Fig 2: MSP and RON are upregulated in tumors from KB1P mice. (A) Mst1 and Mst1r mRNA expression in normal MG (from three individual mice), KP tumors (n = 44) and KB1P tumors (n = 41) analyzed by RNA sequencing. Boxplots represent the median, and 25th and 75th percentiles. (B) Mst1 and Mst1r mRNA expression in KP and KB1P tumors assessed by qRT–PCR (n = 7/group). (C) Western blot analysis of MSP expression in KP and KB1P tumors with densitometric quantification. Numbers above the blot represent individual tumors. Each dot represents one donor tumor from one mouse. (D) MSP serum levels in tumor‐free, WT (n = 5) mice, tumor‐bearing KP (n = 6) mice, and tumor‐bearing KB1P (n = 6) mice assessed by ELISA. (E) Western blot analysis of RON protein expression in KP and KB1P tumors (same samples as in C) with densitometric quantification. Numbers above the blot represent individual tumors. Each dot represents one donor tumor from one mouse. For C and E, β‐actin was used as sample integrity control (same sample, different blot). Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by Mann–Whitney U‐test (A, B, C, E) or one‐way ANOVA followed by Dunn’s post hoc test (D). n.s., not significant.

Fig 3: MSP activation of the RON receptor induces proliferation of KP and KB1P cells in vitro. (A) Proliferation of two KP and two KB1P cell lines in response to 100 ng·mL−1 MSP, 1 µm BMS‐777607, or both reagents. For combined treatments, cells were pretreated with BMS‐777607 for 1 h before the addition of MSP. For all treatments, cells were re‐fed every 24 h with fresh MSP and BMS‐777607 as denoted. DNA content was measured at 72 h post‐treatment. (B) Proliferation of two KP and two KB1P cell lines transduced with control pLKO.1 or shRNA vectors against Mst1r mRNA following 48‐h treatment with recombinant MSP as above. For both A and B, data points shown are technical replicates from 1 representative experiment from three repeated experiments. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 as determined by one‐way ANOVA followed by Dunn’s post hoc test.

Fig 4: MSP stimulates AKT and MAPK signaling pathways through the RON receptor. (A) Mst1r mRNA expression assessed by qRT–PCR in cell lines derived from three KP tumors and four KB1P tumors, respectively. (B) Western blot analysis of RON protein expression in the same cell lines used in A. β‐Actin was used as sample integrity control (same sample, different blot). (C) Western blot analysis of indicated proteins in the same KP and KB1P cell lines used in A, B. Cells were pretreated with 1 μm BMS‐777607 for 1 h prior to 100 ng·mL−1 recombinant MSP for 1 h where denoted. Images are representative of three replicate experiments. Total AKT and ERK were probed on the same blot as sample integrity controls. (D) Two KB1P cell lines were transduced with lentiviral shRNA vectors against Mst1r mRNA (shRON‐1 or shRON‐2) or control pLKO.1 vector. Confirmation of Mst1r mRNA knockdown quantified by qRT–PCR is expressed as relative to two housekeeping genes, Hprt and β‐actin (each dot represents one technical replicate in a given experiment, the experiment was repeated three times for each cell line). (E) Two independent KB1P cell lines transduced with control or shRNA vectors against Mst1r mRNA were treated with 100 ng·mL−1 recombinant MSP for 1 h. Activation of AKT and ERK1/2 was assessed by western blot. Images are representative of three replicate transduction experiments. Data are represented as mean ± SD. **P < 0.01 as determined by one‐way ANOVA followed by Dunn’s post hoc test. n.s., not significant.