Fig 1: CSGALNACT2 is required for the suppression of cell mobility, metastasis, and clonogenic growth in ovarian cancer cells. A Protein and mRNA expressions of CSGALNACT2 in primary and metastatic cell lines of ovarian cancer patients were analyzed by Western Blotting and qRT-PCR. The expression of β-actin is an internal loading control. B Knockdown efficiencies of CSGALNACT2 in HEY and OVCAR8 cells were analyzed by Western blotting and qRT-PCR. shCtrl: cells infected with scrambled shRNA virus; shCSGALNACT2: cells infected with shCSGALNACT2 virus. C Wound-healing assays after CSGALNACT2 knockdown by shRNA in OVCAR8 and HEY cells. Scale bar = 100 μm. D-E Effects of CSGALNACT2 inhibition on cancer cell migration and invasion for OVCAR8 and HEY cells were analyzed; scale bar = 50 μm. And quantification of migrated and invaded cells of CSGALNACT2-silenced cells was presented as mean ± S.D. F Protein and mRNA expression of CSGALNACT2 in CSGALNACT2-overexpressed HEY and OVCAR8 cells was examined by Western blotting and qRT-PCR. G Wound-healing assays after CSGALNACT2 overexpression in OVCAR8 and HEY cells. Scale bar = 100 μm. H The transwell migration and invasion assays of CSGALNACT2 overexpression in OVCAR8 and HEY cells were determined and quantified. Results presented as the mean ± S.D. Scale bar = 50 μm. I-J CCK-8 assays were used to detect changes in cell proliferation after overexpression or knockdown of CSGALNACT2 in OVCAR8 and HEY cells. K-L The expression of proliferation marker CDC25A in cell proliferation after overexpression or knockdown of CSGALNACT2 in OVCAR8 and HEY cells by qRT-PCR. M–N Colony formation assays were used to detect the proliferation ability of cells after overexpression or knockdown of CSGALNACT2 in OVCAR8 and HEY cells. O CSGALNACT2 overexpression and knockdown efficiency validation in A2780 cells by Western blotting and qRT-PCR. P Transwell assays after CSGALNACT2 knockdown in A2780 cells. Scale bar = 50 μm. Q Colony formation assays were used to detect the proliferation ability of cells after the knockdown of CSGALNACT2. R-S Transwell and colony formation assays were used to detect the migration, invasion, and proliferation ability of A2780 cells after overexpression or knockdown of CSGALNACT2. Scale bar = 50 μm. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; no asterisks, P not calculated
Fig 2: CSGALNACT2 suppresses ovarian cancer migration and invasion via DUSP1 modulation of MAPK/ERK pathway. A Volcano plot of the distribution of differentially expressed genes (DEGs) by RNA-seq in HEY cell lines with stable overexpression of CSGALNACT2. Red and blue dots represented statistically significant up- and down-regulated CSGALNACT2, respectively. B Heatmap showed the 20 DEGs with the most significant up- and down-regulation in the upregulated CSGALNACT2 group compared with the control group. C-D qRT-PCR analyzed the expression of core genes, such as RASSF6, DUSP1, TXNIP in HEY cells C and OVCAR8 cells D with up- and down-regulated CSGALNACT2. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not statistically. E The correlation of GO biological process, molecular function, and CSGALNACT2 was analyzed by GSEA via RNA-Seq data. F Reactome analysis of the relationship between various reactions and biological pathways and CSGALNACT2 expression via RNA-Seq data. G-H The correlation of KEGG pathways and CSGALNACT2 was analyzed by GSEA via RNA-Seq data. I-J MAPK signaling pathway related proteins (p-Erk1/2, Erk1/2) were detected by western blotting in HEY, OVCAR8 and A2780 cells
Fig 3: Correlation analysis of CSGALNACT2 with immunotherapy and immune cell infiltration. A Kaplan–Meier curves were plotted based on the different groups of CSGALNACT2/CD8 + T cell enriched or decreased, CSGALNACT2/CD4 + memory T cell enriched or decreased, B CSGALNACT2/Regulatory T cell enriched or decreased C in TCGA cohort. D Survival analyses for high (174 cases) and low (174 cases) CSGALNACT2 expression patient groups in the anti-PD-L1 immunotherapy cohort using Kaplan–Meier curves (IMvigor210 cohort). E Differences in CSGALNACT2 among distinct tumor immune phenotypes in the IMvigor210 cohort. The lines in the boxes represented the median value by the Kruskal–Wallis test. F The number of patients with response to PD-L1 blockade immunotherapy in high or low CSGALNACT2 expression groups. SD, stable disease; PD, progressive disease; CR, complete response; PR, partial response. G The distribution of CSGALNACT2 in different anti-PD-L1 clinical response groups. H The correlation of CSGALNACT2 with immunotherapy response, Wilcoxon rank sum was applied for the significance test
Fig 4: CSGALNACT2 was down-regulated and associated with a better prognosis in ovarian cancer. A The expression of CSGALNACT2 in TCGA-OV from the UALCAN database (https://ualcan.path.uab.edu/). B The protein level of CSGALNACT2 in CPTAC from the UALCAN database. C CSGALNACT2 gene expression level from GEPIA2.0 (http://gepia2.cancer-pku). It includes 426 TCGA-OV samples and 88 GTEx normal samples. D RNA-seq was used to detect relative expressions of CSGALNACT2 in 10 normal and 39 tumor tissues of ovarian cancer. E qRT-PCR was performed to detect relative levels of CSGALNACT2 in normal and tumor tissues (n = 16) of ovarian cancer. F The protein level of CSGALNACT2 in normal ovarian epithelial tissues, ovarian cancer, and ovarian cancer metastatic tissues detected by immunohistochemical (IHC) staining. All micrographs (× 40; scale bar, 50 mm) were imaged from one representative case. G IHC scores between normal ovarian epithelial tissues, ovarian cancer, and ovarian cancer metastatic tissues. H Kaplan–Meier analysis of CSGALNACT2 on progression-free-survival (PFS) of ovarian cancer patients with Paclitaxel treatment from Kaplan–Meier plotter (https://kmplot.com/). *p ≤ 0.05, ***p ≤ 0.001
Fig 5: CSGALNACT2 inhibited the migration, invasion, and clonogenicity of normal Ovarian epithelial cell lines. A-B CSGALNACT2 overexpression and knockdown efficiency validation in IOSE cells by Western blotting and qRT-PCR. C-D Transwell assays detected the migration and invasion ability of cells after the knockdown of CSGALNACT2. Scale bar = 50 μm. E–F Stable knockdown of CSGALNACT2 promoted clonogenic growth in IOSE cell lines. G Transwell assays detected the migration and invasion ability of cells after overexpression of CSGALNACT2. Scale bar = 50 μm. H Colony formation assays were used to detect the proliferation ability of cells after overexpression of CSGALNACT2. I-J CCK-8 assays and the CDC25A expression were used to detect changes in cell proliferation after overexpression or knockdown of CSGALNACT2 in IOSE cells. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001; ns, not statistically
Supplier Page from Abcam for Anti-CSGALNACT2 antibody [EPR13670]