Fig. 7. CRLF2-signaling induces direct interaction between activated PTPN11 and RAS, and PTPN11-activity is required for ALL cell growth.

a Direct interaction between RAS and phosphorylated PTPN11 was monitored via PLA using high-content microscopy. Serum-starved MUTZ-5 cells were induced (or not) with 20 ng/mL TSLP for 10 min. Cells were fixed and permeabilized in a 96 well plate. Antibodies against phosphorylated PTPN11 and pan-RAS were used in conjunction with PLA-probes to allow the amplification and staining of interaction-specific PLA-spots. The negative control (NC) are two cytosolic proteins not expected to interact. Fluorescent-microscopy images show examples of PLA-spots (scale bars = 20 µm). At least 250 cells per well were analyzed using Operetta-CLS automated high-content microscopy platform. The bar-graph shows the averages of three independent experiments (each performed in triplicates). Error bars are SD and P values were determined in one-way ANOVA and post-hoc Bonferroni multiple comparison. b MUTZ-5 cells were pre-incubated with DMSO or 25 µM II-B08 (PTPN11 inhibitor) for 2 h and then stimulated or not with 20 ng/mL TSLP for 10 min before cell lysis. Each cell lysate was split up for RAS-GTP pull-down assay and for WB (whole-cell lysates). c MUTZ-5 cells were treated as in b before fixation. A PLA described in a was performed. d MUTZ-5 cells were seeded at 1.6 × 10⁵/mL density and cultured for 7 days with either 0.5% DMSO (vehicle control), 50 µM Salirasib (indirect pan-RAS inhibitor), 25 µM II-B08, 50 μM Vemurafenib (pan‐Raf inhibitor), 1 μM PD0325901 (MEK1/2-inhibitor), or 5 µM Ruxolitinib (JAK-inhibitor), in presence of 20 ng/mL TSLP. Percentage of viable cells was determined in an NC-250 automated cell counter.