Extended Data Fig. 7: Morpholino-mediated knockdown of the A-hits from the yeast screen show that mknk2b, but not acin1b and drl, affects β-cell regeneration.
From: MNK2 deficiency potentiates β-cell regeneration via translational regulation
a-e, Tg(ins:H2BGFP);Tg(ins:flag-NTR) embryos were injected at the 1-cell stage with a control morpholino (a,c) or a morpholino against mknk2b (b,d), β-cells ablated between 2-3 dpf, and treated with DMSO (a,b) or 2 µM CID661578.6 (c,d) for 2 days. Quantification of regenerated β-cells (e) revealed an increase upon mknk2b knockdown, similar to the chemical inhibition of Mnk2. No additive effect was observed with the combined mknk2b MO and CID661578.6 treatment. Scale bar, 10 µm. n = 24 (Control), n = 26 (mknk2b MO), n = 24 (CID661578.6) and n = 23 (mknk2b MO + CID661578.6). *P = 0.0210 (control vs mknk2b MO) and 0.0459 (mknk2b MO vs CID661578.6), ****P < 0.0001. Data in this graph are pooled from two independent experiments and are presented as mean values ±SEM. f-j, Confocal images of primary islets of Tg(ins:H2BGFP);Tg(ins:flag-NTR) at 5 dpf, after injection at the one-cell stage with control (f), acin1b (g), drl (h) or mknk2b (i) morpholinos. Following the ablation of β-cells, only mknk2b knockdown significantly increased β-cell regeneration (j). Scale bar, 10 µm. n = 12 (Control MO), n = 14 (acin1b MO), n = 11 (drl MO) and n = 9 (mknk2b MO). (j) *P = 0.0410. Data are presented as mean values ±SEM. k-m, Agarose gel images of RT-qPCR validating the knockdown of mknk2b (k), drl (l) and acin1b (m) at two stages (2 dpf and 5 dpf). Amplification of eef1a1l1 was used as control. Arrows point to the band of the expected size. The experiment shown in (k-m) has been repeated twice with similar results. n-r, Single-plane confocal images of Tg(ins:H2BGFP);Tg(ins:flag-NTR) islets from control (n and o) or larvae overexpressing mknk2b in notch-responsive cells (p and q), treated with DMSO (n and p) or CID661578.6 (o and q). Quantification results (r) showed that the stable Tg(tp1:mknk2b) overexpression blocked the effect of CID661578.6 on β-cell regeneration, similar to that observed in the transient overexpression model (see Fig. 3). Scale bar, 10 µm. n = 13 (Control), n = 12 (CID661578.6), n = 14 (Tg(tp1:mknk2b)) and n = 11 (Tg(tp1:mknk2b)+CID661578.6). (r)***P = 0.0001 and *P = 0.0172. Data are presented as mean values ±SEM.
