Figures & data
Figure 1. Experimental workflow employed to analyze the gastric cancer tissue proteome. Equal concentrations of protein from both tumor and non-neoplastic tissue were extracted, digested and labeled with iTRAQ reagents. The labeled samples were pooled and fractionated using strong cation exchange chromatography. The sample fractions were analyzed using a quadrupole time-of-flight mass spectrometer. The MS/MS data was searched against Refseq45 protein database using Sequest and Mascot search algorithms. The overexpressed protein CAMKK2 was validated using immunohistochemistry.
Table 1. A partial list of overexpressed proteins identified in gastric adenocarcinoma
Figure 2. Representative MS/MS spectra of novel overexpressed proteins in gastric adenocarcinoma tissues. (A) Calcium/calmodulin-dependent protein kinase kinase 2 (7-fold). (B) Prolargin (2.6-fold) (C) Filamin-A isoform 1 (2-fold).
Table 2. Summary of the immunohistochemical validation for CAMKK2 in gastric cancer and normal tissues
Figure 3. Validation of CAMKK2 by immunohistochemistry. (A) Representative sections from gastric cancer cases stained with anti-CAMKK2 antibody. (B) Representative sections from normal gastric mucosa stained with anti-CAMKK2 antibody.
Figure 4. Silencing of CAMKK2 decreases cellular proliferation in gastric cancer cells. (A) Western blotting was done using anti-CAMKK2 antibody to study the relative expression level of CAMKK2 in a panel of gastric cell lines. GAPDH was used as a loading control. Gastric cancer cell lines were transfected with CAMKK2 siRNA and cell proliferation was assessed for (B) AGS, (C) KATO-III, (D) SNU-16 and (E) NCI-N87cell lines.
Figure 5. Silencing of CAMKK2 decreases the colony formation ability and invasive property of gastric cancer cell lines. Gastric cancer cell lines were transfected with CAMKK2 siRNA and assessed for either colony formation or invasion ability. (A) Colony formation ability of AGS was decreased post transfection with CAMKK2 siRNA. (B) A graphical representation of the same. (C) Silencing of CAMKK2 led to a decrease in the invasive property of AGS. (D) A graphical representation of the invasive property of AGS and KATO III (KATO III being a suspension line, invasion ability was studied by counting the cells in the upper chamber and the lower chamber of the membrane coated with Matrigel).
Figure 6. Silencing of CAMKK2 decreases AMPK activity in gastric cancer cells Gastric cancer cell line. AGS, KATO-III SNU-16 and NCI-N87 cell lines were transfected with either CAMKK2 or scrambled siRNA. Expression of Total CAMKK2, p-AMPKα (Thr172), Total AMPKα, p-FoxO3a (Ser413) and Total FoxO3a were probed by Western blot. GAPDH was used as control.
