1. Volume 4, Issue 4, Pages 764-775 (August 2013)
Opposing Effect of EGFRWT on EGFRvIII-Mediated NF-κB Activation with RIP1 as a Cell Death Switch 
Vineshkumar Thidil Puliyappadamba, Sharmistha Chakraborty, Sandili S. Chauncey, Li Li, Kimmo J. Hatanpaa, Bruce Mickey, Shayan Noorani, Hui-Kuo G. Shu, Sandeep Burma, David A. Boothman, Amyn A. Habib 
Cell Reports 
Volume 4, Issue 4, Pages (August 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 764-775DOI: (10.1016/j.celrep.2013.07.025)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 EGFRvIII Activates NF-κB
(A) Various cell lines with stable expression of EGFRvIII and endogenous EGFRWT are shown. U1 cells are U87MG cells conditionally expressing EGFRvIII in response to tetracycline (tet). U251vIII cells are U251MG cells conditionally expressing EGFRvIII in response to tetracycline. LN229-V cells are transfected with empty vector, whereas LN229-vIII cells have stable and constitutive expression of EGFRvIII. Endogenous EGFRWT can be detected in cells as a slower migrating band above the EGFRvIII. Expression of EGFRvIII in various cell lines results in increased phosphorylation of the p65 subunit of NF-κB.
(B) Reporter assays show increased transcriptional activity of NF-κB using a luciferase assay. Conditional or constitutive expression of EGFRvIII results in increased NF-κB reporter activity (∗∗∗p < , one-way ANOVA). Error bars indicate SD. RLU, relative luciferase units.
(C) We screened a panel of early-passage primary GBM cultures grown as NSs for the presence of EGFRWT and EGFRvIII.
(D) GBM9-NS cells were treated with either erlotinib (10 μM) or necrostatin (300 nM) overnight followed by western blot for phospho-p65.
(E) A reporter assay shows that the transcriptional activity of NF-κB in tetracycline-treated U251vIII is abolished by exposure to EGF. Data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test to validate the levels of significance between groups and were statistically significant for all groups (∗∗∗p < 0.05). Error bars indicate SD.
(F) Conditional expression of EGFRvIII in U1 cells results in activation of NF-κB reporter activity, which is abolished by addition of EGF (50 ng/ml) (∗∗∗p < , one-way ANOVA). Error bars indicate SD.
(G) EGFRvIII levels in U1 and U251vIII cells are comparable to EGFRvIII levels in resected GBM specimens (GBMs 6 and 10 have high levels of EGFRvIII).
See also Figures S1, S2, and S3.
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4. Figure 2
RIP1 Is Essential for EGFRvIII-Mediated NF-κB Activation and Oncogenicity
(A) RIP1 is essential for EGFRvIII-mediated activation of NF-κB transcriptional activity in U1 cells. siRNA-mediated knockdown of RIP1 blocks the ability of EGFRvIII to activate NF-κB. Addition of tetracycline results in expression of EGFRvIII in both RIP1-silenced and control (scrambled) siRNA control, but NF-κB is activated only in control siRNA cells (∗∗p = , one-way ANOVA). Error bars indicate SD.
(B) Silencing of RIP1 is shown by western blot.
(C) EGFRvIII-mediated transcriptional activity of NF-κB is blocked by necrostatin-1 in U1 cells (∗p = 0.017). Error bars indicate SD.
(D) Stable silencing of RIP1 in U1 cells using RIP1 shRNA is presented. Two independent clones with efficient silencing of RIP1 L11 and S1 are shown along with RIP1 levels in control scrambled shRNA (C)-expressing cells.
(E) Demonstration of doxycycline (doxy) penetrating into the intracranial tumors to induce expression of EGFRvIII. Protein lysates were made from U1-derived intracranial tumors in mice followed by western blot with EGFR. Mice 312 and 314 were exposed to doxycycline in drinking water and food and show expression of both EGFRvIII and EGFRWT, whereas 311 and 313 were not exposed to doxycycline and demonstrate expression of endogenous EGFRWT only.
(F) H&E sections from a representative brain section from the control shRNA group shows the presence of brain tumor, whereas the brain section from the L11 mouse is free of tumor.
(G) Expression of EGFRvIII and RIP1 in tumors from two mice from the control shRNA group is presented. Lysates were prepared directly from tumor tissue followed by western blot.
(H) Kaplan-Meier survival analysis of RIP1-silenced cells (L11) compared to control shRNA expression implanted intracranially in mice (n = 8) is shown. The log rank test was significant (χ2(1) = 15.47; p < ). All mice were exposed to doxycycline in food and water.
See also Figures S4 and S5.
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5. Figure 3
EGFRvIII Recruits Ubiquitin Ligases to RIP1 Resulting in K63-Linked Ubiquitination of RIP1
(A) U1 cells were exposed to tetracycline to express EGFRvIII, followed by immunoprecipitation (IP) with cIAP2 antibodies and western blot with EGFR and RIP1.
(B and C) U1 cells were exposed to tetracycline to express EGFRvIII, followed by immunoprecipitation with TRAF2 or cIAP-1 antibodies and western blot with EGFR and RIP1.
(D)–(F) The same result is shown in U251vIII cells.
(G) U1 cells were exposed to tetracycline, with or without EGF (50 ng/ml) as indicated, followed by IP with RIP1 and western blot with K63-specific ubiquitin antibodies or RIP1 antibodies. An increased K63-linked ubiquitination of RIP1 is detected in tetracycline-treated cells that decreases following exposure to EGF for 1 hr.
(H) The same results are shown in a second U87MG clone conditionally expressing EGFRvIII (U26).
See also Figure S5.
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6. Figure 4 Formation of an EGFRvIII-Associated Signaling Platform
(A) Expression of EGFRvIII in tetracycline-treated U1 cells (left panel) or U251vIII cells (right panel) results in association with RIP1. The EGFRvIII-RIP1 association is abolished by EGF. The experiment was conducted by treating cells to EGF followed by immunoprecipitation with EGFR antibody and western blot with RIP1 antibody. No association with EGFRWT is detected.
(B) Expression of EGFRvIII in tetracycline-treated cells induces association of RIP1 and NEMO and NEMO and EGFRvIII in U1 cells (left panel) and U251vIII cells (right panel). EGF treatment abolishes the RIP1-NEMO association and the NEMO-EGFRvIII association. Immunoprecipitation was conducted with NEMO antibodies followed by western blot with RIP1 antibodies.
(C) Expression of EGFRvIII in tetracycline-treated cells induces association of RIP1 and TAK1 and TAK1 and EGFRvIII in U1 cells (left panel) and U251vIII cells (right panel). EGF treatment abolishes the RIP1-TAK1 association and the TAK1-EGFR association. Immunoprecipitation was conducted with TAK1 antibodies followed by western blot with RIP1 antibodies. EGF treatment was for 15 min in (A)–(C).
(D) In GBM9-NS cells, the RIP1-EGFRvIII association is diminished by necrostatin-1 and to a lesser degree by erlotinib. Immunoprecipitation was conducted with EGFR antibodies followed by western blot with RIP1 antibody.
(E) In GBM9-NS cells, the NEMO-EGFR association is decreased by both necrostatin-1 and erlotinib, whereas the NEMO-RIP1 association is decreased by erlotinib, but not necrostatin-1. Immunoprecipitation was conducted with NEMO antibodies followed by western blot with EGFR or RIP1.
(F) RIP1 was silenced using RIP1 siRNA in U1 (left panel) and U251vIII cells (right panel), followed by immunoprecipitation with NEMO and western blot with EGFR in the presence or absence of tetracycline. The NEMO-EGFRvIII association is lost if RIP1 is silenced.
(G) The EGFRvIII-TAK1 association is unaffected by RIP1 silencing in U1 cells.
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7. Figure 5 Association of RIP1 with FADD and Caspase-8
(A) Association of RIP1 with FADD following EGF exposure in EGFRvIII-expressing U1 cells is shown. FADD becomes associated with RIP1 after EGF exposure for 1 hr in tetracycline-treated cells. Immunoprecipitation was done with FADD antibody followed by western blot with RIP1.
(B) Association of RIP1 with FADD following EGF exposure in U251-vIII cells is presented.
(C) Association of RIP1 and caspase-8 in U1 cells is shown. RIP1 is associated with caspase-8 even in the absence of EGFRvIII in these cells.
(D) In U1 cells, there is no increase in the number of Annexin-positive or PI-positive cells with either tetracycline or EGF exposure (50 ng/ml for 24 hr). Error bars indicate SD.
(E) EGFRvIII becomes constitutively tyrosine phosphorylated in U1 cells following expression. Addition of EGF results in increased tyrosine phosphorylation of EGFRWT but has no effect on EGFRvIII tyrosine phosphorylation.
(F) EGFRvIII and EGFRWT form a complex with each other that does not appear to be influenced by EGF. EGFRWT-Myc or EGFRvIII-HA was transfected in U87MG cells followed by immunoprecipitation with Myc and western blot with HA antibody.
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8. Figure 6
Ligand-Mediated Activation of EGFRWT in U1WT Cells Leads to a RIP1-Dependent Cell Death
(A) U1WT cells were exposed to EGF (50 ng/ml) for 24 hr, and an Annexin-FACS assay was performed. Unstained cells represent viable cells. Annexin-positive cells are undergoing apoptosis. PI-positive cells are undergoing necrosis. In “Control,” cells are treated with control vehicle (ve; PBS). In the absence of EGF, there is no cell death. When EGF is added to tetracycline-treated cells, the number of viable cells is decreased (p < ), and there is evidence of both apoptotic (p < 0.005) and necrotic cell death (p < 0.005). Double-stained cells (Annexin+PI positive) are also shown.
(B) Necrostatin-1 (300 nM) inhibits the EGF-induced cell death, demonstrating the requirement for RIP1 in EGF-induced cell death. There is an increase in viable cells (p < 0.009) and a decrease in apoptotic (p < 0.001) and necrotic cells (p < 0.001). As a control for necrostatin-1, cells were treated with DMSO in EGF+tet cells. Data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison test.
(C) siRNA knockdown of RIP1 in U1WT cells inhibits EGF-induced apoptotic cell death. Cells were transfected with control (scrambled) or RIP1 siRNA followed by EGF exposure for 24 hr and FACS analysis to compare Annexin-positive cells. Exposure to EGF leads to substantial increase in apoptotic (Annexin-positive) cells in control siRNA cells. This apoptotic cell death is inhibited in RIP1 siRNA cells (p < ).
(D) siRNA knockdown of RIP1 in U1WT cells inhibits EGF-induced necrotic cell death. Exposure to EGF (24 hr) leads to substantial increase in necrotic (PI-positive) cells in control siRNA cells. This necrotic cell death is inhibited in RIP1 siRNA cells (p < ).
(E) Double-stained cells (positive for Annexin and PI) for the same experiment are shown.
(F) RIP1 and caspase-8 form a complex with FADD in response to EGF in U1WT cells. Upon addition of EGF, there is a rapid increased association of RIP1 and caspase-8 with FADD. Cells were exposed to tetracycline in all lanes to induce expression of EGFRvIII.
Error bars indicate SD. See also Figures S6 and S7.
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9. Figure 7 RIP1 Expression Correlates with Activation of NF-κB in GBM
(A) Western blot shows expression of EGFRWT, EGFRvIII, and phospho-p65 in a panel of 23 GBMs. Lysates were made directly from resected tumors followed by western blot. EGFRvIII is expressed in only a subset of GBMs (usually with EGFRWT), whereas phosphorylation of p65 can be detected in the majority of GBMs as can RIP1.
(B) RIP1 correlates with phosphorylation of p65 in GBMs. There was a significant positive correlation between the expression of RIP1 and pp65 using Pearson correlation (r = 0.49; p < 0.05).
See also Figure S7.
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10. Figure S1
EGFRvIII Kinase Activity and RIP1 Are Required for EGFRvIII-Mediated NF-κB Activation, Related to Results
(A) U26 cells are derived from U87MG cells and express EGFRvIII conditionally in response to tetracycline. Expression of EGFRvIII in U26 cells results in increased phosphorylation of the p65 subunit of NF-κB.
(B) Activity of a transfected NF-κB luciferase reporter is high in GBM9-NS cells. Both Erlotinib and Necrostatin-1 inhibit NF-κB activity in these cells (p<0.0001, 1way ANOVA). Error bars indicate SD.
(C) U1 cells express EGFRvIII in response to tetracycline. EGFRvIII becomes constitutively autophosphorylated upon expression. We examined the effect of various concentrations of Erlotinib on the kinase activity of EGFRvIII by conducing Western blots for phospho-EGFR (Tyr-1068).
(D) A dose response curve of the effect of Erlotinib on phosphorylation of EGFR. The phospho-EGFR signal on Western blot was quantitated by densitometry.
(E) Tyrphostin AG 1478 is another specific inhibitor of EGFR kinase and was used in a similar experiment at a concentration of 100nM, resulting in complete inhibition of EGFRvIII kinase activity.
(F) A reporter assay showing that the transcriptional activity of NF-κB is increased in tetracycline treated U26 cells. However, the tetracycline induced increase is abolished by exposure to EGF (P <0.0001, 1way ANOVA). Error bars indicate SD.
(G) NF-κB activity is increased in LN229 cells expressing EGFRvIII constitutively compared to LN229 cells transfected with empty vector. Addition of EGF results in a loss of EGFRvIII mediated NF-κB activity (1way ANOVA, p=0.002). Error bars indicate SD.
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11. Figure S2
Activation of EGFRWT Abolishes EGFRvIII-Mediated NF-κB Activation, Related to Results
(A) Electrophoretic mobility shift assay in U1 cells. Exposure of cells to tetracycline (24h) results in EGFRvIII expression and increased NF-κB DNA binding activity. Exposure of cells to EGF (50ng/ml) for 2h or 6h abolishes the EGFRvIII induced NF-κB DNA binding activity.
(B) U251vIII cells express conditionally express EGFRvIII in response to tetracycline. U251vIII also express endogenous EGFRwt. In this experiment we selectively silenced EGFRwt in U251vIII cells by using shRNA directed against exon 3 in EGFRwt (exon 3 is missing in EGFRvIII). We used control (scrambled) shRNA as a control. Western blot showing effective silencing of EGFRwt in U251vIIIshwt cells while the U251vIIIshC cells show the endogenous EGFRwt expressed in these cells.
(C) Next, we conducted a reporter assay examining the transcriptional activity of NF-κB in these cells in response to EGF. Exposure of U251vIIIshC cells to EGF leads to a sharp drop in reporter activity (p≤0.0001, Tukey's Multiple Comparison Test) (EGF 50ng/ml, 6h). However, EGF does not decrease the NF-κB reporter activity in U251vIIIshwt cells, indicating that EGF acts via the EGFRwt in these cells. Renilla luciferase was used as an internal control. Error bars indicate SD.
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12. Figure S3
Coexpression of EGFRWT and EGFRvIII in GBM Cells, Related to Results
Single cell suspensions were generated from neurosphere cultures of GBM9-NS and GBM748-NS. Both these tumors express both EGFRwt and EGFRvIII.
(A) Four clones derived from single cells from GBM9-NS show that in each clone there is co-expression of both EGFRwt and EGFRvIII. Lysates from U251vIII cells with or without tetracycline were run to show the relative positions of EGFRwt (no tetracycline) and EGFRvIII (tetracycline) conditions.
(B) Four clones derived from single cells from GBM748-NS show that in each clone there is co-expression of both EGFRwt and EGFRvIII. The U87MG lane shows the position of EGFRwt while the U251vIII+tet lane shows the position of EGFRvIII in the gel.
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13. Figure S4
RIP1 Is Essential for EGFRvIII-Mediated Activation of NF-κB Transcriptional Activity in U251-vIII Cells, Related to Results
(A) siRNA mediated knockdown of RIP1 blocks the ability of EGFRvIII to activate NF-κB. Addition of tetracycline results in expression of EGFRvIII in both RIP1 silenced and control (scrambled) siRNA control but NF-κB is activated only in control siRNA cells (p=0.001). Error bars indicate SD.
(B) Silencing of RIP1 is shown by Western blot.
(C) EGFRvIII mediated transcriptional activity of NF-κB is blocked by Necrostatin-1 in U251vIII cells (p=0.0001). Error bars indicate SD.
(D) We examined the effect of various concentrations of Necrostatin-1 on the kinase activity of RIP1 by conducing Western blots for phosphorylation of the p65 subunit of NF-κB in U1 cells.
(E) A dose response curve of the effect of Necrostatin-1 on phosphorylation of p65. The p65 signal on Western blot was quantitated by densitometry.
(F) This result was validated with siRNA knockdown of RIP1. Silencing of RIP1 decreases EGFRvIII mediated phosphorylation of the p65 subunit of NF-κB in U1 cells.
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14. Figure S5
RIP1 Regulates EGFRvIII-Mediated Cell Proliferation, Related to Results
(A) A cell counting experiment demonstrating that RIP shRNA clone L11 cells proliferate slowly in cell culture compared to Control shRNA expressing cells (1way ANOVA, p=0.0046). Cell death was excluded by trypan blue exclusion prior to automated cell counting. Error bars indicate SD.
(B) The same experiment was conducted in clone S1 (1way ANOVA, p=0.0011). Error bars indicate SD.
(C) An MTT conversion assay was conducted comparing U1 cells with stably silenced RIP1 (L11) to U1 cells with control shRNA. MTT conversion is higher in control cells compared RIP1 silenced cells suggesting increased proliferation in control cells (two tailed t test, p=0.002). Cell death was excluded by trypan blue exclusion in parallel experiments. Error bars indicate SD.
(D) U251vIII cells were exposed to tetracycline, with or without EGF (50ng/ml) as indicated followed by denaturation and IP with RIP1 and Western blot with K63 ubiquitin antibodies or RIP1 antibodies. The level of K63-linked ubiquitination of RIP1 is high in these cells and does not increase with tetracycline. However, a loss of K63-linked ubiquitination of RIP1 is seen in these cells after prolonged EGF exposure (24h).
(E) U1 cells were engineered to stably and constitutively express high levels of EGFR wild type (U1wt cells). Thus, U1wt cells express high levels of EGFRwt constitutively and EGFRvIII in response to tetracycline (lanes 1-4). Lanes 5 and 6 show EGFR levels in actual GBM tumors (GBM6 and GBM9). Lysates were made directly from resected tumor tissue. Two exposures are shown to demonstrate the separation of EGFRwt from EGFRvIII and also to show the endogenous EGFRwt in U1 cells.
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15. Figure S6
Activation of EGFRWT Leads to a RIP1-Dependent Cell Death in U251vIIIwt Cells, Related to Results
(A) U1wt cells were exposed to EGF for 72 hours. There is no cell death in the absence of EGF. When EGF is added for 72h, there is substantial cell death (tet+EGF) shown by the decrease in the number of viable cells (p<0.0001), with most of the cell death being necrotic as shown by the PI positive cells (p<0.0001). Addition of RIP1 kinase inhibitor Necrostatin-1 inhibits EGF induced necrotic cell death (tet+EGF+necrostatin). As a control for Necrostatin-1 cells were treated with DMSO in tet+EGF cells. Data were analyzed by 1way ANOVA followed by Newman-Keuls Multiple Comparison test. Error bars indicate SD.
(B) Effective silencing of RIP1 in U1 cells was confirmed with a Western blot for the experiment shown in Figures 6C–6E.
(C) Stable overexpression of EGFRwt in U251vIII cells. U251vIII cells were stably transfected with control vector or EGFRwt. U251vIIIwt cells overexpress EGFRwt compared to U251vIIIC cells that express endogenous EGFRwt.
(D) When U251vIIIwt cells are exposed to EGF, they undergo cell death that is largely necrotic (PI positive cells). If the RIP1 inhibitor, Necrostatin-1 is used (300nM), EGF no longer induces cell death. The data were analyzed by one way analysis of variance (ANOVA) followed by Newman-Keuls Multiple Comparison Test. Cell viability is decreased in Tet +EGF condition (p≤0.0001) in comparison to no Tet/Tet and Tet+EGF+Necrostatin conditions. There is a significant increase in PI positive cells after EGF treatment in Tet+EGF group, when compared to no Tet/ Tet and Tet+EGF+Necrostatin conditions (p≤0.0001). Error bars indicate SD.
(E) EGF abolishes EGFRvIII mediated NF-κB activation in U1wt cells. Expression of EGFRvIII by exposure of cells to tetracycline results in NF-κB activation as assessed by a NF-κB reporter assay. Addition of EGF to cells results in a loss of EGFRvIIImediated NF-κB activation. This is consistent with the results found in U1 cells. Error bars indicate SD.
(F) EGF mediates increased association of FADD and RIP1 in U1wt cells for up to 24h. Immunoprecipitation of FADD was followed by Western blot with RIP1 antibodies.
(G) Caspase-8 is complexed with RIP1 in U1wt cells and this association does not change significantly with EGF over time.
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16. Figure S7
FACS Analysis of EGF-Mediated Cell Death and Expression of EGFRvIII in GBMs, Related to Results
(A) FACS plot original data for the graph shown in Figures 6A and 6B.
(B) Quantitative real time PCR was performed to detect EGFRvIII in RNA extracted from individual tumors. Increased EGFRvIII can be detected by qPCR in all tumor samples demonstrating expression of EGFRvIII by Western blot except for GBM4.
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