Supplementary Figure 1

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Bernadette McKinney
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1 Supplementary Figure 1 Constitutive EGFR signaling does not activate canonical EGFR signals (a) U251EGFRInd cells with or without tetracycline exposure (24h, 1µg/ml) were treated with EGF for 15 minutes followed by preparation of lysates and Western blot with the indicated antibodies. (b) U87V or U87EGFR cells were treated with EGF for 15 minutes followed by preparation of lysates and Western blot with the indicated antibodies. (c) Tyrosine phosphorylation of the EGFR at residues 845 and 1173 was examined in U251V cells and U251EGFR cells in the absence of EGF. (d) In this experiment we compared tyrosine phosphorylation of the EGFR at residue 1068 in U251V cells compared to U251EGFR cells. Equal amounts of protein were loaded. (e) shows the same experiment as (d). except that that less protein (15%) was loaded in the U251EGFR lane in order to load equal amounts of EGFR. Tyrosine phosphorylation of the EGFR is still detectable only in U251EGFR cells. Densitometry was performed for A-E for pegfr/egfr and the values are shown. Cells were serum starved overnight for all experiments.

2 Supplementary Figure 2 Regulation of gene-transcription by EGFRwt and mutants (a) An experiment examining mrna levels of EGR2 in U251EGFR cells. In contradistinction to IFIT1 and IFI27, EGR2 is low in the absence of EGF and increased when cells are exposed to EGF (p<0.001). (b) Cetuximab fails to inhibit EGFR mediated induction of IFI27 mrna as detected by real time quantitative PCR. Cells were incubated overnight with either control IgG or Cetuximab (100ug/ml). (c) U251 cells conditionally expressing the mutant EGFRvIII in response to tetracycline were incubated with tetracycline (or no tetracycline) for 24h followed by RNA extraction and qpcr for IFI27. (d) U251 cells were transfected with EGFRwt, a kinase inactive EGFR mutant, or empty vector followed by extraction of RNA and qpcr for IFI27. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. Error bars represent the means±standard deviations of three independentexperiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

3 Supplementary Figure 3 EGFR regulates IFIT1 and IFI27 in MDAMB468 cells (a) Quantitative real time PCR examining IFIT1 mrna levels in MDAMB468 cells. In the absence of EGF, IFIT1 level is high and diminishes in response to EGF exposure (p<0.0001). In addition, use oferlotinib also diminishes IFIT1 mrna suggesting that the high level is EGFR driven (p<0.0001). (b) The same experiment examining IFI27 mrna. (c) A Western blot showing IFIT1 protein levels in MDAMB468 cells which is decreased in cells exposed to EGF for 6h. (d) ELISA for IFI27 levels in MDAMB468 cells shows a high level of IFI27 in the absence of EGF. When EGF is added IFI27 levels are decreased. Exposing cells to Erlotinib also results in a decrease in IFI27 levels. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

4 Supplementary Figure 4 EGFR regulates IFIT1 and IFI27 in glioma cell lines (a) In this experiment we used U251EGFRind cells that express high levels of EGFR conditionally in response to tetracycline and examined the level of IFIT1mRNA by quantitative real time PCR. In the absence of tetracycline IFI27 mrna levels are low with or without EGF. When tetracycline is added (1µg/ml for 24h), there is substantial increase in IFIT1 mrna. When EGF is added to tetracycline treated cells, IFIT1 mrna levels are downregulated (p<0.0001). (b) The same experiment examining IFI27 mrna levels. (c) Quantitative real time PCR examining IFIT1 mrna levels in U87EGFR cells and in empty vector transfected U87V cells. In U87EGFR cells IFIT1 level is high in the absence of EGF and diminishes in response to EGF exposure (p<0.0001). (d) The same experiment was conducted examining IFI27 mrna levels. (e) Western blot showing that the IFIT1 level is high in U87EGFR cells and decreased when cells are exposed to EGF for 6h. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. The EGF concentration used was 50ng/ml. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

5 . Supplementary Figure 5 EGFR regulated IFIT1, IFI27 and TRAIL expression (a) An experiment examining IFIT1 mrna levels in GICs (glioma initiating cells) cultured as neurospheres derived from GBM429 (NS: neurosphere). In the 429NS condition, cells are grown continuously in medium containing EGF and show low levels of IFIT1 mrna. In 429NS-EGF condition, cells are grown in EGF free medium for 24h and show a high level of IFIT1 mrna. In 429NS+EGF, cells are grown in EGF free medium for 24h, then EGF is added for 4h, resulting in a decrease in IFIT1 mrna (p<0.0001). In 429NS+Erlotinib, cells are grown in EGF free medium for 24h in the presence of Erlotinib (10µM) again resulting in a decrease in IFIT1 mrna (p<0.0001). In the absence of EGF, IFIT1 level is high in the absence of EGF and diminishes in response to EGF exposure (p<0.0001). In addition, use of Erlotinib also diminishes IFIT1 mrna suggesting that the high level is EGFR driven (p<0.0001). (b) The same experiment examining IFI27 mrna in 429NS cells. (c) Quantitative real time PCR examining TRAIL mrna levels in U251EGFR cells. In the absence of EGF, TRAIL level is high in U251EGFR cells but not in U251V (vector transfected) cells and diminishes in response to EGF exposure in U251EGFR cells (p<0.0001). (d) The same experiment in U87EGFR cells (p<0.0001). (e) The same experiment in MDAMB468 cells. Since there is no vector control here, we used Erlotinib to demonstrate that TRAIL levels are EGFR driven. Addition of EGF or ErlotinibdownregulatesTRAIL levels in MDAMB468 cells (p<0.0001).error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

6 Supplementary Figure 6 EGFR induced IFIT1 and IFI27 requires IRF3 (a, b) U87EGFR cells were transfected with control (scrambled) sirna or with sirna directed against IRF3 followed by quantitative real time PCR for IFIT1 or IFI27. The mrna levels for both genes are decreased when IRF3 is silenced compared to control sirna. (c) shows silencing of IRF3 by Western blot. (d) The transcriptional activity of IRF3 was examined by using a synthetic pisre-luc reporter in U87EGFR cells with a dual luciferase assay. The activity of IRF3 is high in U87EGFR cells compared to U87V (vector transfected cells). Addition of EGF leads to a statistically significant decrease in reporter activity. Furthermore, Erlotinib also downregulates reporter activity, indicating that the high IRF3 transcriptional activity in the absence of EGF is driven by EGFR. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. (e) The same experiment as (d) was conducted in U87EGFR cell using IFI27-Luc plasmidto examine IRF3 transcriptional activity. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

7 Supplementary Figure 7 Constitutive EGFR signaling activates IRF3 transcriptional activity in MDAMB468 cells. The transcriptional activity of IRF3 was examined by using a synthetic pisre-luc reporter in MDAMB468 cells with a dual luciferase assay. (a) The activity of IRF3 is high in MDAMB468 cells. Addition of EGF leads to a statistically significant decrease in reporter activity. Furthermore, Erlotinib also downregulates reporter activity, indicating that the high IRF3 transcriptional activity in -these cells is driven by EGFR. (b) The same experiment as (a) was conducted in MDAMB468 cells using IFI27-Luc plasmid to examine IRF3 transcriptional activity. Cells were serum starved and cultured in serum free DMEM overnight for all experiments. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

Supplementary Figure 8 EGFR mediated affects on IRF3 promoter occupancy (a) The effect of Cetuximab on EGFR driven transcriptional activity of IRF3 was examined by using a synthetic pisre-luc

8 Supplementary Figure 8 EGFR mediated affects on IRF3 promoter occupancy (a) The effect of Cetuximab on EGFR driven transcriptional activity of IRF3 was examined by using a synthetic pisre-luc reporter in MDAMB468 cells using a dual luciferase assay. The EGFR induced transcriptional activity of IRF3 is not blocked by Cetuximab compared to control IgG in U251EGFR cells. (b) A CHIP assay examining IFIT1 promoter occupancy by IRF3 in MDAMB468 cells with or without EGF. IRF3 promoter binding can be detected in the absence of EGF and decreases by 6h of EGF stimulation and remains decreased 24h after EGF treatment. (c) A CHIP assay examining IFI27 promoter occupancy by IRF3 in MDAMB468 cells with or without EGF. IRF3 promoter binding can be detected in the absence of EGF and decreases by 6h of EGF stimulation coming back to baseline at 24h. (d) A CHIP assay examining IFIT1 promoter occupancy by IRF3 in U87EGFR cells with or without EGF. IRF3 promoter binding can be detected in the absence of EGF and decreases by 6h of EGF stimulation and remains decreased 24h after EGF treatment. (e) A CHIP assay examining IFI27 promoter occupancy by IRF3 in U87EGFR cells with or without EGF. IRF3 promoter binding can be detected in the absence of EGF and decreases by 6h of EGF stimulation and remains decreased 24h after EGF treatment. (f, g) Overexpression of the LDL receptor fails to upregulate IFIT1 or IFI27 mrna in U251MG cells as measured by qpcr. EGFR was used as a positive control. (h) Overexpression of the LDL receptor in U251MG cells detected by Western blot. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

9 Supplementary Figure 9 Constitutive EGFR signaling does not result in increased ER stress (a) U87V cells (transfected with empty vector) and U87EGFR cells were examined for phosphorylation of EIF2α. EGF or Erlotinib were added as indicated followed by lysate preparation and Western blot with phospo-eif2α antibodies. Densitometry was performed to quantitate the peif2α signal. (b) Splicing of XBP-1 was examined by Western blot in U251EGFR cells. Splicing of XBP-1 can only be detected in EGF treated cells. (c) Splicing of XBP-1 was examined at an RNA level by treating U251EGFR or MDAMB468 cells with EGF as indicated followed by RNA extraction and reverse transcriptase PCR. Thapsigargin was used as a positive control. (d) U251EGFR cells were treated with a PERK inhibitor, GSK (1μM) overnight followed by RNA extraction and qpcr for IFI27. GSK fails to prevent EGFR mediated IFI27 induction, (e) GSK fails to prevent EGFR induced activation of IRF3 mediated transcriptional activity assessed using a synthetic pisre-luc reporter. (f, g) MDAMB468 cells were treated with a PERK inhibitor, GSK (1μM) overnight followed by RNA extraction and qpcr for IFIT1 or IFI27. GSK fails to prevent EGFR mediated IFIT1 or IFI27 induction. (h) U251EGFR cells were treated with an ATF6 inhibitor, AEBSF (300μM) overnight followed by RNA extraction and qpcr for IFI27. AEBSF fails to prevent EGFR mediated IFI27 induction.error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

10 Supplementary Figure 10 Inhibition of ER stress fails to block constitutive EGFR signaling (a) GSK fails to prevent EGFR induced activation of IRF3 mediated transcriptional activity in U251EGFR cells assessed using a synthetic pisre-luc reporter. (b) AEBSF fails to prevent EGFR induced activation of IRF3 mediated transcriptional activity in U251EGFR assessed using a synthetic pisre-luc reporter. (c, d) MDAMB468 cells were treated with an ATF6 inhibitor, AEBSF, followed by RNA extraction and qpcr for IFIT1 and IFI27. AEBSF fails to prevent EGFR mediated IFI27 induction. (e) AEBSF fails to prevent EGFR induced activation of IRF3 mediated transcriptional activity in MDAMB468 cells assessed using a synthetic pisre-luc reporter. (f) Silencing XBP-1 fails to prevent EGFR induced induction of IFI27 in U251EGFR cells. (g) Silencing XBP-1 fails to prevent EGFR induced activation of IRF3 mediated transcriptional activity assessed using a synthetic pisre-luc reporter in MDAMB468 cells. (h) Effective silencing of XBP-1 is demonstrated in MDAMB468 cells by Western blot. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

11 Supplementary Figure 11 EGFR associated signaling complexes (a) EGFR co-immunoprecipitates with IRF3 in U87EGFR cells in the absence of EGF. When EGF is added there is a rapid loss of the EGFR-IRF3 association. (b) Similarly, the EGFR coimmunoprecipitates with TBK1 in U87EGFR cells. When EGF is added there is a rapid loss of the EGFR-TBK1 association. (c) EGFR drives TBK1-IRF3 association in U87EGFR cells. TBK1 coimmunoprecipitates with IRF3 in the absence of EGF. When EGF or Erlotinib is added the TBK1-IRF3 association is disrupted. (d) A coimmunoprecipitation experiment examining the EGFR-Shc association in U87EGFR cells. In the absence of EGF there is no association between Shc and EGFR. Shc becomes associated with the EGFR only when EGF is added to U87EGFR cells. For (a-d). Cells were serum starved and cultured in serum free DMEM overnight for all experiments. (e) IRF3 localizes to the cytosolic compartment in U251V cells. Very little IRF3 can be detected in the membrane or nuclear fractions. (f) IKKε is not expressed in U251EGFR cells or U87EGFR cells. MCF7 cells were used as a positive control for IKKε expression. Three independent experiments were done.

12 Supplementary Figure 12 EGFR activates IFN signaling (a) There is a high level of IFNβ mrna in MDAMB468 cells. Addition of EGF or Erlotinib leads to a loss of this increase in IFNβ. (b) There is a high level of IFNα mrna in MDAMB468 cells. Addition of EGF or Erlotinib leads to a loss of this increase in IFNα. (c) High level of IFNβ in supernatants from U251EGFR and MDAMB468 cells. Both cell lines express high levels of EGFR. (d) U251EGFR and U87EGFR cells do not express IRF7 as determined by Western blot. Jurkat cells are used as a positive control. (e, f) Ruxolitinib blocks EGFR induced upregulation of IFIT1 and IFI27 in MDAMB468 cells. Cells were incubated with Ruxolinitib (1uM) overnight followed by extraction of RNA and qpcr for IFI27 or IFIT1. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

13 Supplementary Figure 13 JAK inhibitors block EGFR mediated IFIT1 and IFI27 induction (a) U251EGFR cells were incubated with Tofacitinib (300nM) overnight followed by extraction of RNA and qpcr for IFI27. Tofacitinib blocks EGFR mediated induction of IFI27. (b, c) MDAMB468 cells were incubated with Tofacitinib (300nM) overnight followed by extraction of RNA and qpcr for IFIT1 or IFI27. Tofacitinib blocks EGFR mediated induction of IFIT1 and IFI27. (d, e) JAK inhibitors fail to inhibit ERK and Akt activation induced by EGF in U251EGFR cells. Cells were serum starved and treated overnight with JAK inhibitors. EGF was used for 15 min at a concentration of 50ng/ml. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

14 Supplementary Figure 14 TLR3 in not expressed in glioma cell lines TLR3 is not expressed in U251EGFR and U87EGFR cells as determined by Western blot. MCF10A cells were used as a positive control. The experiment was repeated once.

15 Supplementary Figure 15 Ligand-activated EGFR is more sensitive to virus-induced cell death An MTT conversion assay was undertaken to determine whether EGFR overexpression alters sensitivity to adenovirus infection. (a) Comparison of the effects of adenovirus on U251V cells and U251EGFR cells, with or without EGF. U251EGFR cells are more resistant to adenovirus compared to U251V cells, and this resistance is lost when U251EGFR cells are exposed to EGF. (b) The same experiment performed in U87V and U87EGFR cells. EGF was added for 24h in serum free conditions. Error bars represent the means±standard deviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

16 Supplementary Figure 16 Ligand-activated EGFR is more sensitive to chemotherapy An MTT conversion assay was undertaken to determine whether EGFR overexpression alters sensitivity to temozolomide. (a) Comparison of the effects of temozolomide on U251V cells and U251EGFR cells, with or without EGF. U251EGFR cells are more resistant to temozolomide compared to U251V cells, and this resistance is lost when U251EGFR cells are exposed to EGF. (b) The same experiment performed in U87V and U87EGFR cells. (c) The same experiment was performed in MDAMB468 cells transfected with EGFR sirna or control sirna. In all cell lines EGFR overexpression confers resistance to temozolomide and this resistance is lost if EGF is added. EGF was added for 24h in serum free conditions. Error bars represent the means±standarddeviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

Supplementary Figure 17 Constitutive EGFR signaling in GBM. (a) shows the inverse relationship between TGFα levels and IFIT1 in GBM tumors overexpressing EGFR.

17 Supplementary Figure 17 Constitutive EGFR signaling in GBM. (a) shows the inverse relationship between TGFα levels and IFIT1 in GBM tumors overexpressing EGFR. (b) shows the inverse relationship between TGFα levels and IFI27 in GBM tumors overexpressing EGFR. (c) levels of TGFα, IFIT1 and IFI27 in tumors expressing a low level of EGFR as determined by real time quantitative PCR. (d) EGFR levels were examined in the 27 GBM tumors used in this study by Western blot. Error bars represent the means±standarddeviations of three independent experiments.data were analyzed by 1way ANOVA followed by Tukey s multiple comparison test. (p values and asterisks: 0.05 (*) 0.01 (**), (***).

18 Supplementary Figure 18 Uncropped blots Key uncropped western blot images are shown above.

NIH Public Access Author Manuscript Nat Commun. Author manuscript; available in PMC 2015 June 15.

NIH Public Access Author Manuscript Published in final edited form as: Nat Commun. ; 5: 5811. doi:10.1038/ncomms6811. Constitutive and ligand-induced EGFR signaling triggers distinct and mutually exclusive