Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel

Figures & data

Figure 1 Silencing of SKP2 by siRNA led to decrease of MAD2 expression in A549 and NCI-H1975 cells.

Notes: (A) Human lung cancer A549 and NCI-H1975 cells were transfected with 100 nM control or SKP2-specific siRNA with lipofectamine 2000. After 48 h, total proteins were extracted for the detection of the protein levels Skp2 and Mad2 by Western blotting. GAPDH served as the loading control. (B) Human lung cancer A549 and NCI-H1975 cells were transfected with 100 nM control or SKP2-specific siRNA with lipofectamine 2000. After 48 h, total RNA was extracted for the detection of the mRNA levels MAD2 by RT-QPCR with GAPDH as internal control. Quantitative analysis are expressed as mean ± SEM. n=3, *P<0.01 vs control siRNA-transfected cells. (C) Human lung cancer A549 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 24, 48 and 72 h. Total proteins were extracted for the detection of the protein levels Skp2 and Mad2 by Western blotting. GAPDH served as the loading control. (D) Human lung cancer NCI-H1975 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 24, 48 and 72 h. Total proteins were extracted for the detection of the protein levels Skp2 and Mad2 by Western blotting. GAPDH served as the loading control. (E) Human lung cancer A549 and NCI-H1975 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 24, 48 and 72 h. Total RNAs were extracted for the detection of the mRNA levels MAD2 by RT-QPCR with GAPDH as internal control. Mean value of triplicate is shown. Quantitative analysis results are expressed as mean ± SEM. n=3, *P<0.01 vs control pcDNA3.1-transfected cells.
Abbreviations: SKP2, S-phase kinase-associated protein 2; MAD2, mitotic arrest deficient 2; siRNA, small interfering RNA; mRNA, messenger RNA; SEM, standard error of the mean; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 2 Silencing of SKP2 resulted in increase while overexpression of SKP2 led to decrease of p27.

Notes: (A) Human lung cancer A549 and NCI-H1975 cells were transfected with 100 nM control or SKP2-specific siRNA with lipofectamine 2000. After 48 h, total proteins were extracted for the detection of the protein levels of p27, the phosphorylation of Rb at Ser780 (pRb-S780) and Ser807/811 (pRb-S807/811) by Western blotting. GAPDH served as the loading control. (B) Human lung cancer A549 and NCI-H1975 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 48 h. Total proteins were extracted for the detection of the protein levels of p27, the phosphorylation of Rb at Ser780 (pRb-S780) and Ser807/811 (pRb-S807/811) by Western blotting. GAPDH served as the loading control.
Abbreviations: SKP2, S-phase kinase-associated protein 2; siRNA, small interfering RNA; Rb, retinoblastoma.

Figure 3 Pharmacological inhibition of either CDK1/2 or E2F1 prevented the induction of the expression of MAD2 by SKP2 overexpression.

Notes: (A) Human lung cancer A549 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 48 h, then treated with CDK1/2 inhibitor flavopiridol or E2F1 inhibitor HLM006474 for additional 24 h. Total RNAs were extracted for the detection of the mRNA levels MAD2 by RT-QPCR with GAPDH as internal control. Quantitative analysis are expressed as mean ± SEM. n=3, *P<0.05 vs control. (B) Human lung cancer A549 cells were transfected with 2 μg of vector pcDNA3.1 or pcDNA-SKP2 for 48 h, then treated with CDK1/2 inhibitor flavopiridol or E2F1 inhibitor HLM006474 for additional 24 h. Total proteins were extracted for the detection of the protein levels Skp2 and Mad2 and the phosphorylation of Rb at Ser780 (pRb-S780) by Western blotting. GAPDH served as the loading control. (C) Human lung cancer A549 cells were transfected with 50 nM control or SKP2-specific siRNA with lipofectamine 2000 for 48 h, then treated with CDK1/2 inhibitor flavopiridol or E2F1 inhibitor HLM006474 for additional 24 h. Total proteins were extracted for the detection of the protein levels Skp2 and Mad2 by Western blotting. GAPDH served as the loading control.
Abbreviations: MAD2, mitotic arrest deficient 2; SKP2, S-phase kinase-associated protein 2; mRNA, messenger RNA; SEM, standard error of the mean; Rb, retinoblastoma; siRNA, small interfering RNA.

Figure 4 Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel.

Notes: (A) A549 cells were treated with the indicated concentrations of paclitaxel (0.01–5 μm) for 24 h. Cell viability was assessed by MTT assay. (B) A549 cells were transfected with the indicated concentrations of SKP2 siRNA (10–400 nM) or scrambled siRNA (200 nM) for 48 h. Cell viability was assessed by MTT assay. (C) A549 cells were treated with the indicated concentrations of SMIP004 (0.01–5 μm) for 24 h. Cell viability was assessed by MTT assay. (D) A549 cells were transfected with 50 nM SKP2 siRNA or scrambled siRNA for 24 h, followed by treatment with 100 nM paclitaxel or 50 nM SMIP004 for additional 24 h. Cell viability was assessed by MTT assay. (E) A549 cells were treated as (D); cell proliferation was determined by colony formation assay.
Abbreviations: SKP2, S-phase kinase-associated protein 2; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA, small interfering RNA; SMIP, SMIP004; OD, optical density; PAC, paclitaxel.