Fig 1: Expression of microRNA-376c-3p (miR-376c-3p) was reduced, while SYF2 expression was enhanced in GC cell lines. A, Expression of miR-376c-3p was analyzed by RT-qPCR in GC cell lines. B, Expression of SYF2 was analyzed by Western blot in GC cell lines. Samples were analyzed for three times. miR-376c-3p indicates microRNA-376c-3p; SYF2, SYF2 pre-mRNA-splicing factor; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; GC, gastric cancer.
Fig 2: miR-376c-3p regulates GC cell behaviors through regulating SYF2. A, SYF2 expression, (B) cell proliferation, (C) cell migration, and (D) cell cycle distribution in GC cells transfected with miR-376c-3p mimic, NC-mimic, pSYF2, pcDNA 3.1, or miR-376c-3p mimic and pSYF2. Samples were analyzed 3 times. miR-376c-3p indicates microRNA-376c-3p; SYF2, SYF2 pre-mRNA-splicing factor; GC, gastric cancer; NC-mimic, negative control for miR-376c-3p mimic.
Fig 3: SYF2 was a direct target of miR-376c-3p. A, Binding site between miR-376c-3p and SYF2. B, miR-376c-3p expression levels in GC cells transfected with miR-376c-3p mimic or NC-mimic. C, Luciferase in GC cells transfected with SYF2-wt was inhibited by miR-376c-3p mimic. Samples were analyzed for three times. miR-376c-3p indicates microRNA-376c-3p; SYF2, SYF2 pre-mRNA-splicing factor; GC, gastric cancer; wt, wild type; mt, mutant; NC-mimic, negative control for miR-376c-3p mimic.
Fig 4: RT-PCR validation of DEGs identified from transcriptome analysis. Representative UMAPs and graphs highlighting the gene expression differences of MSMP, C2orf40, SLPI and EPYC between NPnD and iAFnD in (A); SYF2, MT1F, FGFBP2, TAF1D and EPYC between NPD and iAFD in (B). RT-PCR validation of IVD degeneration selected DEGs in the transcriptome data of CHI3L2, PLA2G2A, TNRSF11B and FGFBP2 showing an increase or MT-ND2, MT-CYB, CTGF and TAF1D showing a decrease in iAFD (C) when compared to iAFnD cells from discs of the same individual. mRNA expression of MT2A, UPP1, HMGA1 and TAF1D (increase) and CAPS, SLPI and SPTSSB (decrease) in NPD compared with NPnD (D). Validation of common degeneration markers by RT-qPCR showing an increase or a decrease with degeneration NPD (E) and AFD (F). The values were calculated using the 2-??Ct method: * p < 0.05, ** p < 0.01 and **** p < 0.0001.
Fig 5: Validation of selected cell type and degeneration markers. (A) UMAP highlighting p29 gene expression at the single-cell level in iAF and NP nD and D discs of the same individual. (B) Western Blot analysis of cell lysates from 5 additional individuals normalized to GAPDH (n = 5). (C) Immunohistochemistry staining of p29 (in green), Dapi (in blue) and merged images of cells from 5 additional individuals (scale = 100 µm, n = 5 biological replicates). Graphs are presented as mean ± SEM. * p < 0.05, ** p < 0.01.
Supplier Page from Abcam for Anti-GCIP interacting protein p29 antibody [OTI6A5]