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. Author manuscript; available in PMC: 2018 Jun 19.
Published in final edited form as: Cell Rep. 2018 May 29;23(9):2559–2567. doi: 10.1016/j.celrep.2018.04.113

Figure 2. IMP3 Activation of TAZ Is Mediated by WNT5B.

Figure 2

(A) Control and IMP3-depleted SUM-1315, MDA-435, and MDA-231 cells were blotted for WNT5B protein.

(B) SKBR3 cells were transiently transfected (48 hr) with an empty vector or IMP3-expressing construct (left immunoblot), and WNT5B mRNA was quantified by qPCR using total RNA isolated from these cells (right bar graph).

(C) Control and IMP3-depleted SUM-1315 cells were transiently transfected with an empty vector (EV) or WNT5B-expressing construct for 48 hr. TAZ localization was quantified in these cells by immunofluorescence (left bar graph). Total RNA was extracted and used to quantify the genes indicated in the right bar graph by qPCR.

(D) RNA sequencing result showing increased expression of miR-145-5p upon IMP3 depletion.

(E) Control and IMP3-depleted SUM-1315 cells were transfected with either a control or anti-miR-145 oligonucleotide (20 nM) for 48 hr, and expression of WNT5B and TAZ target genes mRNA was quantified by qPCR.

(F) Total RNA extracted from LPA- (1 µM, 2 hr) treated control and IMP3-depleted SUM-1315 cells was used to quantify TAZ target genes by qPCR. Blot inset shows IMP3 depletion.

(G) Total cell extracts from control, IMP3-depleted, and WNT5B-depleted SUM-1315 and MDA-231 cells were blotted for pLATS1 (T1079), pTAZ (S89), and WNT5B. pTAZ (S89) blots were obtained using a pYAP(S127) antibody that detects both pTAZ (S89) and pYAP.

(H) Control and WNT5B-depleted (shWNT5B-1 and shWNT5B-2) SUM-1315 cells were grown as mammospheres for 7 days, and the number of spheres was quantified (left). Control and IMP3-depleted SUM-1315 cells were transiently transfected with an empty vector (EV) or a construct expressing WNT5B ligand, grown as mammospheres for 7 days, and the number of spheres was quantified (right).

(I) Total extracts from control and TAZ-depleted (shTAZ-1 and shTAZ-2) SUM-1315 cells were blotted for TAZ (left blot). The same cells were grown as mammospheres for 7 days and quantified (right graph).

(J) Control, IMP3, and WNT5B-depleted SUM-1315 cells were transfected with an empty vector or a vector expressing 4SA-TAZ, grown as mammospheres for 7 days, and quantified. p ≤ 0.05.

Relevant data are shown as ± SE.