Extended Data Figure 2: Expression cloning of AxMLP as a myotube cell cycle inducer. | Nature

Extended Data Figure 2: Expression cloning of AxMLP as a myotube cell cycle inducer.

From: MARCKS-like protein is an initiating molecule in axolotl appendage regeneration

Extended Data Figure 2

a, The results of the second-round screen of superpool 9 (see Fig. 1b and Extended Data Fig. 1) and its sub-pooling diagram (right). Sub-pool D and sub-pool 2 showed higher BrdU incorporation activity than the others, identifying pool number 212 as positive (n = 12: 4 biological, 3 technical replicates each; mean ± s.d.). b, The result of the third-round screen of pool number 212 from superpool 9 and its sub-pooling diagram (right). Sub-pool A1 showed activity (n = 6: 2 biological, 3 technical replicates each; mean ± s.d.). c, Fourth-round screen of SP9 identified a single active clone (c1), AxMlp (n = 12: 4 biological, 3 technical replicates each; mean ± s.d.). The pooling diagram is shown on the right side. d, AxMLP supernatant induces an S-phase response in a dose-dependent manner in the newt myotube assay. Different amounts of AxMLP-containing supernatant (30 μl, 20 μl, 10 μl and 5.0 μl, respectively) were provided to the myotube cell culture medium. The myotube BrdU incorporation correlated with the amount of supernatant provided, whereas pCMV-SPORT6 supernatant did not provoke cell cycle entry at any dose (n = 6: 2 biological, 3 technical replicates each; mean ± s.d.). e, f, Newt myotubes treated with purified AxMLP (e) or flow-through (f) were immunostained for BrdU and MHC. More BrdU-incorporated nuclei (red) in myotubes (green) were observed in culture supplied with purified AxMLP compared with flow-through-treated cultures. Scale bar, 1 mm.

Back to article page