Fig. 4

Mutant NRBP1 is required for tumor cell survival and is oncogenic in NIH3T3 cells. Knockdown of mutant NRBP1 expression with shRNA inhibits transformation and survival of HNSCC cell lines. a Western blot analysis of total NRBP1 expression level in HNSCC cell lines. NRBP1 encodes for three different nuclear receptor binding protein isoform: 60 kDa, 51 kDa and 43 kDa in NT8e cells (lane 1), OT9 cells (lane 2). AW13516 cells express only two isoforms (lane 3). b Western blot representation of NRBP1 partial knockdown by two independent hairpins (shNRBP1#1 and shNRBP1#2) in NT8e and OT9 cells. The hairpin constructs inhibit cell survival as assessed by cell counting as described in methods. c and anchorage-independent growth as assessed by colony formation in soft agar d in the NT8e cells harboring an NRBP1 Q73* mutation, but not the OT9 cells, which express WT NRBP1. shGFP, a hairpin specific for green fluorescent protein, was used as a negative control. All results are normalized to survival or colony formation by cells infected with empty vector. Images were taken at 10X magnification. e Western blot analysis of NRBP1 wild type and mutant (Q73*) in NIH-3 T3 cells. Vector control, NIH-3 T3 clones overexpressing wild-type and mutant NRBP1 is shown. NRBP1 Q73* mutation expresses 51 kDa isoform, while two isoform of 60 kDa and 51 kDa can be seen in 3 T3 clones overexpressing wild-type NRBP1. Western blot analysis of total and phosphorylated MAPK in NIH-3 T3 clones expressing NRBP1 wild type (lane 2) and mutant NRBP1 (Q73*). f Representative images (taken at 20x) of soft agar colony formed by NIH-3 T3 cells expressing NRBP1 wild type and Q73*. Bar graph representation of number of soft agar colonies formed by stable NIH-3 T3 clones. NIH-3 T3 cells expressing EGFR VIII was used as positive control and relative comparison of transforming ability of mutant NRBP1. *** P-value <0.0001, ** P-value <0.001