HEK293FT cells were transiently transfected with reporters, N3-ICD construct and

Transcription

1 Supplementary Information Luciferase reporter assay HEK293FT cells were transiently transfected with reporters, N3-ICD construct and increased amounts of wild type or kinase inactive EGFR. Transfections were included with another reporter plasmid encoding Renilla luciferase under the cytomegalovirus promoter (prlcmv) µg as an internal control to measure transfection efficiency. After 48 h of transfection cells were harvested and dual luciferase assays were performed according to manufacturer s instruction (Promega). Luciferase readings were measured using BioTek multipurpose plate reader. The promoter reporter activity values were normalized to transfection control reporter Renilla luciferase readings in the same lysate. The data presented are the average of three assays. sirna transfections On target plus (OTP) sirnas were purchased from Dharmacon against non-targeting control (NTC) and Notch 1(5 -GCGACAAGGUGUUGACGUU, GAUGCGAGAUCGACGUCAA-3, 5 -GGACAUCACGGAUCAUAUG-3 and 5 - GAACGGGGCUAACAAAGAU-3 ) and Notch 3 (5 -CGACCUCACUUACGACUGU- 3, 5 -GAACUGCGAAGUGAACAUU-3, 5 -CCACGAGGAUGCUAUCUGU-3 and 5 -GUACUGCGCCGACCACUUU-3 ) genes and silencer select sirnas against Notch1, 3 and Non-targeting control were purchased from Invitrogen. HCC827 cells were reverse transfected with Dharmafect 2 reagent according to manufacturer s instructions. 24 hours after transfection cells were split 1:3 into dishes and proceeded to drug treatments. Following individual sirnas for Notch1 and Notch3 were purchased from Invitrogen.

2 sirnas: Notch1#1: 5 -GCCUGGACAAGAUCAAUGAtt-3 Notch1#2: 5 -GCCUGUCUGAGGUCAAUGAtt-3 Notch3#1: 5 -GGUGUGAACUGCGAAGUGAtt-3 Notch3#2: 5 -GGUCAGAAUUGUGAAGUGAtt-3 Notch3#3: 5 -GGCUUCACAGGAACCUAUUtt-3 Transfections were done using 5nM of each sirna in an 800 μl of OptiMEM and 20 μl of Lipofectamine RNAiMax (Invitrogen). Expression levels of Notch downstream targets Quantitative RT-PCR (qrt-pcr) was utilized to quantify expression of Notch downstream target genes, Hes1 and Hey1 using primers and method described earlier (26). RNA was extracted with an RNeasy Mini kit and cdna was synthesized using Taqman microrna Reverse Transcription Kit (Life Technologies) with 100 ng total RNA, per 20 μl reaction. Reverse transcription thermocycling parameters were as follows: 25 C for 10 min, 37 C for 60 min, 37 C for 60 min 85 C for 5 min; Reactions were performed on a MyCycler (Bio Rad, CA, USA). Real-time PCR was performed using ABI 7900HT system in 10 μl reaction volumes containing 1 μl of each primer, 5uL of 2 iq SYBR Green Supermix, and 2 μl of nuclease-free water, 1 μl of cdna (total 10 μl) in optical 384-well plates (Applied Biosystems). Cycling conditions: 95 C for 10 minutes, followed by 40 cycles of 95 C for 15 seconds and 60 C for 60 seconds. Triplicate qpcr reactions were performed for each cdna sample for all experiments. The threshold fluorescence level was set manually for each plate using SDS software version 2.3 (Applied Biosystems). Following export of Cycle threshold (Ct) data, further

3 data analysis for both platforms was performed in Microsoft Excel Comparison of slope and R2 values between pre-amplified and non-amplified cdna, as a template on the BioMark arrays was performed as a paired t-test in graph pad. Amplification of endogenous GAPDH was used as an internal control. Supplementary Figure 1. ALDH assay time course in HCC827 cells and gating based on DEAB and DMSO controls. Erlotinib treatment of HCC827, EGFR mutant lung cancer cells but resistant to erlotinib, increases the fraction of ALDH+ cells. Cells were treated with DMSO, 0.1 µm of erlotinib for increased period of time and subjected to aldefluor assay to detect the ALDH+ cells. A, A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate (ALDH negative cells) for flow cytometry analysis. B, Alternative to A, analysis was done gating exclusively based on cells containing high ALDH activity (ALDH positive cells) throughout the experiment. Supplementary Figure 2. Erlotinib treatment of H1650, EGFR mutant lung cancer cells but resistant to erlotinib, no changes in the fraction of ALDH+ cells. Cells were treated with DMSO, 0.1 µm of erlotinib for increased period of time and subjected to aldefluor assay to detect the ALDH+ cells. A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate (ALDH negative cells) for flow cytometry.

4 Supplementary Figure 3. Erlotinib treatment of EGFR wild type lung cancer cells increases the fraction of ALDH+ cells. A, A549 cells were treated with DMSO, 1 µm or 10 µm of erlotinib for three days and subjected to aldefluor assay to detect the ALDH+ cells. A portion of the cells was pre-incubated with the ALDH inhibitor DEAB (+DEAB) to provide a gate (ALDH negative cells) for flow cytometry. B, H358 cells were treated with DMSO, or 1 µm of erlotinib for three and five days and subjected to aldefluor assay to detect the ALDH+ cells. Supplementary Figure 4. Related to Figure 3. EGFR mediated negative regulation of Notch3 transcriptional activity in a tyrosine kinase-dependent manner. HEK293FT cells were transiently transfected with CSL reporter with empty vector, N3DA, EGFR or EGFR(KD) construct to determine the role of EGFR on transcriptional activity of endogenous Notch3 activity. Firefly luciferase activity was determined 48h after transfection and Renilla luciferase activity was used to normalize for transfection efficiency. The data presented are the average of three assays. Supplementary Figure 5, related to figure 5. Validation of Notch1 and 3 expression upon transfection with sirnas. A. HCC4006 and B. HCC827 cells. An equal amount of protein lysates was subjected to western blot analysis using Notch1, Notch3 and actin. Supplementary Figure 6. Validation of EGF-mediated association between EGFR and Notch 3 receptors in HCC2429 cells. HCC2429 cells were stimulated with EGF (25 ng/ml) for the indicated times. A. Cell lysate was prepared from each group and immunoprecipitated with various antibodies (i) EGFR (Ab12), (ii) EGFR (Ab15), and

5 (iii) EGFR (1005) and blotted with Notch3 antibody (top). A fraction of precipitate was also blotted with EGFR antibody (middle). B. An equal amount of each protein lysates was also subjected to western analyses using anti Notch 3, and tubulin to detect the total Notch 3 levels in the lysate. Supplementary Figure 7. EGFR kinase activity is necessary for the tyrosine phosphorylation of the Notch3 receptor. H1650 cells were treated with DMSO or erlotinib (0.1 µm) for indicated periods of time. Cell lysates from each time point were precipitated with a phosphotyrosine antibody followed by a western blot probed with Notch3 antibody (top), Whole cell lysate was also subjected to western analysis to check for of levels Notch3, EGFR-pY1173, and tubulin. Supplementary Table 1. Treatment with erlotinib increases ALDH+ cells in HCC827 cells. Cells were treated with DMSO or erlotinib for the increased periods of the time and subjected to aldefluor assay to determine the ALDH positive cells. Also, after the drug treatment total number of cells was determined and percentage of cell death was counted. Using total number of cells and ALDH+ cells data percentage of ALDH was determined. Calculations were performed based on the data obtained from the supplementary figure 1a and b.