Journal: Molecular biology reports

Article Title: Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

doi: 10.1007/s11033-023-08510-5

Figure Lengend Snippet: Fig. 1 Depletion of eIF5AHyp reduces the activity of a luciferase reporter gene containing a repeated proline motif and reduces endog- enous SIK2 protein level. A Schematic diagram of the reporter constructs with a repeated proline motif (F-pro-FLuc-WT) and the mutant control reporter (F-pro-FLuc-Mut) in FLAG vector. B Fire- fly luciferase activity assay following GC7 or eIF5A-targeting siRNA treatment in the presence of luciferase reporters constructed with repeated proline motifs. ES2 cell lines were transfected with a F-pro- FLuc-WT vector or its mutant vector by electroporation. After 12 h, the transfected ES2 cells were treated with GC7 or eIF5A-targeting siRNA, further incubated for 36–72 h, and harvested to determine the luciferase activity. The firefly luciferase level was normalized to the Renilla luciferase level. Data are mean ± SE of triplicate experiments using GC7 or eIF5A-targeting siRNA. C Using western blotting, the levels of eIF5AHyp, total eIF5A, total SIK2, and β-actin in ES2 cells

Article Snippet: The TOV-112D, CAOV-3, OVCAR-3, and ES2 human ovarian cancer cell lines were purchased from ATCC (Manassas, VA).

Techniques: Activity Assay, Luciferase, Construct, Mutagenesis, Control, Plasmid Preparation, Transfection, Electroporation, Incubation, Western Blot

Journal: Molecular biology reports

Article Title: Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

doi: 10.1007/s11033-023-08510-5

Figure Lengend Snippet: Fig. 2 Validation of p4E-BP1(Ser 65) as SIK2-associated protein. A Cell lysates were prepared from ES2 cells transfected with FLAG- tagged SIK2 and FLAG-EV. A pull-down assay was performed with anti-FLAG agarose beads. The pulled-down samples and unbound fractions were subjected to 4–12% NuPAGE gel and visualized by Ponceau S staining. The pulled-down proteins were analyzed by west- ern blotting with the indicated antibody. Total SIK2, total 4E-BP1, and p4E-BP1(Ser 65) proteins and their molecular weights (MWs) are shown on the right. B ES2 cells were transfected with siRNA to silence the SIK2 or 4E-BP1 genes and harvested 72 h after trans- fection; the cell lysates were analyzed by western blotting with the indicated antibody. The total SIK2 and p4E-BP1(Ser 65) proteins and their MWs are shown on the right. C ES2 cells were transfected with FLAG-EV and FLAG-tagged SIK2 in the absence or presence of GC7. After 12 h, the transfected ES2 cells were treated with GC7

Article Snippet: The TOV-112D, CAOV-3, OVCAR-3, and ES2 human ovarian cancer cell lines were purchased from ATCC (Manassas, VA).

Techniques: Biomarker Discovery, Transfection, Pull Down Assay, Staining, Western Blot

Journal: Molecular biology reports

Article Title: Inhibiting eukaryotic initiation factor 5A (eIF5A) hypusination attenuated activation of the SIK2 (salt-inducible kinase 2)-p4E-BP1 pathway involved in ovarian cancer cell proliferation and migration.

doi: 10.1007/s11033-023-08510-5

Figure Lengend Snippet: Fig. 3 Depletion of eIF5AHyp reduces the activity of the SIK2-p4E- BP1(Ser 65) pathway, which is involved in cell proliferation and migration. ES2 cells prepared by GC7 treatment or siRNA transfec- tion for eIF5A gene silencing and siRNA transfection for SIK2 or 4E-BP1 gene silencing were grown in 96-well, 6-well, and 12-well culture plates and cultured for 36–72 h. In addition, ES2 cells were transfected with FLAG-EV, FLAG-tagged SIK2, or FLAG-tagged 4E-BP1 in the absence or presence of GC7. After 12 h, the trans- fected ES2 cells were treated with GC7 (50 μM) and further cultured for 36 h. A Representative images and quantification of wound heal- ing in ES2 cell monolayers. The relative percentages of wound area

Article Snippet: The TOV-112D, CAOV-3, OVCAR-3, and ES2 human ovarian cancer cell lines were purchased from ATCC (Manassas, VA).

Techniques: Activity Assay, Migration, Transfection, Cell Culture