Fig 2: 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).

Fig 3: The protein expression level of the nine screened hub differentially expressed macrophages-related genes (DEMRGs) in atherosclerosis and myocardial infarction (MI) mice model. (A) The expression level of the nine hub DEMRGs in atherosclerosis. (B) The expression level of the nine hub DEMRGs in MI and SHAM groups in ApoE-/- and C57Bl/6J mice. (C–J) The correlation between left ventricular ejection fraction (LVEF) and the nine DEMRGs expression level in the MI mice model, including SYNJ2 (C), NET1 (D), FZD7 (E), LCP2 (F), EIF4EBP1 (G), HCK (H), GNB2 (I), and PPP4C (J). *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Fig 4: CLOCK/BMAL1 occupancy on target promoters is modulated by PPP4. (A) Chromatin immunoprecipitation experiments with anti-BMAL1 antibody (or control IgG) performed in synchronized U-2 OS cells lentivirally transduced with shRNA constructs targeting the catalytic (PPP4C) or regulatory PPP4R2 subunit of protein phosphatase 4. Chromatin was harvested 24 and 36 h after dexamethasone synchronization, and BMAL1 presence on NR1D1 promoter was analyzed via qPCR. Shown are results from five independent precipitations (mean ± SD). (B) Kinetics of BMAL1-YFP binding to Dbp gene arrays in murine NIH3T3 cells (Stratmann et al. 2012) was analyzed using fluorescence recovery after photobleaching (FRAP) experiments. Shown are representative fluorescence microscopy images of control or PPP4R2-depleted cells before and after photobleaching of BMAL1-YFP located at the Dbp gene arrays (visible as bright spots). Scale bar, 10 µm. (C) Quantification of FRAP experiments described in B. Given are mean (±SEM) relative fluorescence intensities after photobleaching for 25 cells (Ppp2r2 knockdown) and 47 cells (nonsilencing control).

Fig 5: PPP4 interacts with BMAL1 and is rhythmically expressed. (A) Coimmunoprecipitation experiments with lysates from HEK293 cells expressing a BMAL1-luciferase fusion protein. Shown are mean luciferase activity counts after precipitation with specific anti-CRY1, anti-PPP4R2, anti-PPP4C antibodies or unspecific IgG control antibody (mean ± SD, n = 3 independent IPs). Student's t-test: (**) P < 0.01. (B) Western blots with anti-PPP4R2 and anti-BMAL1 antibodies (and anti-ß-Actin antibody as loading control) from liver lysates harvested from mice at the indicated circadian times. (C) Replotted data from a proteomics study performed by Wang et al. (2017). Shown are nuclear abundances of PPP4C and BMAL1 over the course of 1 d. Error bars represent SEM between two biological replicates.