Fig 1: MyoD1 inhibits FUT4 transcription by binding its promoter.a Relative expression levels of MyoD1 and FUT4 in LV-MyoD1-BGC-823 and LV-MyoD1-MKN-45 cells vs. control cells and in SGC-7901 cells in which MyoD1 was silenced MyoD1 expression was measured by qRT-PCR and western blotting. b Significant high expression of FUT4 mRNA in 415 GC vs. 35 normal gastric tissues according to the TCGA database (p < 0.001). c Higher expression of FUT4 in GC than in normal gastric tissues detected by IHC and according to the Human Protein Atlas. d Prediction of the promoter region of FUT4 and design of three pairs of primers (P1–P3) targeting fragments 1–3 (F1–F3) that covered full-length MBS of FUT4. e ChIP-qPCR of P1–P3 performed with anti-MyoD1 antibody. f Design of luciferase reporter for further validation of the direct binding by MyoD1 on MBS of FUT4 gene. Specific motifs in sequences are marked by yellow rectangles. g Luciferase activities for sequence 1, 2, and 3 were compared between pCMV2-GV146-MYOD1-transfected cells and pCMV2-GV146-transfected cells. The significant differences were analyzed according to t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s. not significant).
Fig 2: Knockdown of FUT4 suppresses GC Cell migration and invasion.a qRT-PCR analysis of FUT4 in BGC-823 cells transfected with NC-siRNA, FUT4 siRNA-1, and FUT4 siRNA-2. b The effects of knockdown of FUT4 on BGC-823 cell migration were determined by wound-healing assay at 0, 12, 24, 36, and 48 h. c The effects of knockdown of FUT4 on BGC-823 cell migration (up) and invasion (down) were determined by Transwell assay without (null) or with Matrigel. d Western blotting analysis of the levels of MMP-2, MMP-9, vimentin, and N-cadherin in knockdown of FUT4 on BGC-823 cells compared to the control. The significant differences were analyzed according to t-test (***p < 0.001).
Fig 3: MyoD1 overexpression alters UEA-I glycopattern binding in GC cells.a FUT4 was mainly expressed in intracellular vesicles according to the Human Protein Atlas. b The glycopatterns of total glycoproteins from control and LV-MyoD1-MKN-45 cells defined by lectin microarrays. Fluorescent images were scanned at 70% photomultiplier tube and 100% laser power settings with a Genepix 4000B confocal scanner. A portion of the slide with three replicates of the lectin array is shown. The microarrays revealed increased normalized fluorescent intensities (NFIs, marked with red frames) and decreased NFIs (white frames) when LV-MyoD1-MKN-45 was compared with control MKN-45 cells. c NFIs and recognized glycans of lectins showed increased and decreased signals in LV-MyoD1-MKN-45 vs. control MKN-45 cells from lectin microarray (ratio >1.5 or < 0.67); the significant differences were analyzed according to t test (*p < 0.05, **p < 0.01, ***p < 0.001). d Lectin immunofluorescence approach to validate and investigate lectin binding sites in controls and LV-MyoD1-BGC-823 and LV-MyoD1-MKN-45 cells. The images were acquired with Cy3, DAPI, and merge channels (×120 objective magnification).
Fig 4: Expression of germ cell markers and pluripotency markers in GPT cells. (A) Alkaline phosphatase staining in the testicular cells cultured over 200 days. (B) Alkaline phosphatase staining in GPT cells. (C,D) Gene expression analysis in GPT cells via RT-PCR and western blotting. The cDNA and protein samples included testis, ovary, the testicular cells cultured over 200 days (unselected cells), and GPT cells. The ß-actin was used as an internal control. (E–I) Fluorescent immunostaining analysis of Piwi, Dazl, Ssea1, Nanog, and PCNA in GPT cells. (J–N) Nucleus was counterstained with propidium iodide (PI). (O–S) Merge images. Scale Bars: 50 µm in A and B, 20 µm in (E–S).
Supplier Page from Proteintech Group Inc for FUT4 antibody