Extended Data Fig. 7: Central role of PTPN6 in enabling oncogenic ERK signalling.
From: Signalling input from divergent pathways subverts B cell transformation
a, Levels of NRAS, PTPN6-pY564, PTPN6, ERK1/2-pT202/Y204 and ERK1/2 were assessed by western blotting following doxycycline-induced expression of NRASG12D in mouse B-cell precursors (n = 3 independent experiments). b, ChIP–seq analyses of human B lymphocytes GM12878 (ENCODE) revealed binding of ERK-dependent transcription factors ELK1, CREB1, c-JUN and JUNB to the PTPN6 locus. c, Patient-derived B-ALL cells (PDX2) were treated with BCI-215 (1 μmol l−1) for various times. Levels of STAT5-pY694, STAT5, ERK1/2-pT202/pY204 and ERK1/2 were then measured by western blotting (n = 3 independent experiments). d, Patient-derived B-ALL cells (PDX2) were treated with BCI-215 (1 μmol l−1) for various times, and levels of STAT5-pY694, STAT5, PTPN6-pY564 and PTPN6 were measured by western blotting (n = 3 independent experiments). e, Ptpn6fl/fl B-cell precursors were transduced with 4-OHT-inducible Cre or EV and then induced with 4-OHT for various times and levels of STAT5-pY694, STAT5, ERK1/2-pT202/Y204, ERK1/2 and PTPN6 were measured (n = 3 independent experiments). f, Ptpn6fl/fl B-cell precursors expressing NRASG12D were transduced with GFP-tagged, 4-OHT-inducible Cre or EV. Following induction with 4-OHT, enrichment or depletion of GFP+ cells was monitored by flow cytometry. Shown are average relative changes (means ± s.d.) of GFP+ cells following induction (n = 6 independent biological replicates). g, Quantification (n = 3 independent experiments; means ± s.d.) and representative images from serial replating assays of pre-B cells transformed with NRASG12D following Cre-mediated deletion of Ptpn6. We seeded 10, 000 cells in semisolid methylcellulose and monitored colony formation for 14 days. P-values were determined by two tailed t-test. For gel source data, see Supplementary Fig. 1.
