Fig. 1. PTPN11 genetic depletion reduces MPNST cell growth and alters MEK/ERK activity.

(A) ST8814 and JH-2–002 transduced with doxycycline (Dox) inducible constructs expressing shPTPN11 #818 or #5003, were treated with vehicle or 300 ng/ml Dox for two weeks. Cells were fixed with 10% neutral buffered formalin and then stained with crystal violet. (B) Cells as in A. were treated with vehicle or 300 ng/ml Dox for up to seven days. The phase confluence was monitored by Incucyte real-time imaging system, normalized to corresponding 0-hour scan. (C) Cells as in A. were treated with vehicle or 300 ng/ml Dox for 72 hours, and the indicated proteins were assessed using immunoblot. (D) JH-2–002 transduced with Dox-inducible shPTPN11 #818 was treated with vehicle or 300 ng/ml Dox for 72 hours, and then three replicates were collected for RNAseq, and the fourth replicate was collected for immunoblotting validation. RNAseq result revealed a significant decrease in PTPN11 RNA expression (n= 3, P adj= 0, LFC= −4.69) when comparing shPTPN11 v. ctrl. A total of 51 genes representing MEK/ERK transcriptional output (31), were assessed for their expression after PTPN11 knockdown. A volcano plot demonstrating log2 fold change of shPTPN11 v. ctrl as a function of −log10 (P adj) is shown. Black dots= not significant (ns, P adj> 0.05); red dots= log2 fold change (LFC)> 0 and blue dots= LFC< 0 (P adj< 0.05). 73% (37/51) of genes in the set were significantly transcriptionally downregulated following PTPN11 genetic depletion.