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. 2021 May 19;34(1):302–332. doi: 10.1093/plcell/koab135

Figure 6.

Figure 6

Inducible PI4Kα1 knockdown impacts sporophytic development and has subtle effects on PI4P accumulation at the plasma membrane. A, Pictures and measures of the primary root of 9-day-old seedlings of Col-0 and seedlings expressing the inducible microRNA2 against PI4Kα1 on noninducible (DMSO) and inducible medium (5 μM β-estradiol, β-est) supplemented with sucrose for two independent insertions (#3 in green and #6 in orange). Scale bar: 1 cm. n indicates the number of seedlings measured. Whiskers correspond to the lower and upper quartile, the median and the minimum and maximum (excluding the outliers), respectively. Statistics were done using Wilcoxon test. Bottom, PI4Kα1 protein levels of 9-day-old Col-0 seedlings and seedlings expressing the inducible microRNA2 against PI4Kα1 on noninducible (DMSO) and inducible medium (5-μM β-estradiol) for two independent insertions (#3 and #6). Immunoblot used an anti-PI4Kα1 antibody and an anti-tubuline α (anti-Tub α) as control. The relative density of signal adjusted to the Col-0 DMSO condition is indicated. B, Cytosol/plasma membrane signal intensity ratio of PI4P biosensors mCITRINE-1xPHFAPP1 (P5Y), mCITRINE-1xPHFAPP1-E50A, and mCITRINE-P4MSidM on epidermal root cells of seedlings expressing the inducible microRNA2 against PI4Kα1 on noninducible (DMSO) and inducible medium (5-μM β-estradiol). n indicates the number of seedlings measured. Three cells per seedling were measured. Statistics were done using Wilcoxon test. C, TLC analysis of lipid extract from 9-day-old 32Pi-prelabeled seedlings expressing the inducible microRNA2 against PI4Kα1 on noninducible (DMSO) and inducible medium (5-μM β-estradiol) supplemented with sucrose for two independent insertions (3 and 6). 32Pi labeling was performed overnight (16–20 h), and quantification of 32P-levels in PIP and PA (as control) by phosphoimaging and calculated as percentage of total 32P-lipids. Each sample represents the extract of three seedlings and each treatment was performed in quadruplicate of which averages ± sd are shown.