Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: List of primers used for PCR.

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Amplification

Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: EV was an essential factor in the induction of ISGs by the supernatant from KSHV-infected cells. (A) KSHV EVs isolated by centrifugal filtration induced IFIT1 expression. The supernatant from KSHV-infected cells was separated by centrifugal filter device with a cut-off of 100 kDa. High molecular weight (HMW) proteins (the retained materials by a filter) and low molecular weight (LMW) proteins (the flow-through) was applied to HUVECs, followed by analyzing mRNA expression by RT-qPCR. (B) Induction of ISGs was correlated with the amount of EVs. The same volume of EVs was isolated from the same amount of the supernatant from mock- or KSHV-infected cells. Then, each EV was applied to HUVECs, followed by analyzing mRNA expression by RT-qPCR. ΔCT indicated the normalized CT value of IFIT1 with reference gene, β-actin. (C) Knockdown of Rab27b suppressed the induction of ISGs by KSHV EVs. The expression of Rab27b was suppressed by shRNA in HUVECs. After KSHV infection, mock EVs or KSHV EVs were isolated from the Rab27b-suppressed HUVECs. Each prepared EV was applied to uninfected HUVECs, and mRNA expressions for ISGs were analyzed. The grouping of blots cropped from different gels and full-length blots are included in a . Data are shown as the mean ± SD, n = 6, ** p < 0.01.

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Infection, Isolation, Filtration, Expressing, Molecular Weight, Quantitative RT-PCR, shRNA

Journal: Frontiers in Immunology

Article Title: Extracellular Vesicles From KSHV-Infected Cells Stimulate Antiviral Immune Response Through Mitochondrial DNA

doi: 10.3389/fimmu.2019.00876

Figure Lengend Snippet: Induction of ISGs by KSHV EVs is associated with mtDNA. (A) mRNA expression of IFIT1 in HUVECs treated with DNase I or RNase-treated EVs. (B) Quantification of mtDNA in mock EVs vs. KSHV EVs. Genomic DNA was isolated from the same number of EVs, and mtDNA-related genes were analyzed by qPCR. (C) Western blotting for the cytoplasmic fraction from KSHV-infected HUVECs. Cytoplasmic fraction was extracted by digitonin, and its purity was analyzed by western blot analysis. WCE: whole cell extract, Pel: pellet after extraction of the cytoplasmic fraction, Cyt: cytoplasmic fraction. (D) Quantification for genomic and mitochondrial DNA in the cytoplasmic fraction from mock- vs. KSHV-infected HUVECs. (E,F) Induction of IFIT1 in cGAS or STING-suppressed HUVECs by KSHV EVs. The expression of cGAS (E) or STING (F) was suppressed by shRNAs. Mock EVs or KSHV EVs were treated with each indicated knockdown cell, and the induction in IFIT1 expression was analyzed by RT-qPCR. Data are shown as the mean ± SD, n = 6, ns, not significant, * p < 0.05, and ** p < 0.01.The grouping of blots cropped from different gels and full-length blots are included in a .

Article Snippet: MA), mouse monoclonal anti-KSHV ORF65 , rabbit polyclonal anti-GAPDH (Cusabio, Houston, TX), rabbit polyclonal anti-calnexin (Bioss Antibodies Inc.), mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-mtTFA (Santa Cruz Biotechnology), rabbit polyclonal anti-MX1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT1 (Bioss Antibodies Inc.), rabbit polyclonal anti-IFIT44L (Bioss Antibodies Inc.) and mouse monoclonal anti-β-actin antibodies (Sigma, St. Louis, MO) were used as primary antibodies.

Techniques: Expressing, Isolation, Western Blot, Infection, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A Silencing of circUBAP2 was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting circUBAP2 (si-circUBAP2-1 or si-circUBAP2-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-circUBAP2-1 was chosen for further experiments for its better transfection efficiency. Then, MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for B Cell migration by Transwell assay; C Cell migration by wound healing assay; D The protein levels of Vimentin and Twist was examined by Immunoblotting; E The concentrations of IL-1β and IL-17 in the culture medium by ELISA. F , G The Volcano plot and hierarchical clustering heatmap showing differentially expressed mRNAs in CXCL11-treated MHCC-97H based on RNA sequencing. Upregulated genes were applied for H Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling enrichment analysis and I Gene Ontology (GO) of biological process enrichment analysis. J The expression of IFIT1/IFIT3 in CXCL11-treated and untreated HCC cells based on RNA sequencing data. * P < 0.05, ** P < 0.01, compared with the control group; # P < 0.05, ## P < 0.01, compared with the si-NC group; $ P < 0.05, $$ P < 0.01, compared with the CXCL11 + si-circUBAP2 group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Small Interfering RNA, Transfection, Negative Control, Real-time Polymerase Chain Reaction, Migration, Transwell Assay, Wound Healing Assay, Western Blot, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Expressing, Control

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A The mRNA expression of IFIT1 and IFIT3 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. B The protein contents and distribution of IFIT1 and IFIT3 were examined in nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by IHC staining. C MHCC-97H and Huh-7 cells were transfected with si-circUBAP2 and examined for the mRNA expression of IFIT1 and IFIT3 by real-time PCR. D IFIT1 or IFIT3 silencing was achieved in MHCC-97H and Huh-7 cells by transfecting small interfering RNA targeting IFIT1 or IFIT3 (si-IFIT1-1 or si-IFIT1-2; si-IFIT3-1 or si-IFIT3-2). Si-NC was transfected as a negative control. The transfection efficiency was confirmed by real-time PCR and si-IFIT3-1 and si-IFIT1-2 were chosen for further experiments due to better transfection efficiency. Next, MHCC-97H and Huh-7 cells were transfected with si-NC, si-IFIT1, or si-IFIT3, and examined for E Cell migration by Transwell assay; F Cell migration by wound healing assay; G Cellular protein content and distribution of Vimentin was examined by IF staining; H The protein levels of Vimentin and Twist was examined by Immunoblotting; I The concentrations of IL-1β and IL-17 in the culture medium by ELISA. * P < 0.05, ** P < 0.01, compared with the si-NC group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunohistochemistry, Transfection, Small Interfering RNA, Negative Control, Migration, Transwell Assay, Wound Healing Assay, Staining, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A ENCORI was used to predict miRNAs that circUBAP2 might target, and TargetScan was used to predict miRNAs that might target IFIT1/IFIT3. These two sets of miRNAs intersected at hsa-miR-4756-5p, hsa-miR-1321, hsa-miR-552-3p, hsa-miR-3611, and hsa-miR-24-3p. The expression of miR-4756-5p, miR-1321, miR-552-3p, miR-3611, and miR-24-3p was examined in MHCC-97H and Huh-7 cells with or without CXCL11 treatment. miR-4756 was chosen for further experiments due to its downregulation in both HCC cell lines in response to CXCL11 stimulation. B MHCC-97H and Huh-7 were transfected with si-circUBAP2 and examined for the expression of miR-4756 by real-time PCR. C The expression of miR-4756 was examined in 12 cases of nonmetastatic HCC tissues, metastatic HCC tissues, and para-carcinoma tissues by real-time PCR. D miR-4756 overexpression or inhibition was achieved in MHCC-97H and Huh-7 cells by transfecting miR-4756 mimics or miR-4756 inhibitor; the transfection efficiency was confirmed by real-time PCR. E MHCC-97H and Huh-7 cells were transfected with miR-4756 mimics or miR-4756 inhibitor and examined for the protein levels of IFIT1 and IFIT3 by Immunoblotting. F – H Wild- and mutant-type circUBAP2, IFIT1 3′-UTR, or IFIT3 3′-UTR luciferase reporter plasmids were constructed as described and named as wt-circUBAP2/mut-circUBAP2, wt-IFIT1/mut-IFIT1, and wt-IFIT3/mut-IFIT3. These plasmids were co-transfected in 293T cells with miR-4756 mimics or miR-4756 inhibitor and the luciferase activity was determined. * P < 0.05, ** P < 0.01, compared to si-NC + inhibitor NC group. ## P < 0.01, compared to si-cirUBAP2+miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Over Expression, Inhibition, Western Blot, Mutagenesis, Luciferase, Construct, Activity Assay

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: MHCC-97H and Huh-7 cells were co-transfected with si-circUBAP2 and miR-4756 inhibitor and examined for A The protein levels of IFIT1 and IFIT3 by Immunoblotting; B The concentrations of IL-1β, and IL-17 in the culture medium by ELISA; C Cell migration by Transwell assay; D Cell migration by wound healing assay; E The cellular protein content and distribution of Vimentin was examined by IF staining; F The protein levels of Vimentin and Twist was examined by Immunoblotting. * P < 0.05, ** P < 0.01, compared with the si-NC + inhibitor NC group; ## P < 0.01, compared with the si-circUBAP2 + miR-4756 inhibitor group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Wound Healing Assay, Staining

Journal: Cell Death & Disease

Article Title: Cancer-associated fibroblast-derived CXCL11 modulates hepatocellular carcinoma cell migration and tumor metastasis through the circUBAP2/miR-4756/IFIT1/3 axis

doi: 10.1038/s41419-021-03545-7

Figure Lengend Snippet: A , C The orthotopic implantation tumor model was conducted by injecting Huh-7 cells coinfected with sh-NC/sh-circUBAP2 and anti-NC/anti-miR-4756 lentivirus into the left liver lobe of mice. After 5 weeks, anesthetized mice were sacrificed and livers and lungs were collected for further experiments. B Tumor numbers in mice livers from different groups were counted. D Lung metastatic nodules in mice from different groups were counted. E The histopathological characteristics of mice livers and lungs were evaluated by H&E staining. The protein content and distribution of IFIT1/3 in mice liver tumors were examined by IHC staining. F The expression levels of circUBAP2 and miR-4756 in mice livers from different groups were examined by qRT-PCR. G The protein levels of IFIT1/3, Vimentin, and Twist in mice livers tumors from different groups were examined by Immunoblotting. H A schematic graph of the mechanism. CXCL11 acts as a key mediator integrating CAFs and CAFs-cohabitating HCC cells, and also work as an extracellular remodeler promoting HCC migration and metastasis through the activation of circUBAP2/miR-4756/IFIT1/3 in tumor cells. ** P < 0.01, compared with the sh-NC + anti-NC group; ## P < 0.01, compared with the sh-circUBAP2 + anti-miR-4756 group.

Article Snippet: The membranes were incubated with the following primary antibodies overnight at 4 °C and washed three times with 0.05% TBST for 5 min each time: CXCL11 (CSB-PA06119A0Rb; Cusabio, Wuhan, China), Vimentin (10366-1-AP; Proteintech, Wuhan, China), Twist (CSB-PA025358LA01HU, Cusabio), β-actin (60008-1-Ig, Proteintech), IFIT1 (CSB-PA011018LA01HU, Cusabio), and IFIT3 (CSB-PA011022HA01HU, Cusabio).

Techniques: Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR, Western Blot, Migration, Activation Assay