Fig. 2. Stability of centrin-RNAi in strain N41. (A) The average flagellar number per cell is plotted against the time in days after transformation. Data were recorded from cultures grown continuously in liquid medium (1, ), or inoculated from stock cultures of the same strain maintained in solid medium after 152 (2, ) and 204 days (3, ). The points of time for western blotting (WB), indirect immunofluorescence (IF) and embedding for EM are indicated. Arrows on abscissa mark the start of different cultures in liquid medium. (B) Western blot corresponding to the experiment shown in A comparing the amount of centrin in control cells (lane C) and the three cultures of N41. (C) Indirect immunofluorescence corresponding to A and B using anti-centrin on control cells and on three cultures of N41. Note the decrease in the intensity of centrin-RNAi with time as indicated for example by the presence of centrin on the nucleus and residual NBBCs in N41-1. Accordingly, increased amounts of residual centrin were detected in the corresponding western blot and the average number of flagella per cell was close to wild-type. By contrast, the centrin-RNAi effect was still strong in N41-3, a culture retrieved from slowly growing stocks about two weeks prior to analysis.

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