Figures & data
Figure 1. Restoration of CASZ1 suppresses NB cell proliferation and inhibits the cell cycle. Full-length FLAG-tagged CASZ1a in Tet-on vector was stably transfected into SY5Y (A) and NGP (B) neuroblastoma cell lines that express Tet repressor. Induction of CASZ1a expression by Tet (1 µg/mL) was visualized by immunoblotting whole-cell lysate with anti-FLAG antibody. The fold induction of CASZ1 mRNA at 24 h was detected by real-time PCR in SY5Y (C) and NGP (D). Restoration of CASZ1a in SY5Y (E) and NGP cells (F) inhibited cell proliferation (*P < 0.005; #P < 0.05). FACS analysis documenting the cell cycle distribution of SY5Ytet CASZ1a (G) or NGPtetCASZ1a (H) upon restoration of CASZ1a for 2 d.
Figure 2. CASZ1 regulates the levels of proteins that regulate G1–S phase cell cycle progression. (A) The protein levels of cyclins in SY5Y cells upon CASZ1a induction were visualized by immunobloting whole-cell lysate with indicated antibodies. (B) The protein levels of Cdks in SY5Y cells upon CASZ1 induction were visualized by immunobloting whole-cell lysate with indicated antibodies. The relative densitometric units were normalized to control condition and the ratio shown under each condition. (C) The protein levels of Cdk inhibitors in SY5Y cells were visualized by immunobloting whole-cell lysate with indicated antibodies. (D) The protein levels of cell cycle regulators in NGP cells were visualized by immunobloting whole-cell lysate with indicated antibodies.
Figure 3. Cdk6 and p21 were regulated by CASZ1a shortly after CASZ1 restoration. The protein levels of cell cycle regulators in SY5Y cells (A) and NGP cells (B) were visualized by immunobloting whole-cell lysate with indicated antibody from 8–24 h after induction of CASZ1a. The relative densitometric units were normalized to timed control, and the ratio shown under each condition.
Figure 4. CASZ1a restores pRb activity. CASZ1a restoration leads to de-phosphorylation of pRb at Ser780 and Ser807/811 in SY5Y cells (A) and NGP cells (B) and was visualized by immunobloting whole-cell lysate with indicated antibody. The relative densitometric units were normalized to control condition and the ratio shown under each condition. (C) CASZ1 restoration significantly decreased E2F target gene c-Myc expression in SY5Y cells detected by real-time PCR (*P < 0.005). (D) CASZ1 restoration significantly decreased E2F target gene cyclin D1 expression in NGP cells detected by real-time PCR (*P < 0.01). (E) CASZ1 restoration inhibits E2F activity at both 24 h and 48 h time points as assessed using a E2F-luc reporter assay (*P < 0.005; #P < 0.02).
Figure 5. CASZ1 regulates the levels of proteins that promote G2–M phase cell cycle progression. The protein levels of G2–M cell cycle regulators after induction of CASZ1a in SY5Y cells (A) and NGP cells (B) were visualized by immunobloting whole-cell lysates with indicated antibody. The relative densitometric units were normalized to control condition and the ratio shown under each condition.
Figure 6. CASZ1 modulates transcription of select cell cycle regulators. The mRNA level changes of (A) CASZ1, (B) Cdk6, (C) p21, and (D) Skp2 detected by real-time-PCR in SY5YtetCASZ1a cells treated with or without Tet for 8, 16, and 24 h (*P < 0.005; #P < 0.05).
Figure 7. Model of CASZ1 function in cell cycle regulation. Restoration of CASZ1 in NB cells results in upregulation of p21 a Cdk2/4 inhibitor and downregulation of cyclins such as Cyclin D1 and Cdk2/6. Thus CASZ1 inhibits Cdk enzyme activity, resulting in hypo-phosphorylation of pRb that binds E2F, thus decreasing E2F-mediated transcription and restoring the G1–S checkpoint and inhibition of cell cycle progression.
