Article Figures & Data
Figures
- Figure 1
Rasgrf-1 RNA schematic and its expression in MCL cell line. (A) Domain structure of Rasgrf-1 (full length 1,273 amino acids). CDC 25 is the GEF domain, Ser 929 is the serine residue phosphorylated by BCR activation and microenvironment signaling. The lower line represents the truncated construct (Rasgrf-1rN605, 667 amino acids) with N-terminus deletion. (B) Stably transfected Maver-1, a mantle cell line with the truncated Rasgrf-1 construct. The truncated protein is expressed in-frame with a V5 epitope. Figure shows Western blots (upper) with total Rasgrf-1 antibody and the lower with anti-V5 epitope antibody. Maver-1 Neo (control) and Maver-1-Rasgrf-1rN605 stably transfected cells were analyzed with two different antibodies. Arrows on the right side indicate multiple Rasgrf-1 protein bands of different sizes expressed in the parent cell line. The arrow on the left side points towards the 80 kD additional band in the Maver-1-Rasgrf-1rN605 cells. Lower panel shows identical lysates probed with an anti-V5 epitope antibody and the expression of 80 kD protein band. (C) Growth curve for Maver-1 Neo and Maver-1 Rasgrf-1 expressing cell lines. Viable cell count performed on day 3, 5, 7, 9 and 11. Line graph shows data from two separate experiments (mean±SD).
- Figure 2
Modulation of BCR signaling by the GEF protein Rasgrf-1. (A) and (B) Maver-1 Neo and Maver-1-Rasgrf-1rN605 cell lines were treated with different concentrations of anti-IgM antibody (IgM) for twenty minutes and the lysates were analyzed for ERK. P-ERK (phospho-ERK), Phospho-Rasgrf-1 (Ser 929) and actin. (C) Maver-1 Neo and Maver-1-Rasgrf-1rN605 cell lines were stimulated with anti IgM antibody (10 mg/mL for 20 min) and lysates analyzed for P-ERK, P-Akt Ser 473, total Akt, phospho-PKC alpha and phospho-BTK signals by specific antibodies.
- Figure 3
Modulation of Microenvironment signaling by GEF protein Rasgrf-1. (A) Maver-1 Neo and Maver-1-Rasgrf-1rN605 cells were co-cultured with HS-5 stromal cell line for 24 h. Cells in suspension were collected and lysates were analyzed for P-ERK, total ERK and actin. Maver-1-Rasgrf-1rN605cells demonstrate higher P-ERK signal with HS-5 coculture. (B) Identical lysates were also analyzed for P-Rasgrf-1, total Rasgrf-1 and actin. Western blots show that the co-culture with stromal cells results in phosphorylation of this GEF protein. In Maver-1-Rasgrf-1rN605 cells, there is phosphorylation of the additional truncated Rasgrf-1 band. (C) A similar co-culture experiment in two primary CLL specimens with lysates analyzed for total Rasgrf-1, P-Rasgrf-1, P-ERK, total ERK and actin. CLL specimens are also stimulated by stromal cell co-culture with ERK phosphorylation along with phosphorylation of the GEF, Rasgrf-1.
- Figure 4
Resistance to apoptosis and increased chemotaxis by Rasgrf-1 over-expressing cells. (A) Maver-1 Neo and Maver-1-Rasgrf-1rN605 cells were treated with a chemotherapy drug, bendamustine, an alkylating agent. Cells were either grown in media or in co-culture with HS-5 stromal cells. 48 h after treatment cells were analyzed for apoptosis by the annexin flow cytometry assay. The line diagram represents % apoptosis (above background) at different concentrations of bendamustine. Experiment done twice with similar results. (B) Chemotaxis measured by Transwell migration of Maver-1 Neo and Maver-1-Rasgrf-1rN605 cells with and without SDF-1. SDF-1 was used at a concentration of 200 ng/mL in the bottom well and cells in the bottom well were counted by flow cytometry 4 h after adding them in the Transwell. Bar diagram shows data from three independent experiments.
