Fig 2: The adaptor protein GRB2 is essential for TNFR1 signaling. The GRB2 constitutively bind to PPAP sequence of TNFR1 via its SH3 domain. After TNFa binding to TNFR1, cSRC and JAK2 (which constitutively bind to TNFR1) phosphorylate Y360 and Y401. The phosphorylated tyrosines were represented as single (P) on TNFR1. GRB2 binds to these tyrosine phosphorylated amino acids via its SH2 domain. TNFR1-bound GRB2 binds to RAS and initiate RAS-induced RAS-RAF-ERK and RAS-p110a -AKT pathways.

Fig 3: Sorafenib inhibits FLT3 signaling in 32D cells expressing FLT3-ITD but paradoxically induces Erk activity and proliferation in 32D control cells. (A) Immunoblot analyses of Erk activity in 32D-FLT3-ITD (left panel) and 32D (right panel) cell lines. 32D and 32D-FLT3-ITD cells were incubated with different concentrations of sorafenib for 48 h. Cells with DMSO served as control. Whole cell extracts (20 µg) were analyzed by immunoblotting using an antibody against phosphorylated Erk (phospho Erk, 42 and 44 kDa). Total Erk1/2 and ß-actin served as loading controls. (B) Flow cytometric analysis of BrdU-Alexa Fluor 488/propidium iodide (PI)-stained cells. 32D cells were treated or untreated with sorafenib for 48 h. (C) Cell cycle determination of five independent experiments using the murine 32D cell line. Shown are cells in G1- and S-phase. Data are presented as percent of control without sorafenib (left). The plot is a “min–max” plot and shows an increase of cells in S-phase depending on increasing sorafenib concentration (p = 0.0012).

Fig 4: Altered miRNA expression in p53-deficient spontaneous tumors.(a, b) Hierarchical clustering of miRNA showing the differential expression discriminating between normal skin and mouse tumours (a), and discriminating normal skin, squamous and spindle tumours (b). (c) Box plot showing the overall expression of miR-200 family of miRNAs between squamous and spindle tumours obtained by qRT-PCR. (d) Box plots showing the expression of miR-21 in metastatic and non metastatic mouse tumours (upper panel), in tumours from Rb?EC; p53?EC and p53?EC mice (middle panel), and spindle and squamous cell carcinomas (lower panel) n = number of tumours analysed. e-h') Immunohistochemistry examples showing the expression of Sprouty2 (e, e'), phosphorylated Erk (f, f'), PTEN (g, g') and phosphorylated Akt (h, h') in squamous cell carcinomas (e, f, g, h) and in spindle cell tumours (e', f', g', h'). i) Western blot analysis of squamous, spindle and mixed tumours (tumours showing areas of squamous and spindle cell morphology) showing reduced expression of PTEN and Sprouty 2 in undifferentiated samples in parallel with increased expression of Zeb1 and phosphorylation of Akt and Erk. (j) Quantitative RTPCR analysis of the expression of miR-21 in the same tumor samples used in i.

Fig 5: Effects of sorafenib on the MAPK/ERK signaling pathway in ovarian cancer cells following EGF stimulation. (A) Reverse transcription-polymerase chain reaction analysis of B-Raf mRNA expression levels in EGF-treated ovarian cancer cells. (B) Western blot analysis of Ras, Raf, p- and total MEK protein expression levels in EGF-treated ovarian cancer cells. β-actin served as the loading control. (C) Western blot analysis of EGF-related signaling molecules, including PI3K, p-Akt, total Akt, p-p38, total p38, p-ERK, total ERK, p-JNK and total JNK protein expression levels. Densitometry values of each MAPK and MEK protein were normalized to the total MAPK and MEK protein in the same sample. Densitometry values of Ras and Raf proteins were normalized to β-actin in the same sample. Data are expressed as the mean ± standard error of the mean. *P<0.05 EGF-stimulated Caov-3 vs. SK-OV-3 cells. C, control group; D, DMSO group; E, EGF-treated group; So, sorafenib-treated group; So + E, sorafenib- and EGF-treated group; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; EGF, epidermal growth factor; p-, phosphorylated; MEK, MAPK/ERK kinase; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; JNK, c-Jun N-terminal kinase.