Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C mRNA expression is altered in different human cancers. ( A ) Oncomine database analysis results of SEMA4C mRNA levels in the tumor and normal tissues in human cancers. Note: Red and blue denote upregulation and downregulation of SEMA4C in the tumor tissues, respectively. ( B ) Interactive human body map constructed using the GEPIA webserver shows the status of SEMA4C mRNA expression in the tumor (red) and normal (green) tissue samples from different cancer types based on the TCGA data.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing, Construct

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C mRNA and protein levels are upregulated in the CRC tissues. ( A ) The relative SEMA4C mRNA expression level in normal colorectal and CRC tissues from the TCGA-COAD and TCGA-READ datasets. ( B ) Representative micrographs show SEMA4C immunohistochemical staining of the 83 pairs of colon cancer and adjacent normal colon tissue samples in the tissue microarray (TMA). Note: Scale bars=200 μm; red arrow indicates strong positive SEMA4C protein expression in the colon cancer tissue; blue arrow indicates negative SEMA4C protein expression in the adjacent normal tissue. ( C ) Quantitative analysis of SEMA4C protein expression scores based on the immunohistochemical staining of the TMA with 83 pairs of colon carcinoma and adjacent normal colon tissue samples.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing, Immunohistochemical staining, Staining, Microarray

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: The association between SEMA4C mRNA levels and the clinicopathological variables in the TCGA-CRC dataset.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques:

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: The association between SEMA4C protein levels and the clinicopathological variables in the TMA cohort of colon cancer patients.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques:

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: High SEMA4C expression correlates with poor survival outcomes in CRC patients. ( A , B ) Kaplan-Meier survival curves show OS and PFS of high- and low-SEMA4C expressing CRC patients from the TCGA database. ( C , D ) Kaplan-Meier survival curves show the OS of high- and low-SEMA4C expressing CRC patients belonging to the metastatic and non-metastatic subgroups. ( E , F ) Kaplan-Meier survival curves show OS and TFS of high- and low-SEMA4C expressing CRC patients from the TMA-colon cancer cohort.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of the prognostic significance of SEMA4C mRNA expression and other clinicopathogical parameters in TCGA-CRC patients.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: Univariate and multivariate Cox regression analysis of the prognostic significance of SEMA4C protein expression and other clinicopathological parameters in the TMA cohort of colon cancer patients.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: Functional enrichment analysis of GO terms and KEGG pathways related to SEMA4C and 50 SEMA4C-associated genes in CRC tissues. ( A , B ) The GO terms for the enriched ( A ) biological functions, and ( B ) cellular components related to SEMA4C and 50 SEMA4C-associated genes in the CRC tissues are shown. ( C ) The enriched KEGG pathways related to SEMA4C and 50 SEMA4C-associated genes in the CRC tissues are shown.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Functional Assay

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: GSEA enrichment plots of the TCGA-CRC dataset. GSEA results show the enrichment of gene sets related to epithelial mesenchymal transition (EMT), hedgehog signaling, Wnt/β-catenin signaling, angiogenesis, TGF-β signaling, and notch signaling in CRC patients with high SEMA4C expression. Note: NES, normalized enrichment score.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C expression correlates with the expression of EMT-related genes in CRC patients. Spearman’s correlation analysis results show the association between SEMA4C levels and the expression of EMT-related genes, including N-cadherin, β-catenin, Fibronectin1, Laminin5, COL3A1, COL4A1, CTGF, TGFβ1, ZEB1, ZEB2, SNAIL1, and SNAIL2 in the CRC tissues using the GEPIA database. Note: TPM, transcripts per million; R denotes the Spearman correlation coefficient.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C overexpression correlates with the CMS4 subtype of CRC. ( A ) Box-scatter plot shows the SEMA4C mRNA level in CMS1, CMS2, CMS3 and CMS4 subtypes of CRC patient tissues in the TCGA dataset. ( B ) ROC curve analysis shows that SEMA4C expression levels accurately discriminate between CMS4 and non-CMS4 molecular subtypes of the TCGA-CRC dataset. Note: AUC: area under receiver operating characteristic, CI: confidence interval. ( C , D ) Kaplan-Meier survival curves show the OS in the high- and low- SEMA4C expressing ( C ) CMS1-3 and ( D ) CMS4 subgroups of TCGA-CRC patients. Log-rank test was used to determine the statistical differences between the groups.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Over Expression, Expressing

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C expression negatively correlates with the DNA methylation status of the SEM4AC gene in the CRC patients. ( A ) Spearman’s correlation analysis shows the relationship between DNA methylation status of the SEMA4C gene and the expression levels of SEMA4C mRNA in the CRC samples. Note: R denotes the Spearman’s correlation coefficient. ( B ) Boxplots show the methylation levels of the CpG island shore, cg07166409 (left) and cg00605777 (right) in the SEMA4C gene in CRC patients with low- and high-expression of SEMA4C. ( C ) Kaplan-Meier survival curves show the OS of TCGA-CRC patients with hypomethylation (blue line) and hypermethylation (red line) of the SEMA4C gene in the CpG island shore, cg07166409 (above) and cg00605777 (below).

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing, DNA Methylation Assay, Methylation

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C silencing decreases proliferation, migration, and invasion of the LoVo cells. ( A ) QRT-PCR analysis shows the SEMA4C mRNA levels in the NC-siRNA- or SEMA4C-siRNA-transfected LoVo cells (human colon adenocarcinoma cell line). ( B ) CCK-8 assay results show the viability of control and SEMA4C-silenced LoVo cells. ( C ) Wound-healing assay results show the migration rates of control and SEMA4C-silenced LoVo cells. Note: Scale bars=100 μm. ( D ) Transwell invasion assay results show the invasiveness of control and SEMA4C-silenced LoVo cells. Note: Scale bars=10 μm. H indicates hour. ***, P < 0.001; **, P < 0.01; *, P < 0.05; ns, not significant.

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Migration, Quantitative RT-PCR, Transfection, CCK-8 Assay, Control, Wound Healing Assay, Transwell Invasion Assay

Journal: Aging (Albany NY)

Article Title: High SEMA4C expression promotes the epithelial-mesenchymal transition and predicts poor prognosis in colorectal carcinoma

doi: 10.18632/aging.104038

Figure Lengend Snippet: SEMA4C silencing decreases the expression of EMT markers in the LoVo cells. ( A ) QRT-PCR analysis shows the relative mRNA expression of EMT-related genes in the control and SEMA4C-silenced LoVo cells. ***, P < 0.001; **, P < 0.01; *, P < 0.05. ( B , C ) Representative immunofluorescence images (Scale bars=25 μm) show the levels of E-cadherin (red) ( B ) and Vimentin (green) ( C ) proteins in the control and SEMA4C-silenced LoVo cells. The nuclei are stained with Hoechst (blue).

Article Snippet: The specimens were then blocked in 10% rabbit serum for 30 min and incubated overnight with the sheep anti-human primary SEMA4C antibody (#AF6125, R & D Systems, MN, USA) at 4 o C. Then, we incubated the specimens with rabbit anti-goat horseradish peroxidase (HRP)-conjugated secondary antibody (#305-035-003, Jackson ImmunoResearch, PA, USA) for 30 min at 37 o C. Then, the specimens were incubated with 3, 3’-diaminobenzidine (DAB) and counter-stained with hematoxylin.

Techniques: Expressing, Quantitative RT-PCR, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) qPCR of Pla2g12a in various tissues of 10-week-old C57BL/6 mice. (B) Procedure used for flow cytometry of CD4 + -gated TCRβ + IL-17A + Th17 cells from ex vivo Th17 differentiation culture. (C) qPCR of various PLA 2 enzymes in splenic CD4 + T cells cultured for 3 days under Th0 and Th17 differentiation conditions ( n = 3). (D) Strategy for gene targeting of Pla2g12a . Positions of PCR primers for genotyping are indicated by arrows. (E) PCR genotyping of Pla2g12a +/+ (+/+), Pla2g12a +/– (+/–), and Pla2g12a -/- (–/–) mice. (F) qPCR of Pla2g12a in the skin and spleen of Pla2g12a +/+ and Pla2g12a -/- mice. Values are mean ± s.e.m.. Representative data of two experiments (C) and combined results of two experiments (A, F) are shown. Statistical analysis was performed by Mann-Whitney U test (B, F, G). **, P < 0.01.

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Flow Cytometry, Ex Vivo, Cell Culture, MANN-WHITNEY

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for ex vivo culture of splenic naïve CD4 + T cells with (Th17) or without (Th0) IL-1β, IL-6, IL-23 and TGF-β on anti-CD3ε/CD28-coated plates. (B) qPCR of Pla2g12a in splenic CD4 + T cells cultured for various periods under Th0 and Th17 differentiation conditions. (C) FACS of Th17 (CD4 + -gated TCRβ + IL-17A + ) cells after culture of naïve T cells prepared from Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice for 3 days under Th0 and Th17 differentiation conditions. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (D-F) Scatter plots (D) and heatmap (E) of genes expressed in CD4 + T cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days under Th0 and Th17 differentiation conditions. Cells from 6-7 mice of each genotype were pooled and subjected to microarray analysis. Colors in (E) show the z-score. (F) Pathway enrichment analysis of genes decreased (>2-fold) in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. (G) Heatmap of Th17-related genes in Th17 cells from Pla2g12a -/- mice relative to those from Pla2g12a +/+ mice. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (H) qPCR of Th17 signature genes in Th0 and Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice. (I) Heatmap of lipid-related genes in Pla2g12a -/- Th17 cells relative to Pla2g12a +/+ Th17 cells. Colors indicate fold changes in Pla2g12a -/- cells relative to Pla2g12a +/+ cells. (J) FACS of non-pathogenic Th17 cells from Pla2g12a +/+ and Pla2g12a -/- mice after culture for 3 days with IL-6 and TGF-β. Values are mean ± s.e.m.. Representative data of three experiments (C) and results of one experiment (B, D–I) are shown. Statistical analysis was performed by one way ANOVA with Tukey’s multiple comparisons test (C, J), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H), and two-way ANOVA with Sidak’s multiple comparisons test (B). *, P < 0.05; **, P < 0.01.

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Ex Vivo, Cell Culture, Microarray

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for IMQ-induced psoriasis. (B) qPCR of Pla2g12a in the LNs of WT mice after IMQ challenge over 3 days. (C) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice over 6 days ( n = 6–18). (D, E) HE staining of ear skin sections (D) and quantification of epidermal and dermal thickness (E) in Pla2g12a +/+ and Pla2g12a -/- mice ( n = 35–44) (E). Scale bar, 100 μm. (F) qPCR of Il17a in the LNs and skin of Pla2g12a +/+ and Pla2g12a -/- mice. (G) FACS of Th17 (CD3ε + -gated TCRβ + IL-17A + ) cells in the spleen of Pla2g12a +/+ and Pla2g12a -/- mice on day 6. Representative FACS profiles ( left ) and the proportion and number of Th17 cells ( right ) are shown. (H, I) FACS of Th17 (CD45 + -gated TCRβ + IL-17A + ) cells (H) and γδ T (CD45 + -gated TCRγδ + IL-17A + ) cells (I) in the skin on day 6. Representative FACS profiles ( left ) and the proportions and numbers of Th17 cells (H) and γδ T cells (I) ( right ) are shown. Values are mean ± s.e.m.. Representative data of two (C, F) or four (G) experiments, results from one experiment (H, I), and combined results of two experiments (B, D, E) are shown. Statistical analysis was performed using ordinary one-way ANOVA with Dunnett’s multiple comparisons test (B), one-way ANOVA with Tukey’s multiple comparisons test (G), Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test (H, I), Mixed-effects model with Sidak’s multiple comparisons test (C), and two-way ANOVA with Sidak’s multiple comparisons test (E, F). *, P < 0.05; **, P < 0.01.

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Staining

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A-C) qPCR of cytokines (A, B) and keratinocyte markers (C) in the LNs (A) and skin (B, C) of Pla2g12a +/+ and Pla2g12a -/- mice. (D, E) Procedures used for flow cytometry of CD3ε + -gated TCRβ + IL-17A + Th17 cells from the spleen (D) and CD45 + -gated TCRβ + IL-17A + Th17 cells and TCRγδ + IL-17A + γδ T cells from the skin (E). Values are mean ± s.e.m.. Results of one experiment (A, B) and combined results of two experiments (C) are shown. Statistical analysis was performed using two-way ANOVA with Sidak’s multiple comparisons test (A–C). *, P < 0.05; **, P < 0.01.

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure for IMQ-induced psoriasis. (B, C) Hindlimb swelling ( n = 8–18) (B) and clinical score (n = 4–9) (C) in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice over 42 days after immunization with type II collagen. (D) Representative photos of the hindlimbs of Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. (E) HE and TRAP staining of the knee joints from Pla2g12a +/+ and Pla2g12a -/- mice with (+) or without (–) CIA on day 42. Yellow arrowheads, bone erosion; white arrowheads, inflammatory cell infiltration. Scale bar, 100 µm. (F, G) μCT analysis of the hindlimb bone from Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Yellow arrowheads, bone erosion; white arrowheads, cortical bone; asterisk, trabecular bone. Scale bar, 1 mm. Representative μCT images (F) and tissue mineral density of the cortical bone (G) are shown. (H) FACS of splenic Th17 cells in Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Representative FACS profiles ( left ) and the proportion of Th17 cells ( right ) are shown. (I, J) ELISA of IL-17A (I) and anti-type II collagen (J) in sera of Pla2g12a +/+ and Pla2g12a -/- mice with or without CIA on day 42. Values are mean ± s.e.m.. Representative data of three (B, C, G) or two (H) experiments and combined results of two experiments (I, J) are shown. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test (G, H), Kruskal-Wallis with Dunn’s multiple comparisons test (I, J), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, C). *, P < 0.05; **, P < 0.01.

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Secreted phospholipase PLA2G12A-driven lysophospholipid signaling via lipolytic modification of extracellular vesicles facilitates pathogenic Th17 differentiation

doi: 10.1101/2024.10.27.620543

Figure Lengend Snippet: (A) Schematic representation of the procedure used for antibody treatment in IMQ-induced psoriasis. (B) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A, -IL-17A, or control IgG 1 antibody over 6 days. Antibody treatment started on day 0 (arrow). (C) FACS of splenic Th17 cells in (B) on day 6. (D) IMQ-induced ear swelling in Pla2g12a +/+ (+/+) and Pla2g12a -/- (–/–) mice treated with anti-PLA2G12A or control antibody over 6 days. Antibody treatment started on day 0 (arrow). (E) FACS of splenic Th17 cells in (D) on day 6. (F) IMQ-induced ear swelling in WT mice treated with anti-PLA2G12A or control antibody. Antibody treatment started on day 3 (arrow). (G) FACS of splenic Th17 cells in (F) on day 6. (H) Schematic representation of the procedure used for antibody treatment in CIA. (I, J) Paw thickness (I) and clinical sore (J) in WT mice treated with anti-PLA2G12A, - IL-17A, or control antibody over 42 days. (K) FACS of splenic Th17 cells in (I, J) on day 42. (L) Schematic diagram showing PLA2G12A-driven regulation of Th17 differentiation. PLA2G12A is induced in CD4 + T cells after TCR stimulation and is also supplied from stromal fibroblasts. EV secretion is facilitated by Th17 differentiation driven by Th17-inducing cytokines. PLA2G12A hydrolyzes PE and PC to generate LPE and LPC in EV membranes. The EVs are taken up by Th17-differentiating cells and deliver LPE(1-18:1) intracellularly, which then transactivates RORγt. LPE and LPC are converted to LPA by ATX, which is supplied from Th17 cells, stromal cells ( e.g ., fibroblasts and endothelial cells), and serum. LPA acts mainly on LPA 2 (and to a lesser extent LPA 1 ) on Th17-differentiating cells to augment IL-6/IL23-mediated STAT3 phosphorylation via G 12/13 -Rho-ROCK2 signaling, assisting pathogenic Th17 differentiation in autocrine and paracrine fashions. Gene disruption of PLA2G12A dampens this lipid-orchestrated amplification loop and decreases EV secretion, uptake, and transfer of lipid, miRNA, and protein cargos. Accordingly, antibody-mediated neutralization of PLA2G12A efficiently attenuates psoriasis, arthritis, and probably other Th17-related pathologies such as EAE. Values are mean ± s.e.m.. Results are from one experiment (B–G, I–K). Statistical analysis was performed using unpaired t test (G), ordinary one-way ANOVA with Dunnett’s multiple comparisons test (C, E, K), and two-way repeated measures ANOVA with Sidak’s multiple comparisons test (B, D, F, I, J). *, P < 0.05; **, P < 0.01 versus control IgG 1 .

Article Snippet: The level of IL-17A in mouse serum was determined using a Mouse IL-17A ELISA Kit (Proteintech).

Techniques: Control, Disruption, Amplification, Neutralization

Journal: International Journal of Molecular Medicine

Article Title: Rhodiola rosea suppresses thymus T-lymphocyte apoptosis by downregulating tumor necrosis factor-α-induced protein 8-like-2 in septic rats

doi: 10.3892/ijmm.2015.2241

Figure Lengend Snippet: Primer sequences used for using reverse transcription polymerase chain reaction to validate the microarray analysis.

Article Snippet: The primary antibodies used included rabbit anti-TIPE2 monoclonal antibody (1:200), rabbit anti-Fas monoclonal antibody (1:200), rabbit anti-FasL monoclonal antibody (1:200), mouse anti-Bcl-2 monoclonal antibody (1:200) and were purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Reverse Transcription, Polymerase Chain Reaction, Microarray, Sequencing

Journal: International Journal of Molecular Medicine

Article Title: Rhodiola rosea suppresses thymus T-lymphocyte apoptosis by downregulating tumor necrosis factor-α-induced protein 8-like-2 in septic rats

doi: 10.3892/ijmm.2015.2241

Figure Lengend Snippet: Effect of TIPE2 on the expression of Bcl-2, Fas and FasL in thymus T cells from septic mice. Groups of mice were induced by caecal ligation and puncture for 24 h. Representative western blot showing the levels of Bcl-2, Fas and FasL protein expression in thymus T cells in septic mice. Control group (non-septic group): (A) TIPE2-deficient mice; (B) wild-type mice; (C) TIPE2-deficient mice; (D) wild-type mice. TIPE2, tumor necrosis factor-α-inducible protein 8-like 2; Bcl-2, B-cell lymphoma 2; FasL, Fas ligand.

Article Snippet: The primary antibodies used included rabbit anti-TIPE2 monoclonal antibody (1:200), rabbit anti-Fas monoclonal antibody (1:200), rabbit anti-FasL monoclonal antibody (1:200), mouse anti-Bcl-2 monoclonal antibody (1:200) and were purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Expressing, Ligation, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Rhodiola rosea suppresses thymus T-lymphocyte apoptosis by downregulating tumor necrosis factor-α-induced protein 8-like-2 in septic rats

doi: 10.3892/ijmm.2015.2241

Figure Lengend Snippet: TIPE2 increases the thymus T-lymphocyte apoptosis rate in septic mice. Groups of mice were challenged by caecal ligation and puncture. 24 h later, the thymus T-lymphocyte apoptosis rate was measured using flow cytometry followed by quantitative evaluation. Values are expressed as the mean ± standard error of the mean of three experiments. * P<0.05 vs. wild-type mice. TIPE2, tumor necrosis factor-α-inducible protein 8-like 2.

Article Snippet: The primary antibodies used included rabbit anti-TIPE2 monoclonal antibody (1:200), rabbit anti-Fas monoclonal antibody (1:200), rabbit anti-FasL monoclonal antibody (1:200), mouse anti-Bcl-2 monoclonal antibody (1:200) and were purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Ligation, Flow Cytometry

Journal: International Journal of Molecular Medicine

Article Title: Rhodiola rosea suppresses thymus T-lymphocyte apoptosis by downregulating tumor necrosis factor-α-induced protein 8-like-2 in septic rats

doi: 10.3892/ijmm.2015.2241

Figure Lengend Snippet: Effect of Rhodiola rosea extract on the expression of TIPE2, Bcl-2, Fas and FasL mRNA in the thymus of septic mice. Groups of mice were induced by caecal ligation and puncture for 24 h. Representative gels of reverse transcription polymerase chain reaction products showing the levels of TIPE2, Bcl-2, Fas and FasL mRNA expression in rats. (A) Normal control group; (B) sham operation group; (C) control group; (D) treatment group. TIPE2, tumor necrosis factor-α-inducible protein 8-like 2; Bcl-2, B-cell lymphoma 2; FasL, Fas ligand.

Article Snippet: The primary antibodies used included rabbit anti-TIPE2 monoclonal antibody (1:200), rabbit anti-Fas monoclonal antibody (1:200), rabbit anti-FasL monoclonal antibody (1:200), mouse anti-Bcl-2 monoclonal antibody (1:200) and were purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Expressing, Ligation, Reverse Transcription, Polymerase Chain Reaction, Control

Journal: International Journal of Molecular Medicine

Article Title: Rhodiola rosea suppresses thymus T-lymphocyte apoptosis by downregulating tumor necrosis factor-α-induced protein 8-like-2 in septic rats

doi: 10.3892/ijmm.2015.2241

Figure Lengend Snippet: Rhodiola rosea extract suppresses the expression of TIPE2, Bcl-2, Fas and FasL in thymus T cells from septic mice. Groups of mice were treated with Rhodiola rosea extract and 8 h later challenged by caecal ligation and puncture for 24 h. The expression of TIPE2, Bcl-2, Fas and FasL protein was assessed using western blot analysis and quantified by densitometric analysis. Values are expressed as the mean ± standard deviation of three experiments. * P<0.05, P<0.01 vs. sham-operated group and normal control group; # P<0.05 vs. control group. TIPE2, tumor necrosis factor-α-inducible protein 8-like 2; Bcl-2, B-cell lymphoma 2; FasL, Fas ligand.

Article Snippet: The primary antibodies used included rabbit anti-TIPE2 monoclonal antibody (1:200), rabbit anti-Fas monoclonal antibody (1:200), rabbit anti-FasL monoclonal antibody (1:200), mouse anti-Bcl-2 monoclonal antibody (1:200) and were purchased from Wuhan Boster Biological Technology, Ltd.

Techniques: Expressing, Ligation, Western Blot, Standard Deviation, Control