Grb10 sequence data

This file contains Grb10 exomic sequences from tumors described in the manuscript

<i>Grb10</i> chromosomal position, inheritance, and loss in <i>Nf1</i> mutant mouse tumors.

<p>A. Proportion of tumors showing LOH along chromosome 11 in <i>Nf1</i> mutant tumors. (Carcinomas n = 26, sarcomas n = 26, pheochromocytomas n = 52) B. Breeding schema describing inheritance of mutant and wildtype <i>Nf1</i> genes. C. Microsatellites assessed by PCR-based fragment analysis to verify LOH in <i>Grb10</i> gene and identify parental allele involved (BL6 or 129). (**) indicates loss. Reduced C57Bl/6-derived peak in <i>Nf1</i> mutant cell lines 930, 989, 9223, indicating LOH of maternal C57Bl/6 allele, while cell line 867 has loss of the paternal 129-derived allele. D. Schematic of predominant pattern of <i>Grb10</i> and <i>Nf1</i> loss.</p

<i>Grb10</i> expression in diverse tumors suppresses proliferation.

<p>A. Wildtype <i>Grb10</i> or GFP was expressed in sarcoma line 963, which is wildtype for <i>Nf1</i>. <i>Grb10</i> restoration was confirmed by immunoblotting for the FLAG tag, and pAKT and pERK levels were assessed. B. Cell proliferation was assessed 4 days after plating and was reduced in 963 tumor cells after <i>Grb10</i> restoration (t-test, **p<0.01). C. Wildtype <i>Grb10</i> or GFP was expressed in ^<i>V16</i>HRas-transformed human astrocytes and 881 cells for comparison. <i>Grb10</i> overexpression was confirmed by immunoblotting for Flag, and phosphorylated and total AKT and ERK were assessed. To visualize the markedly different levels of <i>Grb10</i> expression between the cell lines, two exposures (3 minutes and 1 minute) are shown. D. Cell proliferation was assessed 4 days after plating was significantly reduced in 881 (null for <i>Nf1</i>) and ^<i>V16</i>HRas-transformed human astrocytes expressing <i>Grb10</i> (t-test, **p<0.01). E. The <i>NF1</i> mutant human tumor cell lines 02.2, 94.3, 96.2 were assessed for total Grb10 protein, and phosphorylated AKT and ERK by immunoblotting. F. GFP or FLAG-tagged wildtype <i>Grb10</i> was expressed in the human <i>NF1</i> mutant tumor cell line 02.2. Cell lysates were immunoblotted for the FLAG tag, phosphorylated AKT and ERK, total AKT and ERK, and Actin. G. 02.2 cells expressing either GFP or Grb10 were compared for differences in cell proliferation rates after 4 days in culture (t-test, **<0.01). The histogram is representative of 1 of 3 experiments.</p

<i>Grb10</i> silencing in MEFs increases Ras signaling.

<p>A. <i>Nf1</i>^<i>+/-</i> and <i>Nf1</i>^<i>-/-</i> MEFs expressing shRNA against <i>Trp53</i> and shRNA against <i>Luciferase</i> (control) or <i>Grb10</i> were serum starved overnight, then stimulated with insulin. Whole cell lysates were collected at shown timepoints and immunoblotting performed. B. Ras-GTP pulldown MEFs serum starved overnight, then stimulated with insulin. C/D. Wildtype and <i>Nf1</i>^<i>-/-</i> MEFs expressing shRNA against Luciferase (control) or <i>Grb10</i> were serum starved overnight, then stimulated with insulin (panel C) or EGF (panel D). Whole cell lysates were collected at shown time points and immunoblotted.</p

<i>Grb10</i> expression reduces soft agar colony formation by <i>Nf1</i> mutant tumor cell line.

<p>A. Retro-virally expressed HA-Grb10 protein in 989 tumor cells was identified by immunoblotting for the HA tag. B. Soft agar colony formation assay quantified for each cell line. Data from 4 independent experiments, each with replicates of 4. (Student’s t-test, *** p<0.001, *p< 0.05) C. Photographs of representative plate stained for colonies from each group. D. 989 tumor cells expressing either wildtype or mutant Grb10 were serum starved for 18 hours, then stimulated with insulin (150 nM). Whole cell lysates of tumor cells were collected at 0, 5 and 15 minutes after insulin was added, then assessed by immunoblotting for phospho-specific antibodies against activated Ras effectors Akt Ser 473, ERK 42/44 Thr 202/Tyr204, and S6 Ser235/236. Corresponding Ras-GTP pull-down is shown on bottom line.</p

Expression of constitutively activated MEK rescues tumor cells from <i>Grb10</i> mediated suppression of colony formation.

<p>A. Flag-tagged Grb10, MEK DD, or both were expressed in 989 tumor cells. Expression was confirmed by immunoblotting for Flag and phosphorylated Akt and ERK were visualized. Actin is shown as loading control. B/C. 989 tumor cells expressing Flag-tagged Grb10, MEK DD, or both, as shown in panel (A), were grown in soft agar colony formation assay. Photographs of representative plates stained for colonies (B) and quantitation of colony formation (C) are shown.</p

<i>Grb10</i> knockdown in MEFs increases cell proliferation.

<p><i>p53</i> and <i>Grb10</i> expression in MEFs was silenced using lenti-virally expressed shRNAs. A. Immunoblot showing shRNA-mediated silencing of p53 and/or Grb10 in wildtype and <i>Nf1</i> null MEFs. Ren—control shRNA against Renilla, Luc—control shRNA against Luciferase. B. Day 2 post plating. Cell counts performed in triplicate. t-test,*p = 0.03. C. <i>Nf1 44</i>^<i>-/-</i> MEFs in which shRNA against p53 and Luciferase (control) or p53 and Grb10, are expressed. ***p<0.001. D. Increased cell proliferation (day 6) mediated by <i>Grb10</i> loss is genotype-independent (t-test, *p<0.05, ***p<0.001). B, C, D are three independent experiments/transduced sets of cells. E. Three different shRNA against Grb10 were stably expressed in MEFs. Lysates from cells expressing each of the shRNAs and control lysates were immunoblotted for total Grb10 protein and actin. F. Proliferation among MEFs expressing each of Grb10 shRNA are compared (Student’s t-test, ns—not significant, ** p<0.001, ***p<0.0001).</p

<i>Grb10</i> expression is reduced in <i>Nf1</i> mutant tumor cell lines.

<p>A. <i>Grb10</i> underexpression in tumor cell lines (SCCA is a squamous cell carcinoma cell line that arose in an irradiated <i>Nf1</i>^<i>+/-</i> mouse). A heatmap representing data from a Rfdcdfuiop\saaa vb Qiagen PI3K Array comparing expression of PI3K-pathway related transcripts among tumor cell lines and <i>Nf1</i>^<i>+/-</i> MEFs as control. <i>Grb10</i> expression is reduced in tumors compared to MEF control (yellow area at mid-column). B. Western blotting for neurofibromin protein and actin control. C. QPCR analysis of <i>Grb10</i> mRNA normalized to β-Actin shows significant decrease of Grb10 levels in tumor lines derived from <i>Nf1</i>^<i>+/-</i> mice compared to normal adult tissue controls. D. Western blotting for Grb10 in corresponding tumor cell lysates shows uniformly reduced Grb10 protein levels as compared to MEF controls.</p