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. 2020 Jan 23;106(2):153–169. doi: 10.1016/j.ajhg.2019.12.010

Figure 5.

Figure 5

Knockdown of TTC12a Affects the Swimming Velocity of Paramecia

(A) Z projection of track recording paramecia swimming under a dark-field microscope with a 10× objective lens after 24 h or 48 h of RNAi treatment. At 24 h of silencing, swimming behavior was affected in TTC12a- and TTC12a+b-silenced cells, whereas the silencing of TTC12b did not show any swimming defect. At 48 h, TTC12a- and TTC12a+b-silenced cells did not swim anymore, and they died at 72 h.

(B) Analysis of the swimming velocity of TTC12a, TTC12b, and TTC12a+b KD cells as well as control paramecia (i.e., empty vector silenced paramecia). Note that TTC12b-depleted cells swam slightly faster (725mm/s) than control cells (EV, 665 mm/s). The swimming velocity of TTC12a (317 mm/s) and TTC12a+b (282 mm/s) KD cells was reduced to 50% of that of control cells at 24 h. Data are represented as the mean ± SEM. Statistical analyses were performed with GrahPad Prism v5 software through the use of two-tailed unpaired t tests, ∗∗∗p < 0.001, ∗∗p < 0.01, (n = 69–88 per experiment).

(C) Z projection of track recording paramecia swimming under a dark-field microscope equipped with a 10× objective lens. Clones transformed by an RNAi-resistant version of TTC12a (2c and 6c) as well as non-transformed paramecia were grown for 36 h in an empty-vector silencing medium or in a TTC12a-silencing medium. TTC12a-depleted cells barely swam, as shown previously, whereas cells expressing an RNAi-resistant version of TTC12a again swam similarly to control cells. This indicates that TTC12a-depleted phenotypes are rescued by the TTC12a RNAi-resistant version.

(D) Analysis of the swimming velocity of transformed and non-transformed cells grown in an empty-vector silencing medium or in a TTC12a-silencing medium. As expected, TTC12a silencing in non-transformed cells dramatically reduced the swimming velocity (52 mm/s). However, the expression of the TTC12 RNAi-resistant version rescues this phenotype (88% of complementation). Note that the swimming velocity of transformed 2c cells (653 mm/s) was similar to that of non-transformed paramecia (612 mm/s), while transformed 6c cells swam slower (458 mm/s). Data are represented as the mean ± SEM. Statistical analyses were performed with GrahPad Prism v5 software and two-tailed unpaired t tests. ∗∗∗p < 0.001, ∗∗p < 0.01, NS (non-significant) p > 0.1 (n = 26-48 per experiment).

(E) Immunoblot analysis on total protein extract of transformed 2c and 6c cells showing that both clones express TTC12a-GFP. Abbreviations are as follows: EV, empty vector; and KD, knockdown.