Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 2

Figure 2. CRaf RBCRD detects endogenous RasGTP.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) HEK 293T cells were transfected with Venus-RBDCRD, eCFP-CAAX PM reporter (PMem), and mCherry-H2B to label nuclei. Scale bar is 20 µm and applies to all panels. (B) KRas protein levels were reduced in NCI-H727 cells using 2 different siRNA oligos against KRas or control oligo and then transfected with Venus-RBDCRD. Scale bar is 20 µm and applies to all panels. (C) Quantification of RBDCRD PM targeting in NCI-H727 with KRas siRNA. (D) Hec1A isogenic cell ines (Parental, RasWT/- and RasG12D/-) were transfected with Venus-RBDCRD and imaged live. RBDCRD PM targeting was then measured for each cell line. (E) Serum starved HEK 293T cells were stimulated with 100 ng/ml EGF, and imaged every 15 sec, PM targeting of RBDCRD was then measured.

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.
2.
Figure 6

Figure 6. Visualization of the labeled MAPK pathway.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) Example images show induced parental KRasG12D/CRaf cell line, induced and uninduced 4-color cell line where TagBFP-Mek1 and mCherry-Erk2 are constitutively expressed. Scale bar is 20 µm and applies to all panels. (B) Nuclear localization of TagBFP-Mek1 and mCherry-Erk2 was measured in MAPK cell line where eCFP-KRasG12D and Venus-CRaf were either induced or uninduced. (C) MAPK cell line was dosed with Raf, Mek and Erk inhbitors, CRaf PM localization was measured. (D) The nuclear/cytoplasmic ratio of mCherry-Erk1 was measured in MAPK cell line where KRasG12D and CRaf expression were induced. (E) The nuclear/cytoplasmic ratio of mCherry-Erk1 was measured in the MAPK cell line where KRasG12D and CRaf were not induced. (F) Nuclear pERK was measured in the HEK 293 KRasG12D/CRaf/Mek1/Erk2 cell line by immuno-fluorescence 5hrs after inhibitor treatment. (G) Nuclear pERK was measured as in (F) in the HEK 293 cell line expressing TagBFP-Mek1 and mCherry-Erk2 where expression of KRasG12D and CRaf is not induced.

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.
3.
Figure 5

Figure 5. RBDCRD response to MAP-kinase inhibitors.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) RBDCRD PM targeting was measured in Venus-RBDCRD stable cell line after 4 hr treatment with AZD628, GDC0879, PLX4720, Mek-i A, Mek-i B, or Erk-i. (B) phospho-ERK intensity was measured by immuno-fluorescence in cells treated as in (A). (C) RBDCRD PM targeting was measured in cells treated with a constant dose of 250 nM GDC0879 with a dose curve of the other small molecule inhibitor, showing the dominant effect of Mek and Erk inhibitor-induced targeting. (D) Time-lapse experiments show the kinetics of RBDCRD PM targeting and displacement upon treatment with 250 nM GDC0879, 20 nM Mek-i A or the combination of both. (E) Time-lapse experiment as in (D) where GDC0879 and Mek-i A were added sequentially with a 28.5 min. time gap between compound additions. (F) Schematic model describing RBDCRD cellular behavior in the presence of MAPK inhibitors.

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.
4.
Figure 3

Figure 3. Raf priming and MAPK negative feedback inhibition act to recruit CRaf to the PM.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) The Raf inhibitors AZD628, GDC0879 and PLX4720 along with Mek-i A were dosed in eCFP-KRasG12D and Venus-CRaf expressing HEK 293 cells, example images shown. Scale bar is 20 µm and applies to all panels. (B) PM/cytoplasmic CRaf intensity for images such as those in (A) was measured 4 hours after compound addition. (C) Mek-i A was compared to Mek-i B and Erk-i in CRaf redistribution assay. (D) Chart shows EC50 values for both the CRaf PM targeting assay and biochemical kinase activity. (E) Cytoplasmic pERK was measured in cells dosed wih Raf, Mek and Erk inhibitors by immuno-fluorescence. (F) Kinetics of Raf inhibitor priming and feedback were compared by conducting time lapse analysis of cells treated with GDC0879 and Mek-i A, frames acquired every 2 min.

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.
5.
Figure 1

Figure 1. Automated Ras-mediated Raf redistrbution analysis.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) Graphical representation of raw data from Raf-redistribution assay analysis (left, mCherry-KRas in red, Venus-CRaf in green) nuclear and PM masking (center) and radial intensity ratio scanning (right) show image analysis approach. Scale bar is 20 µm and applies to all panels. (B) Schematic of bicistronic vector used to generate stable cell lines. (C) Images of bicsitronic Flp-In stable cell lines expressing Venus-CRaf with eCFP-KRasWT and eCFP-KRasG12D. Cells were stained with Hoechst to demonstrate the percentage of cells that express the fluorescent-fusion proteins (>99% for both cell lines). Scale bars are 20 µm and apply to corresponding magnifications in all panels. (D) CRaf PM/cytoplasmic ratio measurement of KRasWT and KRasG12D stable cell line, p<0.001. (E) eCFP-KRasG12D and Venus-CRaf expressing cells were treated with a serial dilution of MCP110 between 100 µM and 24 nM for 2.5 hrs, example images shown. Scale bar is 20 µm and applies to all panels. (F) CRaf PM/cytoplasmic intensity ratios were measured for MCP110 serial dilution in (E).

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.
6.
Figure 4

Figure 4. Combination of Raf priming with Mek/Erk inhibition has an additive effect on MAPK signaling.. From: Live-Cell Microscopy Reveals Small Molecule Inhibitor Effects on MAPK Pathway Dynamics.

(A) KRasG12D/CRaf HEK 293 cells were dosed with varying concentration of Mek-i A and constant dose of 12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879, as indicated, and CRaf PM targeting was measured after 4 hrs. (B) CRaf PM targeting was measured for the combination of Mek-i B with 12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879. (C) CRaf PM targeting was measured for the combination of Erk-i with12 nM AZD628, 2 µM PLX4720 or 300 nM GDC0879. (D) Top: Immunoblot of Braf:Craf heterodimers in cells treated with GDC-0879 (0.03, 0.1, 0.3, 1 µM) for 4 hours. Heterodimer formation observed with GDC-0879 alone is further increased upon co-administration of 1 µM MEK inhibitor Mek-i A. Bottom: CRaf IP kinase activity assays from lysates of cells treated as above. CRaf was immunoprecipitated from treated cells and kinase activity towards recombinant MEK was tested in vitro. Co-administration of Mek-i A with GDC0879 resulted in a dose-dependent increase in maximal CRaf kinase activity across all GDC0879 doses. For the CRaf kinase activity assays, relative phospho-MEK levels measured with MSD pMEK ELISA assay are shown. (E) Four-day proliferation curves of RasWT/RafWT cells (SW48 colon carcinoma) upon combination treatment of GDC-0879 with increasing concentrations of Mek-i A show induction of viable cell counts compared to MEK inhibitor treatment alone in a dose dependent manner with a maximal effect at co-administration of 1 µM of GDC0879. (F) Model describing the localization effect of Raf, Mek and Erk inhibitors on Raf cellular localization. Ras activation causes CRaf recruitment to the PM, priming of CRaf or inhibition of negative downstream feedback increase CRaf targeting to the PM. Combination of priming and feedback inhibition additively promote CRaf PM targeting.

Daniel J. Anderson, et al. PLoS One. 2011;6(8):e22607.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk