Fig 1.
Establishment of MDCK cells expressing an active Ras or BRaf protein and a FRET biosensor for ERK activity.
(A) A scheme of the Ras-Raf-MEK-ERK pathway and the mode of action of the intramolecular FRET biosensor, EKAREV-NLS. DUSPs, dual-specificity phosphatases; PSPs, protein serine/threonine phosphatases. (B) Parental MDCK cells were stably-transfected with an expression vector for EKAREV-NLS. The transfected cells were then further transfected with the pPBbsr2-lox-tdTomato-oncogene. After the introduction of ERT2CreERT2, the oncogenes were induced by 4OHT. (C) The MDCK-EKAREV-NLS-floxed-tdTomato-oncogene, the name of which is depicted in the first row, was cultured in the presence or absence of 1 μM 4OHT and analyzed by Western blotting with the antibodies listed on the left of each panel.
Fig 2.
Cell density-dependent suppression of ERK activity in MDCK cells expressing an active Ras or BRaf.
(A) MDCK-floxed-tdTomato-NRasG12V cells before 4OHT induction were plated at the cell densities depicted at the bottom of each panel and imaged with a fluorescence microscope. FRET/CFP ratio images in the intensity-modulated display mode indicate the ERK activity. Bars, 50 μm. (B) MDCK-floxed-tdTomato-KRasG12V cells were time-lapse imaged for 450 min. The mean FRET/CFP ratios at low cell density (0.5×104 cells/cm2) and at high cell density (4×104 cells/cm2) are shown by heatmaps. Each row represents time course of FRET/CFP ratio of single cell. The numbers of analyzed cells are 5 for low cell density and 150 for high cell density. (C) Similar experiments were performed after 4OHT induction. (D) MDCK-floxed-tdTomato-oncogene cells before and after 4OHT induction were plated at low and high cell densities and imaged with a fluorescent microscope. The FRET/CFP ratios of 90 cells from three independent experiments are plotted for each condition.
Fig 3.
ERK activity change in the wound-healing assay.
(A) MDCK cells expressing NRasG12V and EKAREV-NLS were plated in Culture-Insert 2 Well (ibidi) at high cell density and imaged with a fluorescence microscope. No difference in ERK activity was detected between cells in contact with and cells apart from the silicon insert. Bar, 200 μm. (B and C) The wound-healing assay was started by removing the silicon insert. Representative consecutive FRET/CFP ratio images. Bar, 50 μm (B). The FRET/CFP ratios in 14 cells at the border were monitored and plotted over time. Mean ± SD (C).
Fig 4.
Increase in tyrosine-monophosphorylated ERK at high cell density.
(A) A schematic representation of the ERK phosphorylation status. np-ERK2, non-phosphorylated ERK2; pT-ERK2, ERK2 monophosphorylated on Thr185; pY-ERK2, ERK2 monophosphorylated on Tyr187; pTpY-ERK2, ERK2 diphosphorylated on Thr185 and Tyr187; DUSPs, dual specificity phosphatases; PSPs, protein serine/threonine phosphatases. (B) MDCK-floxed-tdTomato-HRasG12V cells before and after 4OHT induction were plated at the cell densities depicted at the bottom of each lane and analyzed by Western blotting analysis with Phos-tag gels. The filters were probed with anti-ERK and anti-phospho-ERK (Thr185/Tyr187) antibodies. The yellow bands indicate pTpY-ERK1 and pTpY-ERK2. (C) The fluorescence intensities of ERK2 proteins were quantified and the percentage of each phospho-isoform is plotted. Data obtained from three independent experiments are shown. *p < 0.05. (D) Similar experiments were performed with MDCK cells before and after the induction of NRasG12V, KRasG12V, or BRafV600E as indicated. Data are shown only for pY-ERK2 and pTpY-ERK2 for simplicity. *p < 0.05; **p < 0.01. (E) A schematic of the mechanism of decrease in pTpY-ERK2 at high cell density.
Fig 5.
Cell density-dependent decrease of phosphorylation of Akt and S6 ribosomal protein.
(A and B) MDCK cells before and after KRasG12V or BRafV600E induction were plated at the cell densities depicted at the bottom of each column and analyzed by Western blotting with anti-Akt, anti-phospho-Akt (Thr308), anti-phospho-Akt (Ser473), anti-S6 ribosomal protein antibody, and anti-phospho-S6 ribosomal protein (Ser235/236). (C and D) Quantification of each band in the blotting. Y-axis represents fold change comparing with the pre-induction cells at the low cell density as control.
Fig 6.
Calyculin A-induced decrease of tyrosine-monophosphorylated ERK2 at high cell density.
(A and B) MDCK cells were stimulated with 5 nM calyculin A (A) or 500 nM okadaic acid (B). The mean and upper/lower quantiles of FRET/CFP ratios are plotted before and after 30 min incubation, respectively (n = 50). *p < 0.05; **p < 0.01. (C) Cells were plated at the indicated cell density, cultured overnight, and incubated with 5 nM calyculin A for 30 minutes, followed by Phos-tag Western blot analysis with anti-ERK and anti-phospho-ERK (Thr185/Tyr187) antibodies as shown in Fig 4. (D) The difference between the fractions of calyculin A-treated cells (C) and non-treated cells (Fig 4C) is shown.