Fig 2: Nudt3 is a decapping enzyme that modulates stability of integrin ß6 and lipocalin-2 mRNAs. Stability of the indicated mRNAs was determined following actinomycin D-mediated transcriptional silencing in ConKD and Nudt3KD cells complemented with the indicated proteins. Values are normalized to levels of 18S rRNA and presented relative to their respective levels at time zero. Data were derived from at least three independent experiments. The dotted line demarcates 50%. P-values from comparison of the decay rates are presented with asterisks. (***) P < 0.01 (two-tailed extra sum-of-squares F-test).

Fig 3: Increased FN secretion in cells with down-regulated Nudt3 is essential for increased MCF-7 cell motility. (A,B) FN protein secreted into the medium was isolated by gelatin–sepharose chromatography from cells grown without FBS on tissue culture plastic. Levels of secreted FN in ConKD and Nudt3KD cells and the same cells with down-regulated FN (A) or complemented with GFP, Nudt3, or Nudt3EE/QQ expression constructs (B) were determined by Western blot analysis. (C) Migration of cells depicted in A were determined by scratch-wound healing assays. At least five different wounds were quantified for each time point. Data are representative of three independent experiments ±SD. P-values are denoted by asterisks. (***) P < 0.001 (Student's t-test).

Fig 4: Elevated ITGB6 expression in Nudt3 knockdown cells is essential for increased MCF-7 cells motility. (A) A representative Western blot of integrin ß6 (ITGB6) protein in cell extract from ConKD and Nudt3KD MCF-7 cells is shown. (B) Cell migration of ConKD and Nudt3KD MCF-7 cells in the presence of 10 µg/mL of either avß6-neutralizing antibody (10D5) or nonspecific antibody (IgG2a) in the medium lacking FBS was assessed by scratch-wound healing assays. At least five different wounds were quantified for each time point. Data are representative of three independent experiments ±SD. P-values are denoted by asterisks. (***) P < 0.001 (Student's t-test).

Fig 5: Nudt3 down-regulation increases expression of mRNAs implicated in cell motility. (A) RNA levels of nine mRNAs implicated in cell migration tested in MCF-7 cells expressing nontargeting shRNA (ConKD, open bar), shRNA against Nudt3 (Nudt3KD, filled bar), or shRNA against Nudt4 (Nudt4KD, gray bar) are shown. (B) Western blot analysis of Nudt3 protein levels in ConKD or Nudt3KD MCF-7 cells ectopically expressing GFP, Flag-tagged wild-type Nudt3 (Nudt3), or mutant Nudt3 (Nudt3EE/QQ). The band for Flag-Nudt3EE/QQ runs slightly faster than that for Flag-Nudt3 likely due to differences in protein charge by the introduction of glutamic acid residues into the mutant protein. (C) A subset of the mRNAs implicated in cellular movement derived from the 144 mRNAs detected to increase greater than or equal to twofold by RNA-seq analysis in Nudt3KD are shown. (D) Transcript levels of the indicated mRNAs were determined by qRT-PCR in ConKD or Nudt3KD cells complemented with the indicated proteins. RNA levels in A and D are derived from at least three independent experiments and normalized to 18S rRNA and presented relative to the value in control cells set to one. Error bars represent ±SD. P-values are denoted by asterisks. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001 (Student's t-test).