Overexpression of Spred2 in H1993 cells.

H1993 cells were transfected with Spred2 expression plasmid (OriGene, Rockville, MD, USA) or control plasmid (OriGene) using turbofectin 8.0 (OriGene). (A) Spred2 mRNA expressions after transfection with control plasmid (control) or Spred2 expression plasmid (Spred2) were analyzed by RT-qPCR (n = 3 each). Data is presented as mean ± SEM. *p<0.0001 (unpaired t test). (B) Transfection was carried out on Lab-Tek II Slide (8 Chamber, Electron Microscopy Sciences, Hatfield, PA, USA). The cells were fixed in 95% ethanol and immunostained with anti-Spred2 polyclonal antibody using the polymer method (Polink-2 Plus HRP RABBIT with DAB kit, GBI, Bothell, WA, USA). Spred2 positive cells were shown in brown. (C) Cell extracts after transfection with control plasmid (control) or Spred2 expression plasmid (Spred2) were immunoblotted with indicated primary antibodies (n = 3 each). Upper: Representative photos were shown. Lower: Band densities were digitised and semi-quantitated. Data is presented as mean ± SEM. (D) Ki67 mRNA expressions after transfection with control plasmid (control) or Spred2 expression plasmid (Spred2) were analyzed by RT-qPCR (n = 3 each). Data is presented as mean ± SEM. ^#p<0.05 (unpaired t test). (E) After transfection with control plasmid (control) or Spred2 expression plasmid (Spred2), the cell proliferation was evaluated by CCK-8 assay (n = 3 each). Data is presented as mean ± SE. *p<0.0001 (unpaired t test).

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