferation of the RH and CEP tachyzoites in HFFs was significantly inhibited after IFN-γ stimulation. (D) The overexpression of TRIM21 was detected. (E,F) The overexpression of TRIM21 significantly inhibited the CEP proliferation, but not affected RH proliferation. (G) The si-TRIM21 transfection significantly inhibited the TRIM21 translation. (H,I) The IFN-γ induced inhibition of CEP proliferation was relieved by TRIM21 knockdown. All the experiments were repeated three times. The values were analyzed using the one-way ANOVA and two-way ANOVA. Data were expressed as the mean ± SEM (* p < 0.05; ** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Inhibition, Transfection, Western Blot, Fluorescence, Concentration Assay, Over Expression, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 knockdown relieves the IFN-γ-induced ubiquitin labeling on CEP parasitophorous vacuole membrane (PVM) in HFFs. (A,B) HFFs were transfected with negative control siRNA (si-NC) or siRNA specific against TRIM21 (si-TRIM21), and stimulated with IFN-γ or not as indicated. After T. gondii infection, HFFs were subjected to immunofluorescence assay (IFA). IFN-γ induced ubiquitin labeling on the CEP PVM, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not. (C,D) HFFs were stimulated with IFN-γ or not, and infected with CEP. The cells were then treated with LysoTracker ® (acidic dye) and subjected to immunofluorescence assay (IFA). The result showed us that IFN-γ induced acidification of CEP. On the left, a representative fluorescent image is shown for the T. gondii CEP strain expressing GFP. The yellow box inside each representative image is shown as magnified pictures nearby (A,C) . The percentage of vacuoles stained red with ubiquitin labeling or LysoTracker ® was shown in the right bar diagram (B,D) . Scale bar is 10 μm. The experiments were repeated three times. The values were analyzed using the one-way ANOVA or two-tailed unpaired Student t test. Data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Knockdown, Ubiquitin Proteomics, Labeling, Membrane, Transfection, Negative Control, Infection, Immunofluorescence, Expressing, Staining, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I relieved TRIM21 mediated inhibition of T. gondii proliferation regardless of strain types. (A–D) HFFs were transfected with pcDNA3.1-TRIM21-HA or pcDNA3.1(+) for control, and infected with RH-△ rop18 (A,B) or CEP- rop18 I (C,D) parasites as indicated. Parasitic proliferation was measured at 18 h (RH-△ rop18 ) or 24 h (CEP- rop18 I ) post-infection. The average number of tachyzoites in 100 vacuoles (A,C) or the number of vacuoles containing 1, 2, 4, or 8 parasites (B,D) was determined by fluorescence microscopy. The results indicated that TRIM21 overexpression inhibited the RH-△ rop18 multiplication, but had no significant effect on CEP- rop18 I multiplication. The experiments were repeated three times. The values were analyzed using the two-tailed unpaired Student t test and two-way ANOVA. Data were expressed as the mean ± SEM (** p < 0.01).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Inhibition, Transfection, Control, Infection, Fluorescence, Microscopy, Over Expression, Two Tailed Test

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: TRIM21 mediated NF-κB activation. (A,B) In the HFF cells stimulated with the indicated concentrations of IFN-γ for 24 h, the p-p65 (S536) level was significantly higher than that in the untreated group. (C,D) Western-blot detection of p-p65 (S536) level showed that TRIM21 overexpression significantly elevated p-p65 (S536) phosphorylation in dose-dependent manner. (E,F) In the siRNA-TRIM21 transfected groups, TRIM21 expression was suppressed, but no significant difference was found in the p-p65 (S536) levels between the TRIM21 knockdown group with or without IFN-γ induction. The experiments were repeated three times. The values were analyzed using the one-way ANOVA and the data were expressed as the mean ± SEM (** p < 0.01; *** p < 0.001).

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Activation Assay, Western Blot, Over Expression, Phospho-proteomics, Transfection, Expressing, Knockdown

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: The interaction of p65 and IκB-α was suppressed with TRIM21 overexpression. Interaction of TRIM21 and IκB-α in the total cell lysates of HEK293T cells were analyzed by IP and Western-blot after either treatment with IFN-γ or transfection with pcDNA3.1-TRIM21-HA for the indicated time. (A–C) The IP with the anti-TRIM21 antibody identified the interaction of IκB-α with the overexpressed and the endogenous TRIM21. TRIM21 overexpression promoted TRIM21-IκB-α interaction (A) . IFN-γ induction elevated TRIM21 production and promoted TRIM21-IκB-α interaction (B) . The TRIM21 production induced by IFN-γ increased with the prolonged treating time and promoted TRIM21-IκB-α interaction (C) . (D,E) The IP with the anti-p65 antibody identified the complex of IκB-α-TRIM21-p65 (D) , and the interaction of p65-ubiquitin (E) . The experiments were repeated three times. The TRIM21 band is indicated with “*”, and the IκB-α band is indicated with a black arrow.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Over Expression, Western Blot, Transfection, Ubiquitin Proteomics

Journal: Frontiers in Cell and Developmental Biology

Article Title: Toxoplasma gondii Type-I ROP18 Targeting Human E3 Ligase TRIM21 for Immune Escape

doi: 10.3389/fcell.2021.685913

Figure Lengend Snippet: Tg ROP18 I targets host TRIM21 for immune escape. 1. Host IFN-γ-induced factor TRIM21 restricted T. gondii replication through NF-κB activation and TRIM21 overexpression suppressed the p65-IκB-α interaction to activate NF-κB pathway. 2. Tg ROP18 I which was discharged by T. gondii , interacted with the PRY-SPRY domain of human TRIM21, promoted TRIM21 phosphorylation, and induced TRIM21 degradation via lysosomal pathway. 3. IFN-γ induced ubiquitin labeling on the CEP PVM which resulted in PV acidification and death of parasites, but this labeling was relieved by TRIM21 knockdown regardless of IFN-γ simulation or not.

Article Snippet: HEK293T cells were seeded in T25 flasks to 90% confluence, and transfected with pcDNA3.1-ROP18 I -FLAG using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific) for 24 h. The cell lysates were subjected to immunoprecipitation with anti-TRIM21 rabbit polyclonal antibody (Proteintech, IL, United States) followed by Western blotting.

Techniques: Activation Assay, Over Expression, Phospho-proteomics, Ubiquitin Proteomics, Labeling, Knockdown