Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: BAP31 expression is increased in HCC patients and involved in HCC cell proliferation in vitro . (A) Expression of BAP31 in normal liver and HCC tumor tissues at various clinical stages monitored by IHC analysis. (B) Expression of BAP31 in 12 normal liver and 248 HCC tumor tissue samples. (C) Western blot analysis of BAP31expression in Hep3b and MHCC97h cells after stable transfection using lentivirus. (D) Colony formation assay using Hep3b and MHCC97h cells. Representative images were acquired, and the colonies were counted. (E) Cell viability of Hep3b and MHCC97h cells detected by the cell counting kit-8 assay. * p < 0.05; ** p < 0.01; **** p < 0.0001. BAP31, B-cell receptor-associated protein 31; HCC, hepatocellular carcinoma; IHC, immunohistochemistry.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Expressing, In Vitro, Western Blot, Stable Transfection, Colony Assay, Cell Counting, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Correlation between the expression of BAP31 and clinicopathological parameters in HCC.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: BAP31 promotes HCC cell proliferation in vivo . (A) BAP31 overexpression, BAP31 knockdown, and the corresponding control Hep3b cells were injected into the right flanks of nude mice. Images of tumors were acquired with FUSION FX Spectra after 20 days. (B–D) Images of tumors and tumor weight of nude mice 30 days after subcutaneous injection of Hep3b cells. The results represent the mean ± SD, and ** p < 0.01 was considered significant; Student's t -test was used. (E) Growth curve of xenografts of Hep3b cells transfected with BAP31, vector, BAP31shRNA, and scramble shRNA ( n = 5, mean ± SD). (F) Kaplan–Meier survival curve representing the overall survival of the injected mice with BAP31 overexpression or knockdown and control Hep3b cells ( n = 10, ** p < 0.01 by log-rank test). BAP31, B-cell receptor-associated protein 31; HCC, hepatocellular carcinoma; IHC, immunohistochemistry. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: In Vivo, Over Expression, Knockdown, Control, Injection, Transfection, Plasmid Preparation, shRNA, Immunohistochemistry

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Identification of SERPINE2 as a novel downstream gene regulated by BAP31 in HCC cells. (A) Identification of the genes regulated by BAP31 in HCC cells using RNA-Seq analysis. A heat map was constructed based on the genes differentially expressed between the control and BAP31-knockdown cells. (B) Comparative intersection analysis of differentially expressed genes in the BAP31-knockdown and control Hep3b and MHCC97h cells. (C) qRT-PCR analysis of SERPINE2 expression in the BAP31-knockdown and control HCC cells. (D) Lysates of HCC cells expressing the indicated proteins were immunoprecipitated with anti-BAP31, anti-SERPINE2, or mouse IgG isotype antibody, and the immunoprecipitates were probed with the indicated antibodies. (E,F) Colocalization of BAP31 (green) with SERPINE2 (red) in wild-type (E) and BAP31-overexpression and -knockdown and control (F) HCC cells according to confocal microscopy. BAP31, B-cell receptor-associated protein 31; HCC, hepatocellular carcinoma; SERPINE2, serpin family E member 2. ** p < 0.01.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: RNA Sequencing, Construct, Control, Knockdown, Quantitative RT-PCR, Expressing, Immunoprecipitation, Over Expression, Confocal Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Inhibition of SERPINE2 attenuates BAP31-promoted cell proliferation. (A) Colony formation assay using Hep3b and MHCC97h cells that were transiently transfected with SERPINE2 siRNA or control siRNA. Representative images were acquired, and the colony numbers were counted. (B) Cell viability of transiently transfected Hep3b and MHCC97h cells detected by the cell counting kit-8 assay. (C) Protein levels of SERPINE2, BAP31, β-actin, Erk1/2, phospho-Erk1/2, p38, and phospho-p38 were monitored by western blot. (D) Colony formation assay using BAP31-overexpressing HCC cells that were transiently transfected using SERPINE2 siRNA and control siRNA. (E) Cell viability of transiently transfected Hep3b and MHCC97h cells detected by the cell counting kit-8 assay. ** p < 0.01. BAP31, B-cell receptor-associated protein 31; SERPINE2, serpin family E member 2.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Inhibition, Colony Assay, Transfection, Control, Cell Counting, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Multiplex IHC staining indicates that BAP31 is positively correlated with SERPINE2, LRP1, and Ki67. (A) The HCC tissue microarrays LV2089 and LV1221 were stained using Opal 7-plex technology. (B) The merged-channel multispectral image was captured after four-cycle TSA staining and DAPI counterstain and separated by single channels. Fake colors are assigned as DAPI: blue, BAP31: green, SERPINE2: pink, LRP1: red, and Ki-67: brown (A5 point of LV2089 is shown). (C) The typical analysis process of composite image is as follows: first, at the tissue segment stage, the malignant tumor and tumor stroma are distinguished as red and green, respectively; second, at the cell segment stage, the cell nuclei are identified and labeled with green; third, at the phenotype segment stage, the tumor, interstitial, and immune cells are distinguished as red, green, and blue, respectively; finally, at the scoring stage, blue represents no expression, yellow represents 1+, orange represents 2+, and tan represents 3+; each result was assigned a comprehensive H -score. (D – F) The correlation between the expression levels of BAP31 and SERPINE2, Ki-67, and LRP1 in HCC tumor tissue. (G,H) The correlation between the expression levels of SERPINE2 and Ki-67 and LRP1. (I) The correlation between the expression levels of LRP1 and Ki-67. BAP31, B-cell receptor-associated protein 31; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; SERPINE2, serpin family E member 2; TSA, tyramide signal amplification; DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Multiplex Assay, Immunohistochemistry, Staining, Labeling, Expressing, Amplification

Journal: Frontiers in Cell and Developmental Biology

Article Title: BAP31 Promotes Tumor Cell Proliferation by Stabilizing SERPINE2 in Hepatocellular Carcinoma

doi: 10.3389/fcell.2020.607906

Figure Lengend Snippet: Anti-BAP31 antibody inhibits HCC cell-induced tumor formation. (A) Hep3b cells were injected subcutaneously into the right flank of nude mice on day 0. Starting from day 4, anti-BAP31 antibody, mouse IgG isotype, and PBS were administered intraperitoneally twice a week. Images of the xenograft tumors are shown. (B) Growth curve of xenografts of Hep3b cells treated with anti-BAP31, mouse IgG isotype, and PBS ( n = 5, mean ± SD). (C) Weight of the xenograft tumors was measured. The results represent the mean ± SD, and ** p < 0.01 was considered to be significant according to Student's t -test. (D) Expression levels of BAP31, Ki-67, and SERPINE2 in Hep3b subcutaneous tumors were monitored by IHC analysis. (E,F) Kaplan–Meier survival curve representing the overall survival of mice injected with Hep3b cells and treated with anti-BAP31 antibody, mouse IgG isotype, and PBS ( n = 10, ** p < 0.01 by log-rank test). (G) Hematoxylin and eosin staining of the heart, liver, lung, and kidney in various groups of mice. BAP31, B-cell receptor-associated protein 31; HCC, hepatocellular carcinoma; IHC, immunohistochemistry; SERPINE2, serpin family E member 2; PBS, phosphate-buffered saline.

Article Snippet: After blocking with 5% non-fat milk (BD Biosciences, USA), the membranes were incubated with antibodies against BAP31 (Proteintech, China; 11200-1-AP; 1:2,000), SERPINE2 (Proteintech; 66203-1-Ig; 1:1,000), β-actin (Proteintech; 60008-1-Ig; 1:5,000), Erk1/2, p38 (Cell Signaling Technology, USA; 9926; 1:1,000), and phospho-Erk1/2, phospho-p38 (Cell Signaling Technology; 9910; 1:1,000).

Techniques: Injection, Expressing, Staining, Immunohistochemistry, Saline

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: Establishing an Inflammatory pain model and assessing the expression profiles of IL-27. (A) Course of mechanical hyperalgesia in the mouse pain model (n = 5), two-way ANOVA tested P values ( vs. NC) with Dunnett’s multiple comparisons test. (B–G) The dynamic concentration of IL-27 (IL-27p28 and Ebi3) in the brain, spinal cord (L3–L5), spleen, ipsilateral and contralateral DRG (tested by qPCR), and the ELISA method to measure IL-27p28 in the serum. N = 4, GAPDH was used as an internal reference. (H, I) The mRNA level of Wsx-1 in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. (J, K) The mRNA level of IL-12p35 (IL-35) in the spinal cord (L3–L5) and ipsilateral DRG tissues. N = 4, GAPDH was used as an internal reference. The P value ( vs. NC) was measured using one-way ANOVA with Dunnett’s post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ebi3: Epstein-Barr virus-induced gene 3 protein, Ips-DRG: ipsilateral-dorsal root ganglion, qPCR: quantitative PCR, λ-carr: λ-carrageenan.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Virus, Real-time Polymerase Chain Reaction

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: The identification of the source of IL-27. (A–D) The intracellular co-label staining by flow cytometry was applied to determine the source of IL-27. The four APCs were labeled by dendritic cell (CD11b, CD11c, B), neutrophil (CD11b, Ly6G, C), monocyte (CD11b, Ly6C, A), and macrophage (CD11b, F4/80, D), respectively. IL-27p28 antibody labeled the IL-27. The results indicated that neutrophil/monocyte-derived IL-27 was highly expressed in serum and spleen. IL: interleukin, APCs: antigen-presenting cells, SSC-A: side scatter area.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Staining, Flow Cytometry, Labeling, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: The source of IL-27 in the spinal cord is microglia. (A, B) The cultured primary microglia secret loads of IL-27 in response to LPS (100 ng/mL) insult for 12 hours, while cultured primary astrocytes released minimal IL-27 in the same condition (n = 3). (C, D) IFN-γ-stimulated (20 ng/mL) primary microglia were the main source of IL-27, compared with the astrocytes group (n = 3), which suggests IL-27 was primarily secreted from microglia in the spinal cord. IL: interleukin, LPS: lipopolysaccharide, IFN: interferon.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Cell Culture

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: The knockdown of IL-27 intensified mechanical allodynia in mouse pain models. (A) Schematic diagram showing construction of pAAV2/9-U6-shRNA (IL-27p28)-CMV-EGFP vector. (B) The Sh-1 presents the most effective knockdown of IL-27 at the mRNA level in mouse DRG (L3–L5) tissue and was selected for use in the next operation (n = 3). One-way ANOVA with Tukey’s multiple comparisons test was applied. (C) The experiment procedures for i.t. injection of pAAV2/9-U6-shRNA (IL-27p28) to knock down IL-27 and behavior test in mice. (D) The timelines of mechanical hyperalgesia in WT, λ-carr, and sh-IL-27 mice after tail-vein injection of recombination mouse IL-27 in a hind paw (n = 5). P values ( vs. λ-carr) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E, F) The timelines of mechanical hyperalgesia in mice with WT, sh-IL-27, IL-27 forced expression (IL-27 FE)-treated groups. Mice received the rIL-27 agent (100 ng/kg) at the indicated time (n = 5). P values ( vs. λ-carr) were tested by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, DRG: dorsal root ganglion, i.t.: intrathecal injection, WT: wild type, λ-carr: λ-carrageenan.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Knockdown, shRNA, Plasmid Preparation, Injection, Expressing

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27 induces the differentiation of AAM. (A) The picture shows the procedures of this part. (B–D) Quantitative real-time mRNA levels of M2 indicators, including Arg-1 (B), Chi3l3 (C), Retnla (D), when BMDMs were exposed to IL-27 (100 ng/mL for 24 hours) and IL-4 (20 ng/mL for 24 hours) (n = 3). GAPDH was regarded as a reference. The P value was compared with the IL-27-and IL-4-treated group, using two-way ANOVA followed by Tukey’s multiple comparisons test. (E–G) qRT-PCR levels of M1 indicators, including IL-1β, IL-6, TNF-α, when BMDMs were exposed to LPS (20 ng/mL for 24 hours), IL-27 (100 ng/mL for 24 hours), or LPS (20 ng/mL for 24 hours) + IFN-γ (20 ng/mL for 24 hours) (n = 3). (H, I) IL-27 (H) and IL-4 (I) promote the expression of Arg-1 at the protein level in BMDM with different patterns (n = 3). GAPDH was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. (J) IL-27 has no significant impact on the expression of Arg-1 when IL-27 stimulates microglia in vitro , which indicates that IL-27 doesn’t induce the phenotype switch of microglia (n = 3). ACTB was regarded as a reference. The P value was compared with the NC groups, using one-way ANOVA followed by Dunnett post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data are shown as mean ± standard error of the mean. IL: interleukin, LPS: lipopolysaccharide, IFN: interferon, TNF: tumor necrosis factor, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Quantitative RT-PCR, Expressing, In Vitro, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27 distinctly induces the polarization of AAM from IL-4. (A) The picture of the study pipeline in this part. (B, C, E) Bulk RNA-seq comparing the BMDM treated by NC, IL-27 (100 ng/mL for 12 hours), and IL-4 (20 ng/mL for 24 hours). Data containing the heatmap presenting differential gene expression (B), PCA (C), and a Venn plot to show the shared and non-shared DEGs (E). (D) The knockdown effect of the three candidates’ siRNA was targeted at Wsx-1 at the protein level (GAPDH as a reference). (F) The decreased expression of Arg-1 in the si-Wsx-1 group when BMDM was insulted by IL-27, compared to the IL-4-treated and WT group (n = 3). β-actin as internal reference, P values (WT vs. si-Wsx-1 specifically under IL-27 stimulation) were tested by two-way ANOVA with Sidak’s multiple comparisons test. (G) The mRNA of Arg-1 was inhibited in the IL-27-si-Wsx-1 group, compared to the IL-4-treated and WT group (n = 4). GAPDH was regarded as a reference. (H, I) Compared to the WT and NC groups, the expression level of Chi3l3 induced by IL-27 decreased after si-Wsx-1 treatment (H), whereas the IL-4-treated group (I) remained unaffected (n = 4). The P values (si-Wsx-1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test, n = 4, GAPDH as internal reference. *** P < 0.001, **** P < 0.0001. Data are shown as mean ± standard error of the mean. IL: interleukin, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage, PCA: principal component analysis, DEGs: differential expression analysis of genes, WT: wild type.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: RNA Sequencing, Gene Expression, Knockdown, Expressing, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27-Ucp2 signaling pathway mediates AAM. (A) The schematic illustration of this part. GNP: Genipin explicitly inhibits the protein function of Ucp2. (B) ECAR of glycolysis stress test of BMDMs, either NC or treated with IL-27 (100 ng/mL for 6 hours). (C) The Glycolytic capacity was compared with the NC and IL-27-stimulated group (n = 3), and an unpaired, two-tailed t -test was used. (D) OCR of Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours), n = 3. (E) Proton leak measured from Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours) and IL-4 (20 ng/mL for 6 hours), n = 3, one-way ANOVA followed by Dunnett post-test. (F) OCR of BMDM in IL-27 (100 ng/mL for 6 hours) and IL-4 (20 ng/mL for 6 hours)-treated group. (G) The mRNA analysis of Ucp proteins. GAPDH was used as a reference, n = 4, and a unpaired, two-tailed t -test was used. (H) The mRNA level of Ucp2 was dampened in the si-Wsx-1 group when treated with IL-27, GAPDH was used as a reference, and n = 4, one-way ANOVA followed by Tukey’s multiple comparisons test. (I) OCR of BMDMs either NC or treated with IL-27 (100 ng/mL for 6 hours) and GNP (100 mM for 12 hours). (J) Proton leak measured from Mito Stress Test of BMDM either NC or treated with IL-27 (100 ng/mL for 6 hours) and GNP (100 mM for 12 hours), n = 3, one-way ANOVA followed by Tukey’s multiple comparisons test. (K) Compared with the WT group, si-Ucp2 hampered the IL-27-induced Arg-1 expression at the mRNA level. N = 4, The P values (si-Wsx-1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test, n = 4, GAPDH as internal reference. * P < 0.05, ** P < 0.01, **** P < 0.0001, ns: not significant. Data are shown as mean ± standard error of the mean. IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, ECAR: extracellular acidification rate, BMDM: bone marrow-derived macrophage, OCR: oxygen consumption rate, WT: wild type.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Two Tailed Test, Expressing, Derivative Assay

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: IL-27-Ucp2-mediated AAM and a subsequent activation of the transcription factor FoxO3. (A) A diagram of the experimental process of this section. (B) Volcano plot analysis of differentially expressed proteins (DEPs) comparing IL-27 vs. NC, IL-27 vs. si-Ucp2, and IL-27 vs. GNP-treated groups. The marked Acod1 was indicated. (C) IL-27 induced a higher expression of Irg1 in BMDM, n = 3, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (D) Compared to the WT group, IL-27-induced Arg-1 expression was inhibited in the si-Irg1 treated group. GAPDH was used as a reference, n = 3; the P values (si-Irg1 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E) Transcription factor (TF) prediction for both DEGs and DEPs induced by IL-27-stimulated BMDM. (F) The binding motif of FoxO3 predicted by the JASPAR platform. (G) The mRNA level of FoxO3 in BMDM with either NC, IL-27 (100 ng/mL), and IL-4 (20 ng/mL) treatment. N = 4, GAPDH was used as a reference, and an unpaired two-tailed t -test was applied. (H) Compared to the WT group, IL-27-induced FoxO3 expression was inhibited in si-Irg1/si-Ucp2/GNP-treated group. GAPDH was used as a reference, n = 4; two-way ANOVA tested the P values with Tukey’s multiple comparisons test. ** P < 0.01, *** P < 0.001. Data are shown as mean ± standard error of the mean. IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, GNP: Genipin explicitly inhibits the protein function of Ucp2, BMDM: bone marrow-derived macrophage, WT: wild type, DEGs: differential expression analysis of genes.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Activation Assay, Expressing, Two Tailed Test, Binding Assay, Derivative Assay, Quantitative Proteomics

Journal: The Korean Journal of Pain

Article Title: IL-27-Ucp2-FoxO3 axis mediating the polarization of alternatively activated macrophages and ameliorating inflammatory pain

doi: 10.3344/kjp.25307

Figure Lengend Snippet: FoxO3 controls IL-27-induced AAM differentiation and mitigates inflammatory pain in mice. (A) Immunofluorescence (IF) microscopy of FoxO3 in the nucleus of BMDMs upon stimulation with either NC, IL-27 (100 ng/mL for 12 hours), and IL-4 (20 ng/mL for 24 hours). n = 3, The scale bar indicates 1cm, and (B) the P values were tested by a one-way ANOVA with Tukey’s multiple comparisons test. (C) By comparing the knockdown efficiencies of three candidate siRNAs targeting FoxO3, Si#1 was identified as the most effective and will use it in subsequent experiments. GAPDH was used as a reference, n = 3, and the P values ( vs. NC) were tested by a one-way ANOVA with Dunnett’s post hoc test. (D) Compared to the FoxO3 mRNA in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 4, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (E–G) Three M2 markers (Arg-1, Chi3l3, Retnla) were assessed by qPCR (E) and ELISA methods (F, G) in WT and/or si-Fxox3-treated BMDM in response to IL-27 (100 ng/mL for 12 hours) and IL-4 (20 ng/mL for 24 hours) treatment. GAPDH was used as a reference, n = 3, and the P values (si-FoxO3 vs. WT in NC/IL-27/IL-4 treatment) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (H) Flowchart of the experimental process of the adoptive transfer strategy. (I, J) Course of mechanical hyperalgesia in mice with five different intervention groups. (I) The BMDM transfer time was indicated, n = 5, and P values (M vs. MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. (J) The results of IL-27M-IL-27MSF3 groups. N = 5, and P values (IL-27M vs. IL-27MSF3) were tested by two-way ANOVA with Tukey’s multiple comparisons test. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Data are shown as mean ± standard error of the mean. FoxO3: forkhead box class O3, IL: interleukin, Ucp2: uncoupling protein 2, AAM: alternatively activated macrophage, BMDM: bone marrow-derived macrophage, WT: wild type, M: macrophages, MSF3: macrophage + si-FoxO3, IL-27M: IL-27-primed macrophage, IL-27MSF3: IL-27-primed macrophage+si-FoxO3.

Article Snippet: The forced expression of IL-27 in mice was achieved by injecting recombinant mouse IL-27 protein solution (228a.a HEK293, His, HY- P73200 , MCE) at 100 ng/kg via the tail vein injection (i.v.).

Techniques: Immunofluorescence, Microscopy, Knockdown, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Derivative Assay

Journal: bioRxiv

Article Title: Interleukin-27 is antiviral at the maternal-fetal interface

doi: 10.1101/2025.04.28.651113

Figure Lengend Snippet: A. Representative brightfield images of single trophoblast organoid (TO) culture at 1-, 3-, and 6-days post-passage. 5X magnification, scale bar 1000μm (top). Insets denote area captured at 10X magnification, scale bar 200μm (bottom). B. Expression of IL-27 cytokine ( p28, EBI3 ) and receptor ( WSX1, gp130 ) subunit genes by decidual organoids (DOs) and TOs at day 6 post-passage as determined by quantitative RT-PCR, relative to housekeeping gene HPRT1 . Graphs represent 3 experimental replicates from 3 biological donors (circles, squares, and triangles) for TOs and DOs. C. Conditioned media were collected from TO cultures at 1-, 24-, 48-, and 72-hours post-passage and secreted IL-27 was measured via human IL-27 ELISA. Graph represents 3-5 experimental replicates from 3 biological TO donors (circles, squares, and triangles). D. Representative immunofluorescent images of TOs at day 6 post-passage. 10μm TO cross-sections were stained for DAPI (blue), Ki67 for cytotrophoblasts (white), SDC1 for syncytiotrophoblasts (green), and IL27RA (magenta). White box indicates zoom inset and white dashed line demarcates regions of cytotrophoblasts (CTB) and syncytiotrophoblasts (STB). 20X magnification, Z-stack, merged. Scale bar, 100μm. Images representative of 3 biological TO donors. E. TOs were treated with either isotype control, α-IL-27 or α-IFNλ antibodies at days 3 and 5 post-passage. At day 5 post-passage, TOs then were infected with 10 5 PFU Cambodian ZIKV (ZIKV-CAM) or PBS (Mock) for 24 hours. TO cultures were then collected at day 6 post-passage for RNA analysis. ZIKV viral RNA expression in TOs as determined by quantitative RT-PCR relative to housekeeping gene HPRT1 , normalized to isotype control treated organoids. Graph represents 6 independent infections from 1 TO donor. Statistical analysis performed with Kruskal-Wallis ANOVA, *p<0.05, **p<0.01.

Article Snippet: Whole placental tissues, including maternal decidua, were collected and homogenized, and a mouse IL-27 p28 ELISA (R&D Systems, M2728) was then performed according to manufacturer’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining, Control, Infection, RNA Expression

Journal: bioRxiv

Article Title: Interleukin-27 is antiviral at the maternal-fetal interface

doi: 10.1101/2025.04.28.651113

Figure Lengend Snippet: A. Whole placental lysates (placenta and decidua) were collected from uninfected C57BL/6 mice at E9.5, E13.5, and E17.5 of gestation, and IL-27 protein levels were measured via ELISA. Graph represents placentas from 2 litters per timepoint. B. Schematic represents breeding schemes used to delineate maternal and fetal p28 production in the murine placenta and decidua. Left: p28-GFP -/- dam x p28-GFP +/- sire crosses were used to generate placental tissues that lacked (I., p28-GFP Null ) or expressed fetal-derived p28-GFP (II., p28-GFP F ). Right: p28-GFP +/- dam x p28-GFP -/- sire crosses were used to generate placental tissues that retained maternal p28-GFP expression only (III., p28-GFP M ) or expressed both maternal- and fetal-derived p28-GFP (IV., p28-GFP FM ). Graphics were created with Biorender.com. C. Representative immunofluorescent image of p28-GFP F placenta at E13.5 of gestation. Yolk sac (Ys), labyrinth (Lb), junctional zone (Jz), and maternal decidua (Dc) regions were determined via immunofluorescence staining of endothelial cells (CD31; white) and nuclei (DAPI; blue). 20X magnification, stitched. Scale bar, 1000μm. D. Representative immunofluorescent images of p28-GFP within murine deciduas at E13.5. 10μm placental cross-sections were obtained for all conditions in and stained with anti-GFP (green) and DAPI (blue). Images representative of 4 placentas per condition. 20X magnification, zoomed. Scale bar 100μm. E. Quantification of total p28-GFP signal area (microns 2 ) in decidua and labyrinth regions of placentas, as determined by ImageJ software. Graph represents measurements from 2-4 independent placentas per group, with 3-4 cross-sections obtained per placenta, and 4-8 images acquired per cross-section. Statistical analysis performed with Kruskal-Wallis ANOVA, *p<0.05, **p <0.001, ****p<0.0001.

Article Snippet: Whole placental tissues, including maternal decidua, were collected and homogenized, and a mouse IL-27 p28 ELISA (R&D Systems, M2728) was then performed according to manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Derivative Assay, Expressing, Immunofluorescence, Staining, Software

Journal: Cell death & disease

Article Title: Cellular crosstalk mediated by Meteorin-like regulating hepatic stellate cell activation during hepatic fibrosis.

doi: 10.1038/s41419-025-07734-6

Figure Lengend Snippet: Fig. 4 Metrnl regulates HSCs activation in vitro. A The level of Metrnl in the cultured medium, secreted by LX-2 cells overexpressing Metrnl (OE-Met) and knockdown Metrnl (sh-Met) induced by adenovirus, was measured using ELISA (n = 3 per group). Representative Western blot images and quantification analysis of FN, Col4, and α-SMA expression in LX-2 cells transfected with adenovirus-mediated Metrnl knockdown (B), rMetrnl treatment (200 ng/mL) (C), and adenovirus-mediated Metrnl overexpression (D) combined with TGF-β (2 ng/mL) treatment for 48 h (n = 3 blots). E Volcano plot analysis was performed on the RNA sequencing results of the indicated samples. A stringent set of genes (p < 0.05) was identified in Metrnl overexpressing LX-2 cells compared to vector cells treated with TGF-β for 48 h (n = 3 per group). F Heat map of representative, differentially expressed genes associated with matrix remodeling, inflammation, and fibrosis is shown. The analysis includes genes involved in fibrosis-related signaling pathways (vector and OE-Met, both with TGF-β, n = 3 per group). G Changes in relative mRNA levels of genes related to matrix remodeling, inflammation, and fibrosis were observed in Metrnl overexpressing LX-2 cells. These mRNA levels were normalized to that of GAPDH (n = 3 per group). H Relative mRNA levels of genes related to fibrosis in primary HSCs isolated from C57BL/ 6 mice and treated with TGF-β (2 ng/mL) for 48 h. These mRNA levels were normalized to that of GAPDH (n = 3 per group). All data are mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The PDGFB and FN content of the medium supernatant from cells was measured using a commercially available Human PDGFB ELISA Kit (Proteintech; KE100161) and Human Fibronectin/FN1 ELISA Kit (BOSTER; EK0349) individually, following the manufacturer’s instructions.

Techniques: Activation Assay, In Vitro, Cell Culture, Knockdown, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Transfection, Over Expression, RNA Sequencing, Plasmid Preparation, Protein-Protein interactions, Isolation

Journal: Cell death & disease

Article Title: Cellular crosstalk mediated by Meteorin-like regulating hepatic stellate cell activation during hepatic fibrosis.

doi: 10.1038/s41419-025-07734-6

Figure Lengend Snippet: Fig. 7 Metrnl regulates PDGFB release via EGR1 from hepatocytes to activate PDGFRβ signaling pathway. ELISA-quantified PDGFB levels in culture medium (CM) (A) and mRNA expression level (B) of primary hepatocytes transfected with adenovirus-mediated Metrnl overexpression (OE-Met) and primary hepatocytes from Alb-Metrnl−/−mice (n = 3 per group). The mRNA expression (C) and Western blot images (D) and its quantification analysis (down panel) of α-SMA and Col4 in LX-2 cells after incubating CM from LO2 cells transfected with OE-Metrnl virus, and treated with or without PDGFB neutralization antibody (PDGFB-Nabs) or control IgG, which was used to block PDGFB in the CM (n = 3 per group). E A Venn diagram created by the jvenn tool to display potential transcription factors of PDGFB predicted by TF- Target Finder. F A Venn diagram showing the intersection of seven potential transcription factors (CTCF, EGR1, et.al) with RNA-Seq data from Metrnl-overexpressing LX-2 cells. G The mRNA expression of EGR1 in primary hepatocytes from C57BL/6 mice transfected with adenovirus- mediated OE-Metrnl constructs (n = 5 per group). H The mRNA expression of EGR1 in the liver from CCl4-induced mice injected with AAV- vector or AAV-Metrnl virus (n = 6 per group). Representative Western blot images (I) and quantification analysis of EGR1, also the relative mRNA expression (J) in the liver from WT and Metrnl−/−mice induced by CCl4 for 8 weeks (n = 4 per group). ELISA-quantified PDGFB levels in CM (K) and the mRNA expression levels (L) of primary hepatocytes transfected with OE-Metrnl or OE-EGR1 overexpression virus, or both viruses combined (n = 3–4 per group). M RT-qPCR analysis of PDGFB mRNA levels with EGR1 antibody or IgG antibody respectively by ChIP assay in Metrnl-knockdown or control LO2 cells (n = 3 per group). All data are mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

Article Snippet: The PDGFB and FN content of the medium supernatant from cells was measured using a commercially available Human PDGFB ELISA Kit (Proteintech; KE100161) and Human Fibronectin/FN1 ELISA Kit (BOSTER; EK0349) individually, following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Over Expression, Western Blot, Virus, Neutralization, Control, Blocking Assay, RNA Sequencing, Construct, Injection, Plasmid Preparation, Quantitative RT-PCR, Knockdown

Journal: Cell death & disease

Article Title: Cellular crosstalk mediated by Meteorin-like regulating hepatic stellate cell activation during hepatic fibrosis.

doi: 10.1038/s41419-025-07734-6

Figure Lengend Snippet: Fig. 9 Schematic summary of the possible effect of Metrnl on HSC activation during liver fibrosis. Hepatic Metrnl expression was significantly reduced in the fibrotic liver. The deficiency of Metrnl had a dual effect, resulting in the acceleration of HSC activation. Specifically, the absence of Metrnl in hepatocytes promotes the release of fibrogenic cytokine PDGFB through upregulating transcription factor EGR1, which activates PDGFRβ signaling in HSCs through paracrine regulation. Additionally, the absence of Metrnl in hepatocytes and HSCs results in the downregulation of E3 ubiquitin ligase HECW2, inhibiting K48-linked ubiquitination of FN on proteasomal degradation, thus promoting FN secretion from HSCs. These effects contribute to the deposition of extracellular matrix (ECM) and HSCs activation, ultimately exacerbating liver fibrosis. These findings highlight Metrnl as a novel regulator of liver fibrosis that mediates communication between hepatocytes and HSCs. The mechanistic illustration was created with Adobe Illustrator.

Article Snippet: The PDGFB and FN content of the medium supernatant from cells was measured using a commercially available Human PDGFB ELISA Kit (Proteintech; KE100161) and Human Fibronectin/FN1 ELISA Kit (BOSTER; EK0349) individually, following the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Ubiquitin Proteomics