Journal: Immunity, Inflammation and Disease

Article Title: Inhibition of TMUB1 blocks apoptosis and NF‐κB pathway‐mediated inflammation in recurrent spontaneous abortion

doi: 10.1002/iid3.879

Figure Lengend Snippet: Effects of LPS on apoptosis and inflammation in placental tissues of pregnant mice (A) Representative images of TUNEL staining at magnification X200 (Scale bars: 100 µm) and magnification X400 (Scale bars: 50 µm). (B) The mRNA levels of IL‐6 and TNF‐α were measured with quantitative RT‐PCR. (C) The content of IL‐6 and TNF‐α was quantified by ELISA. (D) Western blot analysis for p‐p65 and p65 (left), and the quantification of the p‐p65/p65 ratio (right). ELISA, enzyme‐linked immunosorbent assay; IL‐6, interleukin‐6; LPS, lipopolysaccharide; mRNA, messenger RNA; RT‐PCR, real‐time polymerase chain reaction; TNF‐α, tumor necrosis factor‐α; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The content of IL‐6 and TNF‐α was measured using the Mouse TNF‐α ELISA Kit, Mouse IL‐6 ELISA Kit, Human TNF‐α ELISA Kit, and Human IL‐6 ELISA Kit (LIANKE Biotech) according to the manufacturer's instructions.

Techniques: TUNEL Assay, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: Immunity, Inflammation and Disease

Article Title: Inhibition of TMUB1 blocks apoptosis and NF‐κB pathway‐mediated inflammation in recurrent spontaneous abortion

doi: 10.1002/iid3.879

Figure Lengend Snippet: Effects of TMUB1 silencing on inflammation in LPS‐induced trophoblast cells. (B) The levels of IL‐6 and TNF‐α in the different treatments were measured by quantitative RT‐PCR and ELISA. (B) The expression of p‐IKKα/β and IKKα was detected by western blot, and the ratio of p‐IKKα/β/IKKα was quantified in different groups. (C) The expression of p‐p65 and p65 was detected by western blot, and the ratio of p‐p65/p65 was quantified in different groups. (D) The protein content of NF‐κB p65 in the cytoplasm and nucleus was detected by western blot. ELISA, enzyme‐linked immunosorbent assay; IL‐6, interleukin‐6; LPS, lipopolysaccharide; RT‐PCR, real‐time polymerase chain reaction; TMUB1, transmembrane and ubiquitin‐like domain containing 1; TNF‐α, tumor necrosis factor‐α.

Article Snippet: The content of IL‐6 and TNF‐α was measured using the Mouse TNF‐α ELISA Kit, Mouse IL‐6 ELISA Kit, Human TNF‐α ELISA Kit, and Human IL‐6 ELISA Kit (LIANKE Biotech) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Immunity, Inflammation and Disease

Article Title: Inhibition of TMUB1 blocks apoptosis and NF‐κB pathway‐mediated inflammation in recurrent spontaneous abortion

doi: 10.1002/iid3.879

Figure Lengend Snippet: Effects of TMUB1 silencing in LPS‐induced mice. Pregnant mice were injected with lentivirus knocking down TMUB1 and then injected intraperitoneally with 0.15 μg/g LPS to induce abortion. (A) Pathological and anatomical observation of uteruses in pregnant mice. (B) The embryo absorption rate and abortion rate were calculated respectively in pregnant mice. (C) TUNEL staining was used to detect apoptosis in LPS‐induced mice with TMUB1 knockdown. Scale bar: 50 µm. (D) The mRNA levels of IL‐6 and TNF‐α were measured by quantitative RT‐PCR. (E) The protein content of p‐p65 and p65 was detected by western blot, and the ratio of p‐p65/p65 was quantified in different groups. DAPI, 4′,6‐diamidino‐2‐phenylindole; LPS, lipopolysaccharide; mRNA, messenger; RT‐PCR, real‐time polymerase chain reaction; TMUB1, transmembrane and ubiquitin‐like domain containing 1; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Article Snippet: The content of IL‐6 and TNF‐α was measured using the Mouse TNF‐α ELISA Kit, Mouse IL‐6 ELISA Kit, Human TNF‐α ELISA Kit, and Human IL‐6 ELISA Kit (LIANKE Biotech) according to the manufacturer's instructions.

Techniques: Injection, TUNEL Assay, Staining, Knockdown, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Ubiquitin Proteomics

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Generation and characterization of WT iPSCs and CADASIL iPSCs. (A) Schematic procedures for establishing iPSC-based CADASIL disease model. Fibroblasts obtained from one CADASIL patient and two healthy controls were reprogrammed into iPSCs. The iPSCs were then differentiated to generate VSMCs and VECs. Changes in disease-associated transcriptional profiling and cellular phenotypes were analyzed. (B) Confirmation of the heterozygous mutation of NOTCH3 (c.3226C>T, p.R1076C) in CADASIL iPSCs by DNA sequencing (right). Phase-contrast images of fibroblasts (left) and fibroblast-derived iPSCs (middle). Scale bar of fibroblasts, 50 μm; Scale bar of iPSCs, 100 μm. (C) RT-PCR of pluripotency markers, SOX2 , OCT4 , and NANOG . Human ESCs (hESCs) were used as positive controls and human fibroblasts as negative controls. (D) Immunofluorescence staining of pluripotency markers, NANOG, SOX2, and OCT4. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (E) Immunofluorescence staining of TUJ1 (ectoderm), α-SMA (mesoderm), and FOXA2 (endoderm) in teratomas derived from WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. (F) DNA methylation analysis of the OCT4 promoter in WT and CADASIL iPSCs. Open and closed circles indicate unmethylated and methylated CpG dinucleotides, respectively ( n = 7). (G) Karyotyping analysis of WT and CADASIL iPSCs. (H) Clonal expansion analysis of WT and CADASIL iPSCs. Representative images of crystal violet staining are shown to the left. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (I) Immunofluorescence staining of Ki67 in WT and CADASIL iPSCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (J) Cell cycle analysis of WT and CADASIL iPSCs. Data are presented as mean ± SD, n = 3. NS, not significant

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Mutagenesis, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Immunofluorescence, Staining, DNA Methylation Assay, Methylation, Cell Cycle Assay

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Transcriptional profiling changes in CADASIL VSMCs. (A) Flow cytometry analysis of VSMC-specific marker CD140b in WT and CADASIL VSMCs. (B) Immunofluorescence staining of VSMC-specific markers, Calponin, SM22 and α-SMA. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (C) Scatter plots showing the correlation between replicates of WT and CADASIL VSMCs. (D) Heatmap illustrating differentially expressed genes in WT and CADASIL VSMCs. (E) Volcano plot showing the number of upregulated (red dot) and downregulated (green dot) genes in CADASIL VSMCs. (F) GO enrichment analysis of upregulated genes in CADASIL VSMCs. (G) Gene set enrichment analysis (GSEA) plots showing representative GO-BP terms enriched in CADASIL VSMCs. (H) Density plot showing Log 2 (fold change) of mRNA expression levels between WT and CADASIL VSMCs for NF-κB target genes. A rightward shift (*** P < 0.001) indicates increased frequency of genes upregulated in CADASIL VSMCs. (I) Heatmap showing upregulated NF-κB target genes in CADASIL VSMCs

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Flow Cytometry, Marker, Immunofluorescence, Staining, Expressing

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Activation of NF-κB in CADASIL VSMCs was related to NOTCH pathway upregulation. (A) Verification of upregulated NOTCH pathway genes and NF-κB target genes in CADASIL VSMCs by RT-qPCR. CADASIL was taken as reference. Data are presented as mean ± SEM, n = 4. *** P < 0.001. (B) Western blot analysis of NF-κB P65 (RelA) and phosphorylated RelA (Ser536) expression levels in WT and CADASIL VSMCs. β-Actin was used as the loading control. Data are presented as mean ± SD, n = 5. NS, not significant. ** P < 0.01. (C) Immunofluorescence staining of NF-κB P65 (RelA) in WT and CADASIL VSMCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of cells with nucleus localized RelA are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. *** P < 0.001. (D) RT-qPCR analysis of NF-κB target genes in CADASIL VSMCs. CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively. Vehicle was taken as reference. Data are presented as mean ± SEM, n = 4. * P < 0.05, *** P < 0.001

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Activation Assay, Quantitative RT-PCR, Western Blot, Expressing, Control, Immunofluorescence, Staining

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: CADASIL VSMCs exhibited hyperproliferation and abnormal cytoskeleton structure. (A) Immunofluorescence staining of Ki67 in WT and CADASIL VSMCs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells (CADASIL was taken as reference) are shown to the right. Data are presented as mean ± SD, n = 8. *** P < 0.001. (B) Clonal expansion analysis of WT and CADASIL VSMCs. Representative images of crystal violet staining are shown to the left, Scale bar, 100 μm. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are shown as mean ± SD, n = 3. *** P < 0.001. (C) Cell cycle analysis of WT and CADASIL VSMCs. Data are shown as mean ± SD, n = 3. *** P < 0.001; NS, not significant. (D) 3D-SIM (top) and confocal microscope images (bottom) of F-actin showing increased aggregation of parallel microfilaments and scattered nodes (arrow heads) in CADASIL VSMCs. Inside the red rectangle is a substantially normal cell. Scale bar of 3D-SIM images, 5 μm. Scale bar of confocal microscope images, 50 μm. The percentages of cells with abnormal F-actin in SIM images are shown. (E) 3D-SIM (top) and confocal microscope images (bottom) showing increased percentage of cells with aggregated vimentin (arrow heads) in CADASIL VSMCs. Inside the red rectangle is a substantially normal cell. Scale bar of 3D-SIM images, 5 μm. Scale bar of confocal microscope images, 25 μm. The percentages of cells with abnormal vimentin in SIM images are shown

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Immunofluorescence, Staining, Cell Cycle Assay, Microscopy

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Transcriptional profiling of CADASIL VECs. (A) Flow cytometry analysis of VEC-specific markers CD31 and CD144 in WT and CADASIL VECs. (B) Phase-contrast images of VECs are shown to the left. Scale bar, 50 μm. Immunofluorescence staining of VEC-specific markers, CD31, vWF, CD144 and eNOS, are shown to the right. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. (C) Immunofluorescence staining of Dil-Ac-LDL in WT and CADASIL VECs. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. (D) Flow cytometry analysis of Dil-Ac-LDL uptake abilities in WT and CADASIL VECs. The relative average fluorescence intensities are shown in the bottom (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (E) The abilities of in vitro tube formation in WT and CADASIL VECs. Scale bar, 100 μm. The relative numbers of tubes are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (F) Flow cytometry analysis of nitric oxide (NO) levels in WT and CADASIL VECs. The relative average fluorescence intensities are shown in the bottom (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (G) Scatter plots showing the correlation between replicates of WT and CADASIL VECs. (H) Heatmap illustrating differentially expressed genes in WT and CADASIL VECs. (I) Volcano plot showing the number of upregulated (red dot) and downregulated (green dot) genes in CADASIL VECs. (J) Gene set enrichment analysis (GSEA) plots showing representative GO-BP terms enriched in CADASIL VECs. (K) GO enrichment analysis of upregulated genes in CADASIL VECs

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Flow Cytometry, Immunofluorescence, Staining, Fluorescence, In Vitro

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Disease-associated phenotypes observed in CADASIL VSMCs were not detected in CADASIL VECs. (A) Immunofluorescence staining of NF-κB P65 (RelA) in CADASIL VECs. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. The relative percentages of cells with nucleus localized RelA are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (B) Western blot analysis of NF-κB P65 (RelA) and phosphorylated RelA (Ser536) expression levels in WT and CADASIL VECs. β-Actin was used as the loading control. Data are presented as mean ± SD, n = 4. NS, not significant. (C) Immunofluorescence staining of Ki67 in WT and CADASIL VECs. Nuclei were stained with Hoechst 33342. Scale bar, 25 μm. The relative percentages of Ki67-positive cells are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 4. NS, not significant. (D) Clonal expansion analysis of WT and CADASIL VECs. Representative images of crystal violet staining are shown to the left, Scale bar, 100 μm. The statistical analyses of relative clonal expansion abilities are shown to the right (CADASIL was taken as reference). Data are presented as mean ± SD, n = 3. NS, not significant. (E) Cell cycle analysis of WT and CADASIL VECs. Data are presented as mean ± SD, n = 3. NS, not significant. (F) 3D-SIM images of F-actin in WT and CADASIL VECs. Scale bar, 5 μm. (G) 3D-SIM images of vimentin in WT and CADASIL VECs. Scale bar, 5 μm

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Control, Cell Cycle Assay

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: CADASIL VSMCs and VECs were more sensitive to inflammatory stimuli. (A) RT-qPCR analysis showing the expression levels of NF-κB downstream genes, IL6 , MCP1 , ICAM1 , in WT and CADASIL VSMCs under basal and TNFα-induced inflammatory conditions. CADASIL treated with TNFα was taken as reference. Cells were treated with or without 10 ng/mL TNFα for 12 h. Data are shown as mean ± SEM, n = 4. *** P < 0.001; ** P < 0.01; NS, not significant. (B) RT-qPCR analysis showing the expression levels of NF-κB downstream genes, IL6 , MCP1 , ICAM1 , in WT and CADASIL VECs under basal and TNFα-induced inflammatory conditions. CADASIL treated with TNFα was taken as reference. Cells were treated with or without 10 ng/mL TNFα for 12 h. Data are shown as mean ± SEM, n = 4. *** P < 0.001; NS, not significant. (C) ELISA assay showing concentration of IL6 in the culture medium of WT and CADASIL VSMCs under basal and 10 ng/mL TNFα-induced inflammatory conditions. The relative concentration of IL6 is shown (CADASIL treated with TNFα was taken as reference). Data are shown as mean ± SD, n = 3. *** P < 0.001; NS, not significant. (D) ELISA assay showing concentration of IL6 in the culture medium of WT and CADASIL VECs under basal and 10 ng/mL TNFα-induced inflammatory conditions. The relative concentration of IL6 is shown (CADASIL treated with TNFα was taken as reference). Data are shown as mean ± SD, n = 3. *** P < 0.001; NS, not significant. (E) Monocyte adhesion to WT and CADASIL VECs under basal and 10 ng/mL TNFα-induced inflammatory conditions. Red arrow heads indicate monocytes. Scale bar, 50 μm. The relative numbers of adhered monocytes are shown to the right (CADASIL treated with TNFα was taken as reference). Data are shown as mean ± SD, n = 3. *** P < 0.001; NS, not significant

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Protein & Cell

Article Title: Modeling CADASIL vascular pathologies with patient-derived induced pluripotent stem cells

doi: 10.1007/s13238-019-0608-1

Figure Lengend Snippet: Schematic drawing of the major cellular phenotypes observed in CADASIL VSMCs. The heterozygous NOTCH3 mutation (c.3226C>T) of VSMCs resulted in increased proliferation ability, cytoskeleton disorganization, activation of NOTCH pathway and NF-κB pathway. However, these disease-associated phenotypes found in CADASIL VSMCs were not observed in CADASIL VECs

Article Snippet: CADASIL VSMCs were treated with 20 μmol/L DAPT (GSI-IX) (Selleck, S2215) and 50 μmol/L Caffeic Acid Phenethyl Ester (CAPE) (Selleck, S7414) for 18 hours respectively.

Techniques: Mutagenesis, Activation Assay

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages.

doi: 10.1007/s00210-023-02911-w

Figure Lengend Snippet: Fig. 4 The M2 polarization could be facilitated by Stefin B. A The level of IL-6 and IL-10 released by macrophages was checked by ELISA. B The percentage of CD206 + mac- rophages was determined by the flow cytometry assay (*p < 0.05 vs Control, #p < 0.05 vs Stefin B OE NC or si-Stefin B NC)

Article Snippet: ELISA kits were utilized for the detection of IL-6 (EK206/3–96, MULTI SCIENCE, China), TNF-α (EK282/4–96, MULTI SCIENCE, China), and IL-1β (E-EL-H0149c, Elabscience, China) released by macrophages, as well as IL-17 (MM-0170M1, MEIMIAN, China) in the serum of mice.

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control

Journal: Naunyn-Schmiedeberg's archives of pharmacology

Article Title: Stefin B alleviates the gouty arthritis in mice by inducing the M2 polarization of macrophages.

doi: 10.1007/s00210-023-02911-w

Figure Lengend Snippet: Fig. 9 Stefin B overexpression suppressed the inflammation in GA mice. A The mRNA level in the ankle joint tissue was checked by RT-PCR assay. B The serum IL-17 level was detected by the ELISA assay (*p < 0.05 vs Control, #p < 0.05 vs GA model, &p < 0.05 vs GAmodel + Stefin B OE NC)

Article Snippet: ELISA kits were utilized for the detection of IL-6 (EK206/3–96, MULTI SCIENCE, China), TNF-α (EK282/4–96, MULTI SCIENCE, China), and IL-1β (E-EL-H0149c, Elabscience, China) released by macrophages, as well as IL-17 (MM-0170M1, MEIMIAN, China) in the serum of mice.

Techniques: Over Expression, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5.

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: Figure 1. UL23 inhibits the expression of chemokines CCL2 and CCL5 during HCMV infection. (a) HFF cells (1×105) were infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 0.1,1,3) for 12 h before qPCR analysis. RT-PCR shows the expression levels of the UL23 mRNA. (b-c) HFF cells (1×105) were infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for different time points before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 mRNA. (d-e) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF- PCDH) (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 and IE1 mRNA. Each assay was conducted thrice. (f, g) HFF cells (1×105) infected with untreated or UV-inactivated HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h before qPCR and ELISA analysis. RT-PCR shows the expression of the UL23 and IE1 mRNA. (h) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-PCDH) (1×105) were infected with HSV-1 (MOI = 1), RSV (MOI = 1) for 12 h before RT-qPCR and ELISA analysis. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 (unpaired, two-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: Secreted CCL2 and CCL5 in supernatants was quantified by human CCL2 ELISA kit (EK187–96, Multisciences) and human CCL5 ELISA kit (EK1129–96, Multisciences) according to the manufacturer’s instructions.

Techniques: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Stable Transfection, Control, Quantitative RT-PCR, Two Tailed Test

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5.

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: Figure 2. UL23 down-regulates the expression of chemokines CCL2 and CCL5 by inhibiting IRF-3 phosphorylation. (a)HFF cells (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for the indicated times. Immunoblots were performed with the indicated antibodies. (b) UL23 stably expressed HFF cells (HFF-UL23) and control cells (HFF-PCDH) (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h. Immunoblots were performed with the indicated antibodies. (c) HFF cells (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 12 h, and then subjected to subcellular fractionation. Cytoplasmic and nuclear extracts were examined by immunoblotting using the indicated antibodies. (d) HFF cells (1×104) were seeded in 96 wells plates and treated with B×795 at the indicated concentrations for 48 h. Cell viability was evaluated using CCK8 assay. (e) HFF cells (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 at an MOI of 1 in the absence or presence of B×795(2.5 μM). At 12 h, total cell lysates were prepared to immunoblot with indicated antibodies. (f) HFF cells (1×105) were un-infected or infected with HCMV-Towne or HCMV-ΔUL23 at an MOI of 1 in the absence or presence of B×795(2.5 μM). At 12 h, total cell supernatants were prepared to ELISA. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 (unpaired, two-tailed Student’s t-test). Data are representative of three independent experiments with similar results.

Article Snippet: Secreted CCL2 and CCL5 in supernatants was quantified by human CCL2 ELISA kit (EK187–96, Multisciences) and human CCL5 ELISA kit (EK1129–96, Multisciences) according to the manufacturer’s instructions.

Techniques: Expressing, Phospho-proteomics, Infection, Western Blot, Stable Transfection, Control, Fractionation, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5.

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: Figure 3. UL23 inhibited immune cell migration by down-regulating the expression of chemokines CCL2 and CCL5. (a) Pattern diagram of migration. (b) Cell culture supernatants from the indicated times-incubated HCMV-Towne or HCMV-ΔUL23-infected (MOI = 1) or mock-infected HFF cells were used to perform a migration assay. THP-1/Jurkat/PBMC cells (1 × 105) were added into the upper chamber of the transwell plates and allowed to migrate for 12 h into the lower chamber. Subsequently, cell numbers in the lower chamber were counted and migration percentage were calculated as described in materials and methods. (c) THP-1/Jurkat/ PBMC cells (1×105) migration assay by the supernatants from HFF-UL23 or HFF-PCDH cells were mock infected or infected with HCMV-ΔUL23 (MOI = 1) for 24 h. After migrating for 12 h into the lower chamber, the number of cells migrating into the lower chamber was counted. (d) THP-1/Jurkat/PBMC cells (1×105) migration assay by the supernatants from HFF cells were un-infected (mock) or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) and treated without or with 2 μM B×795 for 12 h. After migrating for 12 h into the lower chamber, the number of cells migrating into the lower chamber was counted. (E)HFF cells (1×105) were un- infected or infected with HCMV-Towne or HCMV-ΔUL23 (MOI = 1) for 24 h and the culture supernatants were harvested. The culture

Article Snippet: Secreted CCL2 and CCL5 in supernatants was quantified by human CCL2 ELISA kit (EK187–96, Multisciences) and human CCL5 ELISA kit (EK1129–96, Multisciences) according to the manufacturer’s instructions.

Techniques: Migration, Expressing, Cell Culture, Incubation, Infection

Journal: Virulence

Article Title: Human cytomegalovirus UL23 inhibits immune cell migration and blocks antiviral immune cell responses by reducing the expression of chemokines CCL2 and CCL5.

doi: 10.1080/21505594.2025.2500493

Figure Lengend Snippet: Figure 5. The mode of HCMV UL23-mediated immune evasion. During the period of HCMV infection, UL23 decreases the expression of CCL 2 and CCL5 by restraining the IRF-3 phosphorylation and inhibits the migration of immune cells, thereby weakening the ability of the immune system to clear the virus.

Article Snippet: Secreted CCL2 and CCL5 in supernatants was quantified by human CCL2 ELISA kit (EK187–96, Multisciences) and human CCL5 ELISA kit (EK1129–96, Multisciences) according to the manufacturer’s instructions.

Techniques: Infection, Expressing, Phospho-proteomics, Migration, Virus