Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: Identification and functional enrichment of differentially expressed genes. (A) A volcano map of differential expression genes showed 120 upregulated and 81 downregulated genes. Red represents upward regulation in tumor tissue, while blue represents downward regulation. PAPSS2, CYP1A1, and ITPKA were labeled in the figure to indicate their expression trends in tumor samples. In addition, genes with prognostic significance and | logFC | greater than 2 were specifically marked. (B) A heat map diagram of differential gene expression in tumors and normal samples. (C) The significant GO terms were depicted as a heat map with color annotation of up-regulated or down-regulated genes. (D) The results of KEGG enrichment analysis were demonstrated in the circle diagram, each trapezoid representing a KEGG pathway, where red and blue dots represented up-regulated and downregulated genes respectively.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Functional Assay, Quantitative Proteomics, Labeling, Expressing, Gene Expression

Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: The expression levels of CYP1A1 (A) , ITPKA (B) and PAPSS2 (C) in tumor and normal cells. (D – F) Immunofluorescence staining of normal thyroid cells and PTC cells to further confirm differential expression.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative Proteomics

Journal: Scientific Reports

Article Title: 3’-phosphoadenosine 5’-phosphosulfate synthetase 2 (PAPSS2) is a potential diagnostic and prognostic biomarker in colon adenocarcinoma

doi: 10.1038/s41598-026-36388-3

Figure Lengend Snippet: Knockdown of PAPSS2 promote COAD cell proliferation, migration and invasion and downregulates p53/p21. ( A ) The level of PAPSS2 expression in 6 colon cancer cell lines. ( B ) PAPSS2 knockdown efficiency in HCT116 and HT-29 cell lines. ( C , D ) CCK-8 and EdU assays to detect proliferative capacity of HCT116 and HT29. ( E ) Effect of PAPSS2 knockdown on cell migration in HCT116 and HT29. ( F ) Effect of PAPSS2 knockdown on cell invasion in HCT116 and HT29. ( G ) PAPSS2 knockdown affected p53 and p21 levels in HCT116 and HT29 cells.

Article Snippet: The membranes were probed overnight at 4 °C with the following primary antibodies: anti-PAPSS2 ( MG467111 , Abmart), anti-CDKN1A(p21) (10355-1-AP, Proteintech), anti-TP53(p53) (A19585, ABclonal), or anti-β-actin (66009-1-Ig, Proteintech).

Techniques: Knockdown, Migration, Expressing, CCK-8 Assay

Journal: Acta Pharmaceutica Sinica. B

Article Title: Sulfation of chondroitin and bile acids converges to antagonize Wnt/ β -catenin signaling and inhibit APC deficiency-induced gut tumorigenesis

doi: 10.1016/j.apsb.2023.12.006

Figure Lengend Snippet: Sensitization of gut tumorigenesis by intestinal Papss2 ablation is accompanied by the activation of Wnt/ β -catenin and suppression of Wnt repressor TLE3. (A, B) Top 20 hallmark pathways (A) and GSEA (B) of RNA-seq data from the intestinal tumor of Apc Δgut -Het and Apc Δgut -Het Papss2 Δgut mice. (C) Relative mRNA expression of Wnt target genes. (D) Protein expression of β -catenin and TLE3 in the ileum by Western blotting. (E) Representative β -catenin immunostaining (green) of tumor sections with the quantifications of the nuclear-to-cytoplasmic ratio of β -catenin fluorescence shown on the right. n = 5, scale bars: 200 μm. (F) Protein expression of β -catenin and TLE3 in the colon of AOM-treated Papss2 fl/fl and Papss2 Δgut mice by Western blotting. (G) Representative β -catenin immunostaining (green) of colonic tumor with the quantifications of the nuclear-to-cytoplasmic ratio of β -catenin fluorescence shown on the right. n = 5, scale bars: 200 μm. (H) Representative TLE3 immunostaining in the tumor (T) or nontumor (N) regions of Apc Δgut -Het and Apc Δgut -Het Papss2 Δgut mice. Scale bars: 100 μm. (I) Representative H&E and IHC staining of TLE3 on human normal colon and different stages of colon cancer tissue array. Shown on the right is the quantifications of TLE3 positive area. Scale bars: 100 μm. (J) Correlations between the immunohistochemical intensity of PAPSS2 and TLE3 in the same cohort of samples shown in (I). (K–M) Correlations between the expression of PAPSS2 and TLE3 (K), Wnt target genes (L) and β -catenin target genes (M) in human COAD patient cohorts from TCGA using Gene Expression Profiling Interactive Analyses. (N) Luciferase reporter gene assay in HCT116 cells transfected with the TCF/TEF-luciferase reporter and treated with sulfation inhibitor NaClO 3 (left panel), or co-transfected with pCDNA-PAPSS2 expression plasmid (right panel). n = 4–5. (O) Protein expression of β -Catenin and TLE3 in the HCT116 cells transfected with pCDNA3.1 vector or pCDNA-PAPSS2. Shown on the right are the quantifications of protein expressions. n = 3–4. Statistical significance between two groups was assessed by two-tailed Student's t -test. One-way ANOVA with Tukey's test was used for multiple comparisons. The gene expression correlation analysis was performed using the Pearson correlation coefficient. Data are presented as the mean ± SEM or box plots. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: The membranes were incubated with primary antibodies against β -actin (Sigma, A1978), β -catenin (Cell Signaling Technology, #8480), chondroitin sulfate (Abcam, ab11570), PAPSS2 (Santa Cruz, sc-100801), and TLE3 (Proteintech, 11372-1-AP) followed by appropriate secondary antibodies.

Techniques: Activation Assay, RNA Sequencing, Expressing, Western Blot, Immunostaining, Fluorescence, Immunohistochemistry, Immunohistochemical staining, Gene Expression, Luciferase, Reporter Gene Assay, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Acta Pharmaceutica Sinica. B

Article Title: Sulfation of chondroitin and bile acids converges to antagonize Wnt/ β -catenin signaling and inhibit APC deficiency-induced gut tumorigenesis

doi: 10.1016/j.apsb.2023.12.006

Figure Lengend Snippet: Apc Δgut -Het Papss2 Δgut mice exhibit disrupted bile acid homeostasis and inhibition of FXR, which may have contributed to Wnt/ β -catenin activation. Apc Δgut -Het and Apc Δgut -Het Papss2 Δgut mice are the same as described in Fig. 2 (A) Serum total bile acids (BAs) levels. (B) Ileal BAs levels. (C) Relative expression of FXR target genes in the ileum. (D, E) 3D PCoA score plot (D) and heatmap (E) of individual ileal BAs profile. (F, G) Relative CA-sulfate and TCA-sulfate levels (F), and parent CA and TCA levels (G) in the ileum measured by UPLC–QTOFMS. n = 7 per group. (H) Luciferase reporter assay in HCT116 cells transfected with the TCF/TEF-luciferase reporter and treatment with FXR agonist Fex (50 μmol/L) or antagonist T- β -MCA (50 μmol/L). n = 4–5. (I) Schematic summary of the effects of PAPSS2 inhibition on chondroitin sulfation and FXR–TLE3 axis, leading to the activation of Wnt/ β -catenin signaling and sensitization of gut tumorigenesis. Statistical significance between two groups was assessed by two-tailed Student's t -test. Data are presented as the mean ± SEM or box plots. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001. The diagram was created with BioRender.

Article Snippet: The membranes were incubated with primary antibodies against β -actin (Sigma, A1978), β -catenin (Cell Signaling Technology, #8480), chondroitin sulfate (Abcam, ab11570), PAPSS2 (Santa Cruz, sc-100801), and TLE3 (Proteintech, 11372-1-AP) followed by appropriate secondary antibodies.

Techniques: Inhibition, Activation Assay, Expressing, Luciferase, Reporter Assay, Transfection, Two Tailed Test

Journal: Cellular and molecular life sciences : CMLS

Article Title: GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion

doi: 10.1007/s00018-022-04223-3

Figure Lengend Snippet: (A) GRASP KO increases the expression of HS synthesis enzymes while decreases CS synthesis enzymes. Results are based on RNA-Seq analysis of each cell line for indicated genes. (B) GRASP KO increases the protein level of HS synthesis enzymes while decreases that of CS synthesis enzymes. Cell lysate of indicated cells were analyzed for three HS synthesis enzymes EXTL3, EXT1 and EXT2, and a key CS synthesis enzyme GalNAcT1. Note the increased level of EXTL3 and decreased GalNAcT1 level in GRASP KO cells. Results are representative of three independent experiments. (C) Quantification of B. (D) GRASP KO decreases the protein level of the HS sulfation enzyme PAPSS2. Shown are representative Western blots of indicated proteins in the four cell lines from three independent experiments. (E) Quantitation of D. (F) Re-expression of GRASP proteins in GRASP KO cells corrects the expression level of HS and CS synthesis enzymes. Indicated cell lines were transfected with GRASP constructs and probed for EXTL3, GalNAcT1, GRASP65, GRASP55, GFP, and actin. The major enzymes EXTL3 and GalNAcT1 in 55KO and 65KO cells were rescued by expressing GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone (lanes 4 & 7 vs. 3 & 6). (G) Re-expression of GRASP proteins corrects the HS and CS defects in GRASP KO cells. Confocal images of WT HeLa cells and GRASP KO cell lines transfected with indicated constructs followed by HS staining. The level of HS in 55KO and 65KO cells was decreased by the expression of GRASP55-GFP or GRASP65-GFP, respectively, but not by GFP alone. Note the different HS signals in cells expressing GRASP55- or GRASP65-GFP (asterisks) vs. non-transfected cells (arrows). (H-I) Quantification of G. Results are presented as mean ± SEM, statistical analysis was assessed by comparing KO cells to WT cells by student’s t-test. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: After electrophoresis and transfer, nitrocellulose membranes were incubated with antibodies to actin (Sigma, A2066), EXT1 (Santa Cruz, sc-515144), EXT2 (Santa Cruz, sc-514092), EXTL3 (Santa Cruz, sc-271986), GalNAcT1 (Novus, NBP1–81852), GFP (Proteintech, 66002–1-Ig), GRASP55 (Proteintech, 10598–1-AP), GRASP65 (Santa Cruz, sc-374423), or PAPSS2 (Santa Cruz, sc-271429) overnight at 4°C.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Quantitation Assay, Transfection, Construct, Staining

Journal: Cellular and molecular life sciences : CMLS

Article Title: GRASP depletion-mediated Golgi fragmentation impairs glycosaminoglycan synthesis, sulfation, and secretion

doi: 10.1007/s00018-022-04223-3

Figure Lengend Snippet: GRASP KO alters HS and CS synthesis enzymes at both the mRNA and protein levels. WT and GRASP KO cells were analyzed by proteomic and RNA-seq analyses. Shown are the fold changes of the indicated GAG synthesis enzymes in GRASP KO cells compared to that in WT cells. Enzymes in bold were further tested by Western blot.

Article Snippet: After electrophoresis and transfer, nitrocellulose membranes were incubated with antibodies to actin (Sigma, A2066), EXT1 (Santa Cruz, sc-515144), EXT2 (Santa Cruz, sc-514092), EXTL3 (Santa Cruz, sc-271986), GalNAcT1 (Novus, NBP1–81852), GFP (Proteintech, 66002–1-Ig), GRASP55 (Proteintech, 10598–1-AP), GRASP65 (Santa Cruz, sc-374423), or PAPSS2 (Santa Cruz, sc-271429) overnight at 4°C.

Techniques: Western Blot

Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: Identification and functional enrichment of differentially expressed genes. (A) A volcano map of differential expression genes showed 120 upregulated and 81 downregulated genes. Red represents upward regulation in tumor tissue, while blue represents downward regulation. PAPSS2, CYP1A1, and ITPKA were labeled in the figure to indicate their expression trends in tumor samples. In addition, genes with prognostic significance and | logFC | greater than 2 were specifically marked. (B) A heat map diagram of differential gene expression in tumors and normal samples. (C) The significant GO terms were depicted as a heat map with color annotation of up-regulated or down-regulated genes. (D) The results of KEGG enrichment analysis were demonstrated in the circle diagram, each trapezoid representing a KEGG pathway, where red and blue dots represented up-regulated and downregulated genes respectively.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Functional Assay, Expressing, Labeling

Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: The expression levels of CYP1A1 (A) , ITPKA (B) and PAPSS2 (C) in tumor and normal cells. (D – F) Immunofluorescence staining of normal thyroid cells and PTC cells to further confirm differential expression.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: Identification and functional enrichment of differentially expressed genes. (A) A volcano map of differential expression genes showed 120 upregulated and 81 downregulated genes. Red represents upward regulation in tumor tissue, while blue represents downward regulation. PAPSS2, CYP1A1, and ITPKA were labeled in the figure to indicate their expression trends in tumor samples. In addition, genes with prognostic significance and | logFC | greater than 2 were specifically marked. (B) A heat map diagram of differential gene expression in tumors and normal samples. (C) The significant GO terms were depicted as a heat map with color annotation of up-regulated or down-regulated genes. (D) The results of KEGG enrichment analysis were demonstrated in the circle diagram, each trapezoid representing a KEGG pathway, where red and blue dots represented up-regulated and downregulated genes respectively.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Functional Assay, Quantitative Proteomics, Labeling, Expressing, Gene Expression

Journal: Frontiers in Genetics

Article Title: A metabolism-related gene signature for predicting the prognosis in thyroid carcinoma

doi: 10.3389/fgene.2022.972950

Figure Lengend Snippet: The expression levels of CYP1A1 (A) , ITPKA (B) and PAPSS2 (C) in tumor and normal cells. (D – F) Immunofluorescence staining of normal thyroid cells and PTC cells to further confirm differential expression.

Article Snippet: Cells were incubated overnight with primary antibodies at 4°C and secondary antibody (GB21303, 1:300, Servicebio, China) for 1 h. Here are the primary antibodies we used: CYP1A1 (PB9544, 1:200, Boster, China), ITPKA (14270-1-AP, 1:100, Proteintech, China), PAPSS2 (CSB-PA527262LA01HU, 1:100, Cusabio, China).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative Proteomics