Figure 1.
Deletion of both Ets1 and Ets2 ablates HrasG12V transformation of MEFs.
E1+E2+ and E1−E2− MEFs were infected with HRasvG12V or empty vector control retrovirus, then selected with Hygromycin for at least 5 days before functional examination of the four generated cellular genotypes, as shown. A) Western blotting analysis of 20 µg protein lysates probed with antibodies against proteins shown, with α-tubulin as loading control. B) Representative images and C) Quantification of the cellular colonies that grew in soft agar from the four different genetic groups Asterisk indicates P<0.05 as determined by the Student t test. D) Cells were injected subcutaneously into nude mice (106 cells per injection site) and after 3 weeks the tumors were harvested. The ratios represent the percentage of tumors that grew from the total number of injections for each of the different genotypes.
Figure 2.
Ets1 and Ets2 activate c-Myc expression in HrasG12V transformed MEFs.
A) Fold change of c-Myc gene expression by real time rt-PCR in the 4 different genetic groups examined. B) Western blotting analysis of 20 µg protein lysates probed with antibodies against MYC and α-tubulin. C) Schematic illustration of the c-Myc promoter showing conserved Ets binding sites (box) and the ChIP primers used (arrows) relative to the P2 promoter. D) ChIP performed on MEFs with indicated genotypes using anti-ETS1 and ETS2 antibodies with IgG as control. The threshold value for the promoter being studied was normalized to that of input values and represented as relative enrichment. Asterisk indicates P<0.05.
Figure 3.
Overexpression of c-Myc in Ets1/Ets2-null MEFs rescued HrasG12V transformation.
E1−E2− control and E1−E2−/H-RasvG12V cells were infected with either MSCV-GFP empty control or MSCV-GFP-c-Myc vector, and cells were sorted for GFP expression by FACS. A) 20 µg of protein lysates from E1−E2−/H-Rasv12/MSCV-GFP control and E1−E2−/H-Rasv12/MSCV-GFP-c-Myc cells analyzed by western blot against MYC and β-Actin (protein loading control) antibodies. B) Graph representing the percentage of tumors formed over the total number of injections for the indicated cellular genotypes. N/A indicates that there were no tumors observed for the specified group. C) Representative pictures of all tumors derived from E1+E2+/H-Rasv12 and E1−E2−/H-Rasv12/MSCV-GFP-c-Myc cells. D) Graph showing individual and average volumes of E1+E2+/H-Rasv12 and E1−E2−/H-Rasv12/MSCV-GFP-c-Myc tumors. Asterisk indicates P<0.05.
Figure 4.
mir17-92 expression depends on Ets1 and Ets2 in HrasG12V transformed MEFs.
A) Fold change of pre-mir17-92 and B) individual microRNAs of the mir17-92 cluster: in the indicated genetic groups relative to control cells. Asterisk indicates P<0.05. C) Schematic illustration of the mir17-92 promoter showing conserved ETS(box) and MYC (triangle) binding sites, as well as the ChIP primers used (arrows) relative to the start RNA start site. D) ChIP performed on the indicated MEFs genotypes using IgG control and specific antibodies as indicated. Asterisk indicates P<0.05.
Figure 5.
Ets1, Ets2 and c-Myc activate miR17-92 transcription.
C-Mycf/f MEFs were infected with pBabe-Cre retroviral vector or control pBabe-empty vector, selected with puromycin before functional examination. A) Protein analysis of 20 µg lysates from MEFs by western blot using antibodies as indicated B) Relative expression of Ets1, Ets2 and c-Myc mRNAs after 48 hrs of transfection of expression vector. Asterisk represents P<0.05. C and D) Fold change relative real time PCR gene expression of pre-mir17-92 (C) and individual miRs of the cluster (D) after 48 hrs transient transfection of the indicated vectors in c-Myc−/− MEFs relative to control empty vector. Asterisk indicates P<0.05.
Figure 6.
MiR-17-92 overexpression in Ets1/Ets2-null MEFs rescue HrasG12V transformation.
E1−E2−/pBabe control and E1−E2−/H-Rasv12 were infected with either MSCV-puro empty control or MSCV-puro-miR-17- 92 vector and cells were selected by Puromycin before further functional analysis. A) pre-mir17-92 cluster expression in the indicated genotypes relative to control vector. Asterisk indicated P<0.05. B) Graph demonstrating the percentage of tumors formed over the total number of injections for the different cellular groups. N/A indicates that there were no tumors observed for the specified group. (C) Representative images showing the total E1+E2+/H-Rasv12 and E1−E2−/H-Rasv12/MSCV-puro-miR-17-92 derived tumors. D) Graph indicating individual and average volume of E1+E2+/H-Rasv12 and E1−E2−/H-Rasv12/MSCV-puro-miR-17-92 tumors.