Journal: Heliyon

Article Title: Overexpression of RBM15 modulated the effect of trophoblast cells by promoting the binding ability between YTHDF2 and the CD82 3′UTR to decrease the expression of CD82

doi: 10.1016/j.heliyon.2024.e30702

Figure Lengend Snippet: Overexpression of RBM15 inhibited CD82 expression and its potential regulation mechanism. (A) qPCR was used to detect the expression levels of NOTUM and CD82 in the placental NC and PE groups. N (biological replicates) = 7 each group. (B–C) Western blotting (B) and immunofluorescence (C) were used to detect CD82 abundance in the placenta of the NC and PE groups. TPGB is the markers of trophoblast cells. N (biological replicates) = 3 each group. (D) qPCR analysis of CD82 expression following RBM15 overexpression. The pCDH-RBM15 vector and its control vector, pCDH-NC, were transfected into HTR8/SVneo and JEG-3 cells for 24 h. (E) Western blotting was used to detect CD82 abundance after RBM15 overexpression. pCDH-RBM15 vector and its control vector pCDH-NC were transfected into cells for 48 h. (F) SRAMP predicted the m 6 A modification site of CD82 mRNA 3'UTR. (G) Effect of overexpression of RBM15 on the stability of the CD82 3’UTR was detected by luciferase activity. pmirGLO-CD82 3′UTR WT (pmirGLO-WT) or pmirGLO-CD82 3′UTR mut (pmirGLO-mut) vector and pCDH-RBM15 (or pCDH) were co-transfected into 293T cells for 48 h. (H) YTHDF2-RIP detection was used to evaluate the binding ability between YTHDF2 and the CD82 3′UTR. pCDH or pCDH-RBM15 were transfected into HTR8/SVneo for 48 h **p value less than 0.01, ***p value less than 0.001, ****p value less than 0.0001. PE, pre-eclampsia; NC, normal pregnancy placenta; CD82, cluster of differentiation 82; NOTUM, palmitoleoyl-protein carboxylesterase; RBM15, RNA-binding protein 15; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pCDH-NC, vector.

Article Snippet: The primary antibodies and secondary antibodies included UltraPolymer Goat anti-Mouse IgG (H&L)-HRP (proteintech, #PR30012), G3BP1 (proteintech, #13057-2-AP), RBM15 (proteintech, #10587-1-AP), YTHDF2 (proteintech, #24744-1-AP), MMP2 (proteintech, #10373-2-AP), NOTUM (proteintech, #14663-1-AP), CD82 (proteintech, #66803-1-Ig), GAPDH(proteintech, #10494-1-AP), UltraPolymer Goat anti-Rabbit IgG (H&L)-HRP (proteintech, #PR30011), and MMP9 (N-terminal) (proteintech, #10375-2-AP).

Techniques: Over Expression, Expressing, Western Blot, Immunofluorescence, Plasmid Preparation, Control, Transfection, Modification, Luciferase, Activity Assay, Binding Assay, RNA Binding Assay

Journal: Heliyon

Article Title: Overexpression of RBM15 modulated the effect of trophoblast cells by promoting the binding ability between YTHDF2 and the CD82 3′UTR to decrease the expression of CD82

doi: 10.1016/j.heliyon.2024.e30702

Figure Lengend Snippet: RBM15 participates in the effect of trophoblast cells via regulation CD82 in HTR8/SVneo and JEG-3 cells. (A) qPCR was used to detect the expression of RBM15 and CD82 after overexpression of RBM15 and CD82. pCDH, pCDH-RBM15, or pCDH-CD82 were transfected into cells for 24 h. (B) Western blotting was used to detect the abundance of RBM15, CD82, MMP-2, and MMP-9 after the overexpression of RBM15 and CD82. Cells were transfected with pCDH, pCDH-RBM15, or pCDH-CD82 for 48 h. (C) Transwell assays were used to detect changes in cell migration after overexpression of RBM15 and CD82. Cells were transfected with pCDH, pCDH-RBM15, or pCDH-CD82 for 48 h. (D) Transwell assays were used to detect changes in cell invasion after overexpression of RBM15 and CD8. pCDH, pCDH-RBM15, or pCDH-CD82 were transfected into cells for 48 h *p value less than 0.05; **p value less than 0.01; ***p value less than 0.001; ****p value less than 0.0001. RBM15, RNA-binding protein 15; MMP-2, matrix metallopeptidase 2; MMP-9, matrix metallopeptidase 9; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pCDH-NC, pCDH vector; CD82, cluster of differentiation 82.

Article Snippet: The primary antibodies and secondary antibodies included UltraPolymer Goat anti-Mouse IgG (H&L)-HRP (proteintech, #PR30012), G3BP1 (proteintech, #13057-2-AP), RBM15 (proteintech, #10587-1-AP), YTHDF2 (proteintech, #24744-1-AP), MMP2 (proteintech, #10373-2-AP), NOTUM (proteintech, #14663-1-AP), CD82 (proteintech, #66803-1-Ig), GAPDH(proteintech, #10494-1-AP), UltraPolymer Goat anti-Rabbit IgG (H&L)-HRP (proteintech, #PR30011), and MMP9 (N-terminal) (proteintech, #10375-2-AP).

Techniques: Expressing, Over Expression, Transfection, Western Blot, Migration, RNA Binding Assay, Plasmid Preparation