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. 2020 Apr 22;44(1):156–164. doi: 10.3892/or.2020.7592

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Copyright: © Sun et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 2. — Construction of a series of truncated Cav2.1 mt molecules and addressing their effects on cell proliferation. (A) Diagram of the structures of seven truncated Cav2.1 mt molecules, an ~263 kDa N-terminus of Cav2.1 without polyQ repeat (Cav2.1N), a ~91 kDa C-terminus of Cav2.1 mt with 26 polyQ repeats (Cav2.1dm1), an ~44 kDa C-terminus of Cav2.1 mt with 26 polyQ repeats (Cav2.1dm2), an ~22 kDa C-terminus of Cav2.1 mt beginning with Q26 (Cav2.1dm3), an ~19 kDa C-terminus of Cav2.1 mt without polyQ repeat (Cav2.1C), an ~25 kDa C-terminus of Cav2.1 mt ending with Q26 (Cav2.1dm4) and a reversely assembled Cav2.1dm3 ending with Q26 (Cav2.1dm5). (B) mRNA expression levels of Cav2.1N, Cav2.1dm1, Cav2.1dm2, Cav2.1dm3, Cav2.1C, Cav2.1dm4 and Cav2.1dm5 in the transfected cells were confirmed by qPCR (t-test). (C) Comparison of proliferation of the SH-SY5Y cells transfected with a series of truncated Cav2.1 mt molecules. The WST-1 result indicated that Cav2.1N and Cav2.1C, containing no polyQ repeat did not affect the cell proliferation of the SH-SY5Y cells. Conversely, the forced expression of Cav2.1dm1, Cav2.1dm2 and Cav2.1dm3, containing 26 polyQ repeats significantly impaired cell proliferation. **P<0.01, a significant difference in cell proliferation was observed between EV/Cav2.1N/Cav2.1C and Cav2.1dm1/Cav2.1dm2/Cav2.1dm3, and Cav2.1dm1/Cav2.1dm2 and Cav2.1dm3. NS, no difference of cell proliferation was observed among EV, Cav2.1N and Cav2.1C, and Cav.1dm1 and Cav2.1dm2 (Tukey's test). (D) Comparison of the proliferation of the SH-SY5Y cells transfected with Cav2.1dm3 and its two variations. The result indicated that only those molecules composed with integrity Q26 and C-terminus of Cav2.1 harbored cytotoxicity (Tukey's test). (E) Partial amino acid sequences of Cav2.1 wt-Q13 and Cav2.1dm3. (F) Comparison of the 3D structure of Cav2.1 wt and Cav2.1dm3. White arrows indicate the differences on the secondary structure between Q13 and Q26. **P<0.01. Cav2.1mt, mutant-type Cav2.1; Cav2.1 wt, wild-type Cav2.1; EV, empty vector; NS, no significance.