Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Primers used in cloning in this study

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Cloning, Sequencing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Quantitative RT-PCR primers used in this study

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Quantitative RT-PCR, Sequencing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: PRRSV infection induces SOCS1 expression in porcine AMs and Marc-145 cells. (A–C) Porcine AMs were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected to assess SOCS1 mRNA levels (A) using qRT-PCR or SOCS1 protein levels (B) by Western blotting and ELISA (C). For ELISA, data were expressed as picograms per microgram of total protein. (D) Porcine AMs were infected or mock infected with HN07-1 at different MOIs. At 12 hpi, the cells were collected for analysis of SOCS1 mRNA levels using qRT-PCR. (E and F) Marc-145 cells were infected or mock infected with HN07-1 at an MOI of 1. At the indicated time points, the cells were collected, SOCS1 mRNA levels (E) were assessed using qRT-PCR, and SOCS1 protein levels (F) were assessed using Western blotting. (G and H) Porcine AMs were infected or mock infected with HNhx (G) or BJ-4 (H) at an MOI of 1. At the indicated times, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. (I) Porcine AMs were infected or mock infected with HN07-1, heat-inactivated HN07-1, or UV-inactivated HN07-1. At the indicated time points, the cells were collected, and SOCS1 mRNA levels were assessed using qRT-PCR. All experiments were performed with at least three independent replicates. The results obtained were compared with 0 h (medium control) using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: SOCS1 enhances PRRSV replication. (A–D) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against SOCS1 (an equimolar mixture of two siRNAs) for 24 h before infecting them with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested, SOCS1 mRNA levels (A) were assessed using qRT-PCR, and SOCS1 protein levels (B) were assessed using ELISA. The cells were collected for PRRSV ORF7 (C) quantification using qRT-PCR or viral enumeration (D) using a TCID50 assay. (E and F) Marc-145 cells (E) and CRL2843-CD163 cells (F) were transfected with pcDNA3.1-Myc/his_A or pcDNA3.1-SOCS1-His and then infected 24 h later with HN07-1 (MOI = 0.1). At 24 hpi, the cells were collected, and SOCS1 protein levels were assessed using Western blotting with an anti-His Ab. Viruses (E) were enumerated using a TCID50 assay, and PRRSV ORF7 (F) was quantified using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significance was denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, TCID50 Assay, Infection, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Induction of SOCS1 expression by PRRSV is not completely dependent on type I IFNs. (A and B) Porcine AMs were treated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, porcine AMs were harvested and analyzed for SOCS1 mRNA (A) quantification using qRT-PCR and SOCS1 protein (B) quantification using ELISA. (C–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNA against IFNAR1 for 24 h and then infected with PRRSV (MOI = 1). At 24 hpi, porcine AMs were harvested and analyzed for IFNAR1 (C) and SOCS1 mRNA (D) quantification using qRT-PCR or SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated or mock treated with B18R for 1 h and then treated with medium alone (control), HN07-1 (MOI = 1), or poly(I:C) at a final concentration of 10 μg/ml. Twenty-four hours later, the cells were harvested and analyzed for SOCS1 mRNA quantification using qRT-PCR. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

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

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: Cloning and sequence analysis of porcine SOCS1 promoter and mapping of potential transcriptional regulatory elements. (A) Schematic representation of the porcine SOCS1 promoter. SOCS1 promoter–truncated mutants were subcloned into pGL4.17-basic vector and the resulting constructs denoted as −2000/100-Luc, −1359/100-Luc, −381/100-Luc, and −58/100-Luc. The relative lengths and positions of the 5′ ends of these fragments were indicated. (B and C) Marc-145 cells (B) or CRL2843-CD163 cells (C) were transfected with a series of SOCS1 promoter–truncated mutants or pGL4.17-basic vector and a pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, they were infected or mock infected with HN07-1 (MOI = 1). Another 24 h later, the cells were harvested to determine luciferase activity. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Cloning, Sequencing, Plasmid Preparation, Construct, Transfection, Luciferase, Infection, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: PRRSV N protein induces SOCS1 production. (A and D) Marc-145 cells (A) or CRL2843-CD163 (D) was cotransfected with a series of plasmids that encode PRRSV nsps and structural protein (nsp1α, nsp1β, nsp2, nsp4, nsp5, nsp11, and N) or pcDNA 3.1-Myc/his_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. (B) Marc-145 cells were transfected with N protein expression vector at doses of 0.5, 1.0, and 2.0 μg per well (six-well plate) and were harvested at 48 h posttransfection. Western blotting using an anti-SOCS1 Ab was performed to assess SOCS1 protein levels. (C and E) Marc 145 cells (C) or CRL2843-CD163 (E) was cotransfected with PRRSV N protein deletion mutants or pcDNA3.1-Myc/His_A vector, −381/100-Luc promoter, and pRL-TK Renilla luciferase reporter plasmid. After 24 h, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Western Blot

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: AP-1 binding element in the porcine SOCS1 promoter is indispensable for SOCS1 production. (A) Schematic diagram representing porcine SOCS1 promoter deletion mutants (−308ΔIRF3-Luc, −188ΔSp1-Luc, −160ΔSp1-Luc, −116ΔIRF3-Luc, −104ΔAP-1-Luc, and −381/100-Luc). (B and D) Marc-145 cells (B) or CRL2843-CD163 cells (D) were cotransfected with a series of SOCS1 promoter mutants and pRL-TK Renilla luciferase reporter plasmid for 24 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for luciferase activity analysis. (C and E) Marc-145 cells (C) or CRL2843-CD163 cells (E) were cotransfected with a series of SOCS1 promoter mutants, N protein, and pRL-TK Renilla luciferase reporter plasmid. Twenty-four hours later, the cells were harvested for luciferase activity analysis. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Binding Assay, Luciferase, Plasmid Preparation, Infection, Activity Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The AP-1 signaling pathway is involved in SOCS1 upregulation by PRRSV. (A) Porcine AMs were pretreated with DMSO or SR11302 for 1 h and then infected or mock infected with HN07-1 (MOI = 1) in the presence of inhibitor. The cells were harvested at 24 hpi for analysis of SOCS1 mRNA levels using qRT-PCR. (B–E) Porcine AMs were transfected with 20 nM NC siRNA or siRNAs against porcine c-Jun and c-Fos using RNAiMAX and then infected after 24 h with PRRSV HN07-1 (MOI = 1). After 24 h, the cells were harvested and analyzed to measure knockdown efficiency (B and C). Or the cells were harvested for analysis of SOCS1 mRNA levels (D) using qRT-PCR or lysed for SOCS1 protein (E) quantification using ELISA. (F) Marc-145 cells were pretreated with DMSO or SR11302 for 1 h and transfected with a vector that encodes the PRRSV N protein. After 24 h, the cells were harvested for SOCS1 protein level and p–c-Jun/c-Jun and p–c-Fos/c-Fos level quantification using Western blotting. (G) Porcine AMs were infected with HN07-1 (MOI = 1) and then harvested at the indicated time points for analysis of SOCS1, p–c-Jun/c-Jun, and p–c-Fos/c-Fos levels, using Western blotting. (H) Marc-145 cells were transfected with a vector that encodes the PRRSV N protein at doses of 0.5, 1.0, and 2.0 μg per well (six-well pates) and then harvested 48 h later for analysis of SOCS1 and p–c-Jun/c-Jun levels using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to β-actin or GAPDH levels. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Infection, Quantitative RT-PCR, Transfection, Knockdown, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The p38 and JNK MAPK signaling pathways are required for PRRSV upregulation of SOCS1 expression. (A and B) Porcine AMs were pretreated with DMSO, p38 inhibitor SB203580 (SB), ERK inhibitor PD98059 (PD), or JNK inhibitor SP600125 (SP) for 1 h and then infected or mock infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of SOCS1 mRNA levels (A) using qRT-PCR and SOCS1 protein levels (B) using ELISA. All experiments were performed with at least three independent replicates. Differences between groups were assessed using Student t test, and statistical significances were denoted by **p < 0.01.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Protein-Protein interactions, Expressing, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Porcine Reproductive and Respiratory Syndrome Virus Enhances Self-Replication via AP-1–Dependent Induction of SOCS1

doi: 10.4049/jimmunol.1900731

Figure Lengend Snippet: The p38/AP-1 and JNK/AP-1 signaling pathways are involved in PRRSV-induced SOCS1 production. (A–C) Porcine AMs were pretreated with DMSO or p38 inhibitor SB203580 (SB) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (A), p–c-Fos/c-Fos (B), and SOCS1 levels (C) using Western blotting. (D–F) Porcine AMs were pretreated with DMSO or JNK inhibitor SP600125 (SP) for 1 h and then infected with HN07-1 (MOI = 1). At 24 hpi, the cells were harvested for analysis of p–c-Jun/c-Jun (D), p–c-Fos/c-Fos (E), and SOCS1 levels (F) using Western blotting. (G and H) Marc-145 cells were pretreated with DMSO, p38 inhibitor SB, or JNK inhibitor SP for 1 h, and they were then transfected with plasmid that encodes the PRRSV N protein at a dose of 2.0 μg per well (six-well plates) for 48 h. The cells were harvested for analysis of SOCS1 and p–c-Jun/c-Jun expression using Western blotting. Relative expression levels shown below the images were evaluated as fold changes after normalization to GAPDH levels.

Article Snippet: The cells were transfected with an SOCS1 promoter luciferase reporter plasmid (−2000/100-Luc, truncated mutant plasmids or element deletion mutants of SOCS1 promoter) and a pRL-TK Renilla luciferase reporter plasmid (an internal control reporter vector from Promega).

Techniques: Protein-Protein interactions, Infection, Western Blot, Transfection, Plasmid Preparation, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p directly inhibits the expression of SOCS1. A Venn diagram indicated target candidate genes of miR-6794-5p using TargetScan and miRWalk, which are miRNA target prediction sites. B, C After overexpression of the miR-6794-5p mimic in U251 ( B ) and A549 ( C ) cells, the mRNA expression of each of the candidate genes was confirmed by qRT-PCR. The values were normalized to GAPDH. D After miR-6794-5p was overexpressed in both cells, the level of SOCS1 protein was confirmed by Western blot analysis. β-actin was used for normalization. The experiment was repeated with triplicates and representative Western blotting images are shown. E, F Dual luciferase activity was examined after A549 cells were co-transfected with wild-type (WT) or mutant (Mut) vectors of the SOCS1 3’UTR in the presence or absence of the miR-6794-5p mimic, respectively. G Ago2-RNA immunoprecipitation (Ago2-IP) assay was performed in negative control (NC) or miR-6794-5p overexpressed A549 cells, and SOCS1 enrichment was confirmed by qRT-PCR. The values were normalized to GAPDH. H The mRNA expression of SOCS1 in plasma of normal and patients with lung cancer (normal, n = 23; lung cancer, n = 23) was analyzed by qRT-PCR. I Kaplan-Meier plots were used to compare survival rates between normal groups and lung adenocarcinoma patients. All data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. Student’s t-test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Transfection, Mutagenesis, Immunoprecipitation, Negative Control

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p promotes EMT, cell mobility, invasiveness, and stemness maintenance by suppressing SOCS1. A - D U251 and A549 cells were co-transfected with miR-6794-5p or SOCS1 overexpressing vectors. A The expression of EMT and stemness marker proteins in the indicated cells was confirmed by Western blot analysis. β-actin was used for normalization. The experiment was repeated with triplicates and representative Western blotting images are shown. Wound healing ( B ), matrigel coating invasion ( C ), and sphere formation assays ( D ) were performed to confirm cell mobility, invasiveness, and stemness maintenance in the indicated cells. Scale bar is 200 μm. The data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. One-way ANOVA followed by bonferroni comparison test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Transfection, Expressing, Marker, Western Blot, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p induces macrophage M2 polarization via JAK1/STAT3 pathway. A Exosomes were isolated from the conditioned media of U251 cells overexpressing Bcl-w. After treatment of THP-1-derived macrophages with exosomes, the mRNA levels of CD163, CD206, and CD11b were measured by qRT-PCR analysis . B, C After THP-1-derived macrophages were treated with conditioned media from U251 and A549 cells transfected with miR-6794-5p mimics, respectively, mRNA levels of macrophage M2 markers (CD163, CD206, and CD11b) were measured by qRT-PCR analysis. D, E mRNA ( D ) or protein ( E ) levels of macrophage M2 markers (CD163, CD206, and CD11b) in THP-1-derived macrophages overexpressing the miR-6794-5p mimic were verified by qRT-PCR or Western blot analysis, respectively. F Using the TCGA dataset, expression of CD206 was compared with normal ( n = 5) and GBM patients ( n = 167) and displayed as box and whisker plots. G The expression level of CD206 in the lung tissues at each stage of lymph node metastasis of patients with lung cancer was confirmed by IHC. H Kaplan-Meier overall survival curves of patients with lung squamous cell carcinoma according to the expression of CD163, CD206 (MRC1), and CD11b (ITGAM). I, J Expressions of p-STAT3, STAT3, p-JAK1, JAK1, and SOCS1 in miR-6794-5p-overexpressed ( I ) or SOCS1-knockdown ( J ) THP-1-derived macrophages were shown by Western blot analysis. K Expression of STAT3 protein after transfection with STAT3 against siRNA in macrophages overexpressing miR-6794-5p (left). Expression mRNA levels of macrophage M2 markers (CD163, CD206, and CD11b) and IL-10 were checked in the indicated cells (right). L After overexpressing empty vector, miR-6794-5p and miR-6794-5p + SOCS1 in THP-1-derived macrophages, the expression of SOCS1 protein was confirmed by Western blot analysis (left), and the mRNA expression levels of macrophage M2 markers (CD163, CD206, and CD11b) were confirmed by qRT-PCR (right) in indicated cells. β-actin was used for normalization in Western blot analysis. The values were normalized to GAPDH in qRT-PCR. The experiment was repeated with triplicates and representative Western blotting images are shown. The data are presented as the mean ± S.D. after triplicate. * P < 0.05; ** P < 0.01; *** P < 0.001. A - D Student’s t-test. K , L One-way ANOVA followed by bonferroni comparison test

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: Isolation, Derivative Assay, Quantitative RT-PCR, Transfection, Western Blot, Expressing, Whisker Assay, Plasmid Preparation, Comparison

Journal: Cell Communication and Signaling : CCS

Article Title: Tumor-derived miR-6794-5p enhances cancer growth by promoting M2 macrophage polarization

doi: 10.1186/s12964-024-01570-5

Figure Lengend Snippet: miR-6794-5p increases metastasis by inducing M2 polarization in vivo. A - B C57BL/6 mice were injected via tail vein with negative control (NC), miR-6794-5p, or miR-6794-5p + SOCS1 overexpressing LLC1 cells ( n = 5; 5 X 10 5 cells/mouse). Lung tissue was harvested by sacrifice at 4 weeks after cell injection. A Lung tissue images of each group were subjected to H&E and IHC staining with anti-SOCS1. Scale bar is 100 μm. B IHC staining was performed with anti-CD206 using the lung tissues of each group. Scale bars are 100 μm (top) and 50 μm (bottom). C - F Negative control (NC) and miR-6794-5p overexpressed LLC1 cells were subcutaneously injected into the right flank of C57BL/6 mice ( n = 4; 2 X 10 5 cells/mouse). After tumorigenesis, the expression levels of M1 macrophages, M2 macrophages and activated CD8+ T cells in the tumor tissues of the two groups were analyzed by representative flow cytometry. C, D Respective percentages of M1 macrophages (CD45+ F4/80+ CD11b + MHCII+ CD206- cells) and M2 macrophages (CD45+ F4/80+ CD11b + MHCII- CD206+ cells) isolated from tumor tissues developed in negative control (NC) and miR-6794-5p overexpressing mice were analyzed by flow cytometry. E The M1/M2 ratio was calculated based on the percentages of M1 and M2 macrophages analyzed by flow cytometry. F The proportion of activated CD8+ T cells (CD45+ CD8+ CD3+ CD25+ cells) isolated from tumor tissue developed in negative control (NC) and miR-6794-5p overexpressing mice was analyzed by flow cytometry. G Schematic diagram illustrating the mechanism of exosomal miR-6794-5p secreted from tumor cells on surrounding macrophages

Article Snippet: The pLPC-flag-SOCS1 vector was provided by Addgene (Plasmid #129514).

Techniques: In Vivo, Injection, Negative Control, Immunohistochemistry, Expressing, Flow Cytometry, Isolation