Figure 5.

KHSRP affects p27 mRNA abundance via the 3ʹUTR and modulates CP sensitivity of MCF7 cancer cells. (a) Extracts prepared from HEK293 cells expressing GFP-tagged KHSRP (lane 1) were incubated with biotinylated RNAs comprising a fragment containing AREs of the 3ʹUTR of LDRL mRNAs (lane 2), the 3ʹUTR of p27 mRNA (lane 3), and RASM as a negative control (lane 4). RNA was captured with streptavidin beads and monitored for the presence of GFP-KHSRP, ELAVL1 and actin by immunoblot analysis with GFP, ELAVL1, and actin antibodies, respectively. (b) HEK293 cells expressing GFP-T and GFP-T-p27(3ʹUTR) were transiently transfected with siKHSRP or scr (siRNA control) for 48 h and treated with 20 µM CP for the last 15 h. The level of GFP in CP-treated (+CP) versus untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin. (c) MCF7 cells were transiently transfected with siRNAs targeting KHSRP (siKHSRP) and Scr control oligos. P27 mRNA levels of CP-treated (+CP, 24 h) relative to untreated cells (-CP) was assessed by RT-qPCR normalized to β-actin. An immunoblot showing knock-down of KHSRP is depicted below. (d) Cell proliferation of MCF7 cells was determined by Trypan Blue assay at the indicated time points after CP treatment. Error bars represent SEM, n = 3. *P < 0.05, ***P < 0. 001, two-tailed student's t-test.