Journal: bioRxiv

Article Title: MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein

doi: 10.64898/2026.01.09.698592

Figure Lengend Snippet: MOV10 promoted TRAF3 K63-linked polyubiquitination dependent on E3 ligase TRIM24. (A) HEK293T cells were co-transfected with Myc-hMOV10 and HA-TRIM24 for 24 h. Cell lysates were harvested and subjected to immunoprecipitation using HA Ab. (B) Co-localization of MOV10 and TRIM24. Plasmids expressing Myc-MOV10 (red) and HA-TRIM24 (green) were co-transfected into Vero E6 cells, which were then infected or not with SeV for 9 h. Merged images show co-localization of these proteins. Nuclei are highlighted by DAPI staining (blue). (Bar: 5 µm). (C) HEK293T cells were transfected with plasmids expressing Myc-hMOV10 or EV and infected or not with SeV. Protein expression of TRIM24, LaminA/C, and GAPDH in cytoplasmic (CE) and nuclear (NE) fractions was assessed by western blotting. The density of TRIM24 bands relative to GAPDH was calculated using grayscale analysis. (D) HEK293T cells were co-transfected with plasmids expressing HA-TRIM24, Flag-TRAF3, and Myc-hMOV10. Cells were harvested for Co-IP using HA Ab, with IgG Ab as the negative control. (E) Plasmids encoding HA-TRIM24, Flag-TRAF3, or Myc-hMOV10 (0, 1 or 3 μg) were co-transfected into HEK293T cells. Cell lysates were collected and then subjected to immunoprecipitation using HA Ab. (F) HEK293T cells were transfected with either siTRIM24 or siNC (50 nM) for 36 h, followed by co-transfection with Flag-TRAF3, HA-K63, or Myc-hMOV10. Lysates were subjected to immunoprecipitation using Flag Ab. (G and H) WT (TRIM24 +/+ ) and TRIM24 knockout (TRIM24 −/− ) HEK293T cells were co-transfected with Flag-TRAF3, HA-K63, or Myc-hMOV10. Lysates were subjected to immunoprecipitation using Flag Ab. (I and J) TRIM24 +/+ and TRIM24 −/− HEK293T cells were transfected with either siMOV10 or siNC, followed by co-transfection with Flag-TRAF3 and HA-K63. Lysates were subjected to immunoprecipitation using Flag Ab.

Article Snippet: Abs against MOV10 (10370-1-AP), TRAF3 (18099-1-AP), TRIM24 (14208-1-AP) and HA (51064-2-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Transfection, Immunoprecipitation, Expressing, Infection, Staining, Western Blot, Co-Immunoprecipitation Assay, Negative Control, Cotransfection, Knock-Out

Journal: bioRxiv

Article Title: MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein

doi: 10.64898/2026.01.09.698592

Figure Lengend Snippet: SADS-CoV N protein degraded TRAF3 by catalyzing K48-linked polyubiquitination. (A) IPI-2I cells were infected with SADS-CoV (MOI = 0.1). Cells were collected at 6, 12, 24, and 36 h and detected by western blotting using Abs against TRAF3, MOV10, TRIM24, SADS-CoV N protein and GAPDH. (B) TRAF3 interacted with SADS-CoV N protein. HEK293T cells were co-transfected with Flag-TRAF3 and GFP-N. Cell extracts were subjected to immunoprecipitation using Flag Ab. (C) Co-localization of TRAF3 and SADS-CoV N protein. Plasmids expressing Flag-TRAF3 (red) and Myc-N (green) were transfected or co-transfected into Vero E6 cells. Merged images show co-localization of these proteins. Nuclei are highlighted by DAPI staining (blue). (Bar: 5 µm). (D) Plasmids encoding GFP-N, HA-pMOV10, and Flag-TRAF3 were transfected or co-transfected into HEK293T cells, respectively. Lysates were subjected to immunoprecipitation using HA Ab. (E) HEK293T cells were transfected with plasmids encoding Myc-N or EV. Lysates were subjected to immunoprecipitation using MOV10 Ab, with IgG Ab as the negative control. (F) IPI-2I cells were infected with SADS-CoV (MOI = 0.1) and treated with CQ, MG132, Z-VAD-FMK, or DMSO (all 20 μM) for 5 h before sample collection. Western blotting was used to detect expression of TRAF3, SADS-CoV N protein and GAPDH. (G) HEK293T cells were transfected or co-transfected with plasmids encoding Flag-TRAF3, HA-Ub, and Myc-N. Lysates were collected and then subjected to immunoprecipitation using Flag Ab. (H) HEK293T cells were co-transfected with plasmids encoding Flag-TRAF3, Myc-N, HA-K48 or HA-K63. Lysates were collected and then subjected to immunoprecipitation using Flag Ab. (I) HEK293T cells were co-transfected with Myc-N, HA-K48, Flag-TRAF3 or Flag-TRAF3 MATH. Lysates were subjected to immunoprecipitation using Flag Ab.

Article Snippet: Abs against MOV10 (10370-1-AP), TRAF3 (18099-1-AP), TRIM24 (14208-1-AP) and HA (51064-2-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Infection, Western Blot, Transfection, Immunoprecipitation, Expressing, Staining, Negative Control

Journal: bioRxiv

Article Title: MOV10 inhibits SADS-CoV replication by enhancing TRIM24-mediated K63-linked TRAF3 ubiquitination and this inhibition is antagonized by viral N protein

doi: 10.64898/2026.01.09.698592

Figure Lengend Snippet: Models of MOV10 mechanisms in counteracting SADS-CoV and viral N protein antagonizing host innate immunity. To inhibit SADS-CoV replication, the host factor MOV10 enhanced the catalysis of K63-linked polyubiquitination of TRAF3 by E3 ubiquitin ligase TRIM24, thereby activating the RIG-I-mediated antiviral signaling pathway. On the other hand, the SADS-CoV N protein suppressed RIG-I-mediated antiviral signaling by promoting K48-linked polyubiquitination, leading to degradation of TRAF3.

Article Snippet: Abs against MOV10 (10370-1-AP), TRAF3 (18099-1-AP), TRIM24 (14208-1-AP) and HA (51064-2-AP) were purchased from Proteintech (Wuhan, China).

Techniques: Ubiquitin Proteomics

Journal: Food & Function

Article Title: Punicalagin is the key pomegranate polyphenol inhibiting gut microbial trimethylamine (TMA) production from l -carnitine in an in vitro human colon model

doi: 10.1039/d5fo04781a

Figure Lengend Snippet: Effects of pomegranate polyphenols and gum Arabic at 2 mg mL −1 on in vitro l -carnitine metabolism. Average percentages of (A) l -carnitine, (B) γ-butyrobetaine (γ-BB), and (C) trimethylamine (TMA) relative to initial l -carnitine concentration are displayed over 48 hours. Results are shown as mean ± SD from 2–5 donors with 1–4 biological replicates each. Statistical analysis employed linear mixed models, including treatment as fixed effects with random intercepts for donors (* p < 0.05, ** p < 0.01, *** p < 0.001), to measure significant differences between the polyphenol treatments (high-throughput model) and the control. High-throughput and batch colon models were inoculated with 1% faecal inoculum from a healthy donor, 2 mM l -carnitine, and the treatment. After collection, samples were directly stored at −80 °C until LC-MS/MS quantification using isotope-labelled internal standards. The dashed line indicates the results obtained for treatments at a comparable dose of pomegranate extract (22.8 mg mL −1 ) and l -carnitine but in a pH-controlled in vitro batch colon model, as described in a previously published report.

Article Snippet: Individual pomegranate polyphenols were obtained from different suppliers: punicalagin was sourced from BOC Science (CAS 65995-63-3), punicalin from Apollo Scientific (CAS 65995-64-4), and Merck provided ellagic acid (CAS 476-66-4) and gallic acid (CAS 149-91-7).

Techniques: In Vitro, Concentration Assay, High Throughput Screening Assay, Control, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 1 | CXCL10 expression is increased in chronic prostatitis patients and experimental autoimmune prostatitis (EAP) mice. (A) CXCL10 expression in the serum of chronic prostatitis patients and healthy volunteers. (B) CXCL10 expression in the serum of chronic prostatitis patients with and without pain symptom. (C) Pearson’s correlation coefficient analysis for CXCL10 expression and National Institutes of Health Chronic Prostatitis Symptom Index. (D) The diagnostic efficiency of CXCL10 expression in the serum for chronic prostatitis patients. The expression levels of CXCL10 in prostate (E) and serum (F) of EAP mice. (G) RT-qPCR was used to assess CXCL9, CXCL10, CXCL11, and CXCR3 expressions in prostate from EAP mice. (H) IHC staining for CXCL10 and CXCR3 in prostate from EAP mice. Quantification of IHC staining of CXCL10 (I) and CXCR3 (J) in prostate from EAP mice. Data are shown as mean ± standard deviation (SD) by one-way ANOVA analysis (B), or unpaired, two-tailed Student’s t-test analysis (A, E, F, G, I, J), or Pearson’s correlation (C). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Expressing, Diagnostic Assay, Quantitative RT-PCR, Immunohistochemistry, Standard Deviation, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 2 | The potential source of CXCL10 in the prostate of EAP mice. Representative photographs of immunofluorescence staining for CXCL10 and markers for CD4+ T cells (A), macrophages (B), and prostatic stromal cells (C–E) in the prostate of EAP mice. (F) Quantification of CXCL10 immunofluorescence intensity in the experiments of (A–E). High-expressed levels of IFN-g in the serum (G) and prostate (H) in EAP mice. The expression levels of CXCL10 in WPMY-1 cells were evaluated with RT-qPCR (I) and ELISA (J) when treated with IFN-g, IL-17A, and IFN-g and IL-17A. Data are shown as mean ± SD by unpaired, two-tailed Student’s t-test analysis (F–H), or one-way ANOVA analysis (I, J). “ns” indicates P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Staining, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 3 | CXCL10 deficiency ameliorates EAP severity. (A) Histological evaluation for the degree of inflammation for mice in the normal, EAP, sh-NC, and sh-CXCL10 groups. (B) The histopathological scores for mice in the normal, EAP, sh-NC, and sh-CXCL10 groups. (C) Pain response test for mice in the normal, EAP, sh-NC, and sh-CXCL10 groups. Data are shown as mean ± SD by unpaired, two-tailed Student’s t-test analysis. **P < 0.01; ***P < 0.001; ****P < 0.0001; #P < 0.05; ###P < 0.001; ####P < 0.0001.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 4 | Flow cytometry and immunofluorescence analysis for macrophages in the prostate of EAP mice. (A) Representative photographs of immunofluorescence staining of CXCR3 and macrophage markers in the prostate of normal, EAP, sh-NC, and sh-CXCL10 mice. Cells were stained for CD11b (red) and CXCR3 (green). (B) The number of CD11b+CXCR3+ cells in the prostate of the normal, EAP, sh-NC, and sh-CXCL10 groups was counted at ×400 magnification. (C, D) The results of flow cytometry for macrophages in the prostate of EAP mice. Data are shown as mean ± SD by unpaired, two-tailed Student’s t-test analysis. *P < 0.05; **P < 0.01; ****P < 0.0001.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Flow Cytometry, Staining, Cytometry, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 6 | CXCL10 promotes the migration of macrophages via CXCR3-mediated Erk and p38 MAPK activation. (A) Representative photographs of migration assay for bone marrow-derived macrophages (BMDMs) stimulated with rmCXCL10 (100 ng/ml) for 24 h after pretreating with AMG487 (1 mM) for 1 h. (B) The results of migration assay for BMDMs stimulated with rmCXCL10. (C) BMDMs were plated on m-Slide and analyzed for 10 h in the presence of CXCL10 gradient (100 ng/ml). (D) Cell speeds were compared for BMDMs between untreated cells and cells pretreated with AMG487 (1 mM) for 1 h. (E, F) The viabilities of cells treated with CXCL10 or AMG487 were evaluated with CCK-8 assays. (G) Cells were pretreated with AMG487 (1 mM) for 1 h, then with rmCXCL10 (100 ng/ml) for the indicated time, and the phosphorylated p38 MAPK and Erk1/2 levels in BMDMs were detected. (H, I) Representative photographs and the results of migration assay for BMDMs stimulated with CXCL10 (100 ng/ml) for 24 h after pretreating with Erk1/2 inhibitor or p38 MAPK inhibitor for 1 h. Data are shown as mean ± SD by one-way ANOVA analysis (B, E, I) and by unpaired, two-tailed Student’s t-test analysis (D, F). “####” indicates P <0.0001 compared between the CXCL10 group and the CXCL10 and AMG487 groups. “ns” indicates P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Migration, Activation Assay, Derivative Assay, CCK-8 Assay, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 7 | CXCL10 promotes secretions of inflammatory mediators of macrophage via CXCR3-mediated ERK and p38 MAPK activation. The mRNA expression levels of IL-6 (A) and MCP1 (B) in BMDMs were calculated 3 h after LPS treatment. BMDMs from EAP mice were pretreated with AMG487 (1 mM) for 1 h, then stimulated with CXCL10 (100 ng/ml) for 12 h, and then with LPS (50 ng/ml) for 3 h. Cells were collected for detecting the mRNA expression levels of IL-6 (A) and MCP1 (B). (C) Western blot analysis of the phosphorylation of the ERK1/2, and p38 MAPK signaling pathways in LPS-induced macrophages for 3 h. The expression levels of IL-6 in the prostate (D) and serum (E) for mice in the normal, EAP, sh-NC, and sh-CXCL10 groups. The expression levels of IL-6 in the prostate (F) and serum (G) for mice in the AMG487 and vehicle groups. The expression levels of MCP1 in the prostate (H) and serum (I) for mice in the normal, EAP, sh-NC, and sh-CXCL10 groups. The expression levels of MCP1 in the prostate (J) and serum (K) for mice in the AMG487 and vehicle groups. Data are shown as mean ± SD by one-way ANOVA analysis (A, B) or unpaired, two-tailed Student’s t-test analysis (D–K). *P < 0.05; **P < 0.01. ns, no significance.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Activation Assay, Expressing, Western Blot, Phospho-proteomics, Protein-Protein interactions, Two Tailed Test

Journal: Frontiers in immunology

Article Title: Pathogenic Roles of CXCL10 in Experimental Autoimmune Prostatitis by Modulating Macrophage Chemotaxis and Cytokine Secretion.

doi: 10.3389/fimmu.2021.706027

Figure Lengend Snippet: FIGURE 8 | Schematic diagram of the pathogenic roles of CXCL10 in EAP. During EAP, CXCL10 recruits macrophages to prostate and induces the secretions of proinflammatory mediators via the Erk1/2 and p38 MAPK signaling pathways, leading to the exacerbation of EAP. Prostatic stromal cells, T cells, and macrophages are potential sources of CXCL10.

Article Snippet: The primary antibodies used were rabbit anti-CXCL10 antibody (NBP2-67004; Novus), rabbit anti-CXCR3 antibody (DF7113; Affinity), rat anti-CD11b antibody (ab8878, Abcam), mouse anti-CD4 antibody (SC-19641, Santa Cruz), mouse anti-a smooth muscle actin (a-SMA) antibody (SC-53142, Santa Cruz), mouse anti-desmin antibody (SC-23879, Santa Cruz), and mouse anti-Vimentin antibody (SC-6260, Santa Cruz).

Techniques: Protein-Protein interactions