Figures & data
Table I. Expression pattern of CDKL1 in gastric cancer tissues and adjacent tissues. Immunoblot results were scored in four categories: negative expression, low-level expression, middle-level expression and high-level expression.
Figure 1. Images of immunohistochemical expression of CDKL1 in human gastric cancer compared with matched adjacent tissues. The specimens were viewed with an Olympus CKX41 microscope at magnification of 20 ×. (A, B, C) Representative photos of high-level, middle-level and low-level expression of CDKL1 protein in human gastric cancer. (D, E) Representative photos of low-level and negative expression of CDKL1 protein in adjacent tissues.
Figure 2. Infection of gastric cancer cells by CDKL1-siRNA-expressing lentivirus. (A) Analysis of CDKL1 protein in gastric cancer cell lines (GES, SGC7901, MKN45, MGC803) by Western blot assay. (B) The lentiviral vector containing CDKL1 siRNA or a scrambled sequence was constructed and respective lentivirus was packaged. SGC-7901 cells were infected with CDKL1 siRNA lentivirus or control lentivirus for three days and observed under fluorescence microscopy in bright field (left panel) and fluorescent field (right panel) with 400 × magnification. (C, D) Expression levels of mRNA were measured by qPCR. In comparison with control, CDKL1 siRNA lentivirus infection resulted in 76.4% and 82.2% decrease in the expression level of CDKL1 mRNA in SGC7901 (C) and MGC-803 (D) cells, respectively. (E, F) Expression levels of protein were measured by Western blot. CDKL1 protein was significantly downregulated in CDKL1 siRNA lentivirus infected SGC7901 (E) and MGC-803 (F) cells. *, p < 0.05 and ***, p < 0.001.
Figure 3. The effect of CDKL1 knock down on the proliferation and colony formation of gastric cancer cells. (A, B) Analysis of cell viability of CDKL1 siRNA lentivirus or control lentivirus infected cells for one to five days. The proliferation of both SGC7901 (A) and MGC-803 (B) cells was significantly inhibited after CDKL1 knock down. (C, D) Colony formation in lentivirus infected cells was assayed 14 days after culture. Statistical results of colony number showed that the colony-forming ability was impaired in CDKL1-siRNA lentivirus infected SGC7901 (C) and MGC-803 (D) cells. *, p < 0.05 and ***, p < 0.001.
Figure 4. Detection of apoptotic cells by flow cytometry. After seven days of lentivirus infection, SGC7901 (A) and MGC-803 (B) cells were cultured in six-well plates and subjected to Annexin V staining/flow cytometric analysis. Results showed that CDKL1 knock down resulted in inducing the apoptosis of both gastric cancer cells. **, p < 0.01 and ***, p < 0.001.
Figure 5. Analysis of CDKL1 downstream target genes by Western blot. Expression of proliferation and apoptotic markers was observed in CDKL1-siRNA lentivirus infected SGC7901 cells by qPCR (A) and Western blot (B). The reduction of CDKL1 with its siRNA stimulates the activation of apoptotic molecules, such as Bik and p21 Waf1/Cip1 and attenuated the expression of Bcl-2 and PCNA. *, p < 0.05 and **, p < 0.01.
