Fig. 4

Targeted disruption and complementation of the mlf gene using strategy 4. A Targeted disruption of the mlf gene in the MLF KD cell line resulted in decreased cyst generation during encystation. Cyst number was counted from the control and MLF KD cell lines cultured in encystation medium for 24 h (Enc). **, p < 0.01. B The CWP1 protein level decreased by targeted disruption of the mlf gene in the MLF KD cell line during encystation. The control and MLF KD cell lines cultured in encystation medium for 24 h (Enc) were subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-MLF, anti-CWP1, and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. The band intensity from triplicate Western blots was quantified using Image J as described in Fig. 1E. *, p < 0.05. **, p < 0.01. C Targeted disruption of the mlf gene in the MLF KD cell line resulted in decreased expression of cwp1 and cwp2 during encystation. The control and MLF KD cell lines cultured in encystation medium for 24 h (Enc) were subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in Fig. 2C. **, p < 0.01. ***, p < 0.001. ns, p > 0.05, not significant. D Complementation of the disrupted mlf gene by transfection of mlf expression plasmid increased cyst formation during vegetation growth. Cyst number was counted from the control and complement cell lines cultured in growth medium. *, p < 0.05. E Complementation of the disrupted mlf gene by transfection of mlf expression plasmid increased cwp1 and cwp2 gene expression during vegetation growth. The control and Complement cell lines cultured in growth medium were subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in Fig. 2C. **, p < 0.01. ns, p > 0.05, not significant. F Complementation of the disrupted mlf gene by transfection of mlf expression plasmid increased the CWP1 protein level during vegetative growth and encystation. The control and Complement cell lines were cultured in growth (Veg) or encystation medium for 24 h (Enc) and then subjected to SDS-PAGE and Western blot analysis. The blot was probed with anti-HA, anti-MLF, anti-CWP1, and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. The band intensity from triplicate Western blots was quantified using Image J as described in Fig. 1E. *, p < 0.05. **, p < 0.01. ***, p < 0.001