Skip to main content
Figure 4 | BMC Genomics

Figure 4

From: Reversible, interrelated mRNA and miRNA expression patterns in the transcriptome of Rasless fibroblasts: functional and mechanistic implications

Figure 4

Characterization of cell cycle-related parameters in Rasless cells. (A) FACS analysis of cell-cycle stages in cultures of proliferating K-Raslox MEFs and derived growth-arrested cultures treated with 4OHT for 6-days or 12-days (Rasless). Barplot of a representative experiment including untreated, control DU315-6 cells (K-Raslox) (empty bars) and the same cells after treatment with 4OHT for 6-days (grey bars) or 12-days (black bars). (B, C) Hierarchical clustering of cell-cycle-related (panel B) and E2F-target (panel C) genes differentially expressed in Rasless MEFs. Heatmaps analyzing absolute expression values of a group of 73 probesets relevant for cell cycle regulation (selecting genes annotated to cell-cycle GO term 0007049 in our GeneCodis functional analyses), and a set of 30 probesets for E2F transcription factor targets (identified from available E2F literature [35, 36]) that were repressed in Rasless MEFs and rescued by BRAF and MEK1 expression (listed in Additional file 1: Table S1, FDR = 0.01). Red: overexpression. Green: repression. Black: unchanged expression. (B) Lanes 1–7: Rasless cells. 12-d 4OHT-treated cell lines DU315-6 (1–3, 6,7) and DU244-1 (4, 5). Lanes 14–20: K-Rasloxcell lines DU315-6 (14, 16–20) and DU244-1 (15). Lanes 8–13: BRAF-rescued cell line LG7-6 (8, 9, 13) and MEK1-rescued cell line MCL1-6 (10–12). (C) Lanes 1–7: Rasless cell lines 12-d 4OHT-treated cell lines DU315-6 (3–7) and DU244-1 (1, 2). Lanes 8–14: K-Rasloxcell lines DU315-6 (9, 10, 12–14) and DU244-1 (8, 11). Lanes 15–20: BRAF-rescued cell line LG7-6 (15–17) and MEK1-rescued cell line MCL1-6 (18–20). (D) Immunoblot validation of transcriptional data confirming upregulation of Cdkns (p15INK4b, p16INK4a and p21CIP1) and downregulation of cell-cycle-related proteins (Myc, Dusp6, Pcna and different cyclins and Cdks) after 6-days or 12-days 4OHT treatments of two representative K-Raslox cell lines (DU315-6 and DU244-1) and a control double-knockout (H-Ras-/-; N-Ras-/-) MEF cell line (A624-8).

Back to article page