Journal: Science signaling

Article Title: A PI3K- and GTPase-independent Rac1–mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration

doi: 10.1126/scisignal.aba8627

Figure Lengend Snippet: (A) Transwell migration assays performed with wild-type or M1268T MET-expressing cells transfected with a negative control or siRNA targeting p110α, p110β, or both, and treated with DMSO (control), A66 (500 nM) or TGX221 (TGX, 40 nM) either alone or combined. Data are means ± SEM, N=3 or 4 independent biological replicates. Student’s t test (compared to control, or as indicated): NS, non-significant; **P<0.01, ***P<0.005. (B) Transwell migration by U87MG cells treated with DMSO (N=8), PF-02341066 (100 nM, N=3), A66 (500 nM, N=3), TGX221 (40 nM, N=3) or A66 and TGX221 combined (A66+TGX, N=4). Data are means ± SEM from independent biological replicates. Student’s t test (compared to control, or as indicated): NS, non-significant; *P<0.05, **P<0.01, ***P<0.005.One-way ANOVA test followed by Tukey’s multiple comparisons test (compared to control): NS, non-significant; *P<0.05, ***P<0.005. (C and D) Wild-type and M1268T MET-expressing cells were transfected with negative control (RNAi control) or p110α and p110β combined (RNAI p110 α+ β) siRNAs. The percentage of cells with Rac1 at the plasma membrane (C) or lacking stress fibres (D) was counted, following immunostaining with an antibody against Rac1 or rhodamine-phalloidin, respectively. Data are means ± SEM from N=3 independent biological replicates. Student’s t test: NS, non-significant; *P<0.05, **P<0.01, ***P<0.005.

Article Snippet: Mouse monoclonal antibodies against murine MET (B-2; 1:500, #sc-8057), HSC70 (1:5000, #sc7298), rabbit polyclonal antibodies against p110β (1:500, #sc-602) and Vav2 (1:1000, #sc-H200) were purchased from Santa Cruz Biotechnology.

Techniques: Migration, Expressing, Transfection, Negative Control, Immunostaining

Journal: Science signaling

Article Title: A PI3K- and GTPase-independent Rac1–mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration

doi: 10.1126/scisignal.aba8627

Figure Lengend Snippet: (A and B) Western blots for phosphorylated p70-S6K (P-p70-S6K), p70-S6K (A and B), phosphorylated ERK1/2 (P-ERK1/2), ERK1/2 and HSC70 (B) were performed on M1268T MET-expressing cells treated with DMSO, dynasore (80 μM) or UO126 (10 μM), as indicated. Data below blots are mean ± SEM, phospho:total ratio, obtained by densitometry of N=3 independent biological replicates. (C) Western blots for phosphorylated p70-S6K (P-p70-S6K), p70-S6K, HSC70 and Rac1 were performed on M1268T MET-expressing cells transfected with negative control or Rac1 siRNA (“RNAi”). Below are mean levels of indicated phosphorylated protein over total protein ± SEM, obtained by densitometry of Western blots. N=3 independent biological replicates. (D and E) Relative colony area of (D) wild-type and M1268T MET-expressing cells and (E) U87MG cells transfected with negative control or Rac1 RNAi and grown in soft agar. N=3 independent biological replicates. Data (A to E) are mean values ± SEM. P values were obtained with the Student’s t test. NS: non-significant, *P<0.05, ***P<0.005.

Article Snippet: Mouse monoclonal antibodies against murine MET (B-2; 1:500, #sc-8057), HSC70 (1:5000, #sc7298), rabbit polyclonal antibodies against p110β (1:500, #sc-602) and Vav2 (1:1000, #sc-H200) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Negative Control

Journal: Science signaling

Article Title: A PI3K- and GTPase-independent Rac1–mTOR mechanism mediates MET-driven anchorage-independent cell growth but not migration

doi: 10.1126/scisignal.aba8627

Figure Lengend Snippet: (A) Western blots for phosphorylated mTOR on Ser2481 (P-mTOR), mTOR, HSC70 and Rac1 of M1268T Met-expressing cells transfected with negative control (RNAi control) or Rac1 (RNAi Rac1) siRNAs. Below, the mean levels of indicated phosphorylated protein over total protein ± SEM obtained by densitometry of Western blots. N=3 independent biological replicates. (B) Colony area of M1268T Met-expressing cells grown in soft agar and treated with DMSO, Ehop-016 (4 μM) or NSC23766 (NSC) (100 μM). N=3 independent biological replicates. (C) Colony area of M1268T MET-expressing cells transfected with control, Vav2 or Tiam1 siRNAs grown in soft agar. N=4 independent biological replicates. (D) Colony area of M1268T-MET-expressing cells transiently transfected with GFP-Rac1-T17N dominant-negative construct. After flow cytometry separation, the cells expressing GFP-Rac1-T17N or the GFP negative cells (no GFP) were grown in soft agar and were treated with DMSO or PHA-665752 (PHA) (100 nM), N=4 independent biological replicates. Data (A to E) are mean values +/- SEM. P values were obtained with the Student’s t test. NS: non-significant, **P<0.01, ***P<0.005.

Article Snippet: Mouse monoclonal antibodies against murine MET (B-2; 1:500, #sc-8057), HSC70 (1:5000, #sc7298), rabbit polyclonal antibodies against p110β (1:500, #sc-602) and Vav2 (1:1000, #sc-H200) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Dominant Negative Mutation, Construct, Flow Cytometry