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. 2016 Jul 27;7(36):57783–57797. doi: 10.18632/oncotarget.10860

Figure 6. Knockdown of SATB2 in pancreatic cancer cell lines inhibits epithelial-mesenchymal transition, and markers of cell proliferation, pluripotency and stem cells.

Figure 6

(A) Cell Motility Assay. Pan CSCs/Scrambled, Pan CSCs/SATB2 shRNA, AsPC-1/Scrambled, AsPC-1/SATB2 shRNA, PANC-1/Scrambled, PANC-1/SATB2 shRNA cells were grown in petri dishes. After 18 hours of incubation, cells were scratched with the fine pipette tips. Phase contrast images of scratched cells were captured at 0 h, 24 h and 48 h time points. (B) Transwell Migration Assay. Transwell migration assay was performed in Pan CSCs/Scrambled, and Pan CSCs/SATB2 shRNA cells as described in Materials and Methods. Data represent mean ± SD. * = significantly different at P < 0.05. (C) Transwell Invasion Assay. Transwell invasion assay was performed in Pan CSCs/Scrambled and Pan CSCs/SATB2 shRNA as described in Materials and Methods. Data represent mean ± SD. * = significantly different at P < 0.05. (D) Expression of EMT-related genes/proteins. Upper panel, RNA was isolated and the expression of E-cadherin, N-cadherin, and Zeb1 in Pan CSCs/Scrambled, and Pan CSCs/SATB2 shRNA cells was measured by qRT-PCR. GAPDH was used as an internal control. Data represent mean (n = 4) ± SD. * = significantly different between groups (P < 0.05). Lower panel, Protein expression of E-Cadherin, N-cadherin, Zeb1 and Snail. Cell lysates were collected, and the expression of E-Cadherin, N-cadherin, Zeb1 and Snail was measured by the Western blot analysis. β-Actin was used as a loading control. (E) Expression of cell proliferation/survival genes/proteins. Upper panel, RNA was isolated and the expression of Bcl-2 and XIAP in Pan CSCs/Scrambled, and Pan CSCs/SATB2 shRNA cells was measured by qRT-PCR. GAPDH was used as an internal control. Data represent mean (n = 4) ± SD. * = significantly different between groups (P < 0.05). Lower panel, Protein expression of Bcl-2 and XIAP. Cell lysates were collected, and the expression of Bcl-2 and XIAP was measured by the Western blot analysis. β-Actin was used as a loading control. (F) Expression of pluripotency maintaining factors. Upper panel, RNA was isolated and the expression of c-Myc, Nanog, and Oct-4 in Pan CSCs/Scrambled, and Pan CSCs/SATB2 shRNA cells was measured by qRT-PCR. GAPDH was used as an internal control. Data represent mean (n = 4) ± SD. * = significantly different between groups (P < 0.05). Lower panel, Protein expression of c-Myc, Nanog, and Oct-4. Cell lysates were collected, and the expression of c-Myc, Nanog, and Oct-4 was measured by the Western blot analysis. β-Actin was used as a loading control. (G) Expression of stem cell markers. Upper panel, RNA was isolated and the expression of CD44 in Pan CSCs/Scrambled, and Pan CSCs/SATB2 shRNA cells was measured by qRT-PCR. GAPDH was used as an internal control. Data represent mean (n = 4) ± SD. * = significantly different between groups (P < 0.05). Lower panel, Protein expression of stem cell markers. Cell lysates were collected, and the expression of CD24, CD44 and CD133 was measured by the Western blot analysis. β-Actin was used as a loading control.