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. 2016 May 26;15(10):2251–2265. doi: 10.1016/j.celrep.2016.04.090

Figure 2.

Figure 2

DENR Regulates Cell Migration in Concert with MCTS1

(A) qRT-PCR analysis of Neuro2A cells transiently transfected with either a control (non-targeting) shRNA construct or Denr shRNA construct revealed a significant decrease in Denr mRNA levels.

(B) Parallel immunoblotting studies show depletion of Denr protein levels upon Denr shRNA treatment.

(C) In utero electroporation studies of E14.5-labeled cortical cells within the E17.5 cortex following treatment with control (non-targeting shRNA vector with GFP-only vector), Denr shRNA (together with GFP-only vector), and forced expression of mouse Denr WT or human DENR WT (non-targeting shRNA vector with GFP expression vector comprising a DENR expression cassette).

(D) Denr knockdown as well as overexpression of mouse or human DENR resulted in a significant reduction in the proportion of GFP+ cells in the CP of E17.5 cortices (control 0.426 ± 0.045; Denr shRNA 0.287 ± 0.039; Denr WT 0.231 ± 0.028; DENR WT 0.324 ± 0.027; two-way ANOVA F(4,33) = 3.980; p < 0.0096 with Bonferroni multiple comparison test; ∗p < 0.05; n = 3–5).

(E and F) The defective migration of Denr shRNA-treated cells can be restored by co-delivery of human DENR WT, which is refractory to knockdown, but co-treatment with Mcts1 siRNAs abolishes this effect (Denr shRNA 0.287 ± 0.039; Denr shRNA + DENR WT 0.435 ± 0.036; Denr shRNA + DENR WT + Mcts1 siRNA 0.334 ± 0.042; two-way ANOVA F(2,30) = 2.897; p = 0.0386 with Bonferroni multiple comparison test; ∗p < 0.05; n = 3–5).

(G and H) Whereas forced expression of DENR WT disrupts cell migration, co-delivery of Mcts1 siRNAs abolishes this effect, observed as a significant increase in the proportion of GFP+ cells that arrive within CP (two-way ANOVA F(2,30) = 14.12; p < 0.0001; DENR WT + control siRNA 0.299 ± 0.02 versus DENR WT + Mcts1 siRNAs 0.525 ± 0.056; Bonferroni multiple comparison test; ∗∗∗p < 0.005; n = 6), together with a concomitant reduction in the proportion of GFP+ cells within the IZ (DENR WT + control siRNA 0.453 ± 0.018 versus DENR WT + Mcts1 siRNA 0.317 ± 0.04; Bonferroni multiple comparison test; ∗p < 0.05).

Graphs plot mean ± SEM. The scale bars represent (C) 25 μm and (E and G) 50 μm.