Journal: Genes & Development

Article Title: Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity

doi: 10.1101/gad.348622.121

Figure Lengend Snippet: Protein phosphatase 4 (PPP4) is essential for normal circadian rhythms. ( A ) RNAi-based screen for catalytic subunits of all known Ser/Thr-phosphatases important for circadian dynamics. Human U-2 OS cells harboring a Bmal1-luciferase reporter construct were lentivirally transduced with three to 10 shRNA constructs per indicated catalytic subunit, synchronized with dexamethasone, and monitored for 5–7 d in a luminometer. Shown is the mean period deviation (±SD) from nonsilencing controls of three independent experiments performed in a 96-well plate format. ( B ) Trend-eliminated oscillation dynamics of U-2 OS reporter cells lentivirally transduced with shRNA constructs targeting the catalytic subunit of protein phosphatase 4 (PPP4C). Knockdown efficiency was quantified using qPCR. Shown are results from three experiments (mean ± SD). ( C ) Trend-eliminated oscillation dynamics of U-2 OS reporter cells lentivirally transduced with expression constructs for PPP4C, a dominant-negative variant of PPP4C (PPP4C mut), or tGFP as an overexpression control. Shown are results from three experiments (mean ± SD).

Article Snippet: Cell lysates were homogenized by vortexing and luciferase activity was measured by using the dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol in a 96-well plate-reading luminometer (Orion II, Berthold Detection System).

Techniques: Luciferase, Construct, Transduction, shRNA, Knockdown, Expressing, Dominant Negative Mutation, Variant Assay, Over Expression, Control

Journal: Genes & Development

Article Title: Protein phosphatase 4 controls circadian clock dynamics by modulating CLOCK/BMAL1 activity

doi: 10.1101/gad.348622.121

Figure Lengend Snippet: PPP4R2 is the PPP4 regulatory subunit crucial for normal rhythms in human cells and Drosophila . ( A ) RNAi-based screen for all known PPP4 regulatory subunits important for circadian dynamics. Human U-2 OS cells harboring a Bmal1 -luciferase reporter construct were lentivirally transduced with one to nine shRNA constructs per indicated catalytic subunit (number depended on availability in our laboratory RNAi construct library), synchronized with dexamethasone, and monitored for 5–7 d in a luminometer. Shown is the mean period deviation (±SD) from nonsilencing controls of three independent experiments performed in a 96-well plate format. ( B ) Trend-eliminated oscillation dynamics of U-2 OS reporter cells lentivirally transduced with shRNA constructs targeting the regulatory subunit of protein phosphatase 4, PPP4R2. Knockdown efficiency was quantified using qPCR. Shown are results from three to five experiments (mean ± SD). ( C ) Double-plotted actograms showing behavioral activity of male flies of the indicated genotype during LD (days 1–5) and DD (days 6–12) at constant 25°C. The tim-Gal4:27 line used here also contains the UAS-Dicer construct to enhance RNAi efficiency. For overexpressing PPP4R2r , the P{EP}PPP4R2r EP307 line with an UAS insertion immediately upstream of the transcription start site was employed. ( D ) Statistical analysis of differences between control flies and those down-regulating or overexpressing Drosophila PPP4R2r. The individual period values of the indicated genotypes are plotted. The mean ± SD are shown as vertical colored lines and the mean is indicated as a gap in the line. (RNAi-1) UAS-PPP4R2r-RNAi TRiP BL26296 , (RNAi-2) UAS-PPPR2r v105399 /+, (tim) tim-gal4:27 UAS-Dicer , (Pdf) Pdf-gal4 , (EP) P{EP}PPP4R2r EP307 . P- values were determined by Student's t -test: (****) P < 0.00005, (***) P < 0.0005, (**) P < 0.005, (*) P < 0.05.

Article Snippet: Cell lysates were homogenized by vortexing and luciferase activity was measured by using the dual-luciferase reporter assay system (Promega) according to the manufacturer's protocol in a 96-well plate-reading luminometer (Orion II, Berthold Detection System).

Techniques: Luciferase, Construct, Transduction, shRNA, Knockdown, Activity Assay, Control