Fig. 4: PLUM interacts with L1td1 and regulates its function in pluripotency. | Nature Communications

Fig. 4: PLUM interacts with L1td1 and regulates its function in pluripotency.

From: Microprotein PLUM encoded by Lin28b uORF is a cytoplasmic determinant of pluripotency and embryonic development

Fig. 4

a Flag-PLUM IP-MS in EpiSCs. Candidate PLUM-interacting proteins are ranked by Mascot score. b L1td1 co-IP with Flag-PLUM in EpiSCs with or without RNase A treatment. c Schematic of L1td1 domain-deletion mutants. d Flag-PLUM co-IP with L1td1 domain-deletion mutants. e Numbers of Oct4-GFP+ colonies in EpiSCs transfected with CTRL, shL1td1-1/2 or L1td1. Significant differences were detected between CTRL and shL1td1-1 (P = 0.0043), shL1td1-2 (P = 0.0105), L1td1 (P = 0.0015). f Numbers of Oct4-GFP+ colonies in PLUM KO EpiSCs transfected with CTRL, shL1td1-1/2 or L1td1. Significant differences were detected between CTRL and shL1td1-1 (P = 0.0002), shL1td1-2 (P = 7.77 × 10−5), L1td1 (P = 0.0017). g Number of Oct4-GFP+ colonies in EpiSCs transfected with control, L1td1 or L1td1 mutant (∆620–782 and ∆620–713). Significant differences were detected between control and L1td1 (P = 0.0012), Δ620–782 (P = 0.0159), Δ620–713 (P = 0.0202). h Left: Representative images of mCherry-L1td1 in WT and KO EpiSCs with or without PLUM overexpression. Scale bar, 5 µm. Right: Number of L1td1 condensates per cell. Significant differences were detected between KO and WT (P = 1.63 × 10⁻¹¹). i Schematic of L1td1 condensates formation through PLUM binding. j Representative images of L1td1-GFP in EpiSCs treated with 1,6-Hexanediol for five minutes. Scale bar: up, 5 µm; down, 1 µm. k FRAP analysis of L1td1-GFP condensates in EpiSCs. Scale bar, 1 µm. Significant differences were detected between 2 s and 60 s (P = 2.53 × 10⁻8). l Representative images of mCherry-L1td1, ∆620–782 and ∆ 620-713 in EpiSCs. Scale bar, 5 µm. The experiments in (b, d, j, l) were repeated independently three times with similar results. Representative data are shown. Data in (e–h, k) are mean ± s.d., n = 3 biological replicates (e–g), n = 20 clones (h), 25 clones (k). P-values were calculated by two-tailed unpaired student’s t test, *P < 0.05, **P < 0.005, ***P < 0.001, ****P < 0.0001. Source data are provided as a Source Data file.

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