Fig. 1

CCNL1 plays a regulatory role in CM proliferation and heart repair after MI, which is partially related to its LLPS behavior. a Western blot analysis of CCNL1 expression and localization in the nucleus and cytoplasm of the heart tissues from 1-day-old and 7-day-old mice. Lamin B was served as a loading control for the nucleus and GAPDH for the cytoplasm. Data were represented as means ± SEM (n = 9; ***P < 0.001). b The neonatal CM was transfected with small interfering RNA-targeting CCNL1 (CCNL1 siRNA) and corresponding control siRNA (CTL siRNA), and the CM proliferation was evaluated by immunofluorescence staining of EdU, pH3 and Actinin (marked CM). The arrows point to EdU/pH3-positive signal in CM. Data were represented as means ± SEM (EdU: n = 6; pH3: n = 7; **P < 0.01). Scale bar: 50 µm. c The cardiac function of adult MI mice 28 days after administration of AAV-9 CCNL1 shRNA was analyzed by echocardiography. ANOVA test was used (n = 10; ***P < 0.001). d Quantification of the percentage of Ki67/pH3⁺ CM in the heart tissue of MI mice 28 days after administration of AAV-9 CCNL1 shRNA. Data were represented as means ± SEM (n = 6; ***P < 0.001). e The expression and localization of CCNL1 in the CM of 1-and 7-day-old mice were analyzed by immunofluorescence staining. Scale bars: 5 µm. f The CM was transfected with EGFP-CCNL1 for 48 h and FRAP was performed. Quantitative FRAP data are shown as mean ± SEM. A typical FRAP recovery curve of puncta of EGFP-CCNL1 averaged from n = 6 biological replicates. g Time-lapse imaging of the fusion behavior of puncta of EGFP-CCNL1 in the nucleus of CM. The time interval is 60 s. n = 4 biological replicates. Scale bars: 5 µm. h The effect of 1,6-hexanediol (5%) treatment on the formation of puncta in neonatal CM transfected with exogenous EGFP-CCNL1. The red dotted circle indicates the nucleus. Scale bars: 5 µm. i The effect of 1,6-hexanediol on droplet formation of CCNL1 (with IDRs) protein purified in vitro in the presence of salt solution (150 mM NaCl) or crowding agent (10% PEG 8000). Representative differential interference contrast (DIC) images of the droplets and quantification of the size of droplets are shown. Each dot represents a field (8.67 × 8.67 cm²). n = 3 biological replicates. Scale bars: 20 µm. j The effect of the IDRs of CCNL1 on the formation of puncta in neonatal CM. After transfection of EGFP-CCNL1 and EGFP-CCNL1^1–300 plasmid in neonatal CM, the images of puncta formation were collected by Live-cell imaging. The number of puncta is quantified. Each dot represents the number of puncta in the CM nucleus under a field. Data were represented as means ± SEM (n = 14; ***P < 0.001). Scale bars: 5 µm. k Venn diagram indicates the overlapping and unique proteins identified from the two groups (CCNL1-IgG and CCNL1-IP) by Co-IP/LC-MS analysis. l Cluster of Orthologous Groups (COG) analysis of proteins using Co-IP/LC-MS analysis data. PPP1CA is enriched in the category of “Signal Transduction mechanisms”. m Western blot analysis of PPP1CA expression and localization in the nucleus and cytoplasm of the heart tissues from 1-and 7-day-old mice. Lamin B was served as a loading control for the nucleus and GAPDH for the cytoplasm. Data were represented as means ± SEM (n = 6; *P < 0.05, **P < 0.01). n, o EGFP-CCNL1 plasmid and PPP1CA-overexpressing plasmid (mcherry-PPP1CA) were transfected into neonatal CM, and the co-localization of EGFP-CCNL1 (green) and mcherry-PPP1CA (red) was observed by confocal microscopy. The fluorescence intensity trace (yellow line) is shown on the right. The quantification of the Rr and Overlap_R is shown in bar graph format (13 cells). Scale bar: 5 µm. Rr: Pearson’s_Rr, indicates Pearson’s correlation; Overlap_R: overlap coefficient. p, q Western blot analysis of PPP1CA and Yap expression and localization in the nucleus and cytoplasm of CM transfected with EGFP-CCNL1. Lamin B was served as a loading control for the nucleus and GAPDH for the cytoplasm. Data were represented as means ± SEM (n = 3–5; *P < 0.05, **P < 0.01, ***P < 0.001)