Fig 2: Mechanism whereby Lgl regulates hepatocyte polarization.A, apical lumen formation and protein expression level of 3D-cultured HepG2 cells that overexpressed GFP-tagged Lgl proteins. Cells expressing the indicated Lgl protein were grown in 3D Matrigel culture for 24 h. Cell clusters (n ≥ 100 for each condition) were scored for lumen formation in three independent experiments, and values are presented as the mean ± SD (upper panels). ∗∗∗p < 0.001 (one-way ANOVA followed by the Tukey–Kramer test). Cell lysates were analyzed by immunoblot with the indicated antibodies in three independent experiments (lower panels). B and E–G, interaction of Lgl with aPKC. Proteins in the lysate of human embryonic kidney 293T (HEK293T) cells expressing the indicated proteins (lysate) were immunoprecipitated (IP) with the anti-FLAG M2 antibody or control IgG and then analyzed by immunoblot with the indicated antibodies in four independent experiments. The numbers for HA–Par3 and FLAG–Lgl1 in (G) indicate the relative amount of the plasmid DNA used for the transfection (0, 1, 3, or 10 times). Positions for marker proteins are indicated in kilodalton. C and D, representative confocal images of HepG2 cells expressing the indicated Lgl1 protein. Cells were grown in 3D culture for 24 h, followed by staining with the indicated antibodies and Hoechst. The scale bars represent 10 μm.

Fig 3: Role for Lgl in regulation of lateral membrane integrity.A, domain organization of human Lgl1 and Lgl2 and sequence alignment of the PBR-containing loop in Lgl proteins from various species. The amino acid sequences of the loop region between the 10th and 11th WD40 blades are aligned: human, Homo sapiens; mouse, Mus musculus; chicken, Gallus gallus; frog, Xenopus tropicalis; Medaka fish Oryzias latipes; and fruit fly, Drosophila melanogaster. The basic residues lysine and arginine are shown in magenta, and the aPKC-phosphorylated serine residues are shown in blue. Numbers indicate amino acid positions in human Lgl1. B, depletion of Lgl1 and/or Lgl2 in HepG2 cells. Lysates of HepG2 cells transfected with the indicated siRNAs were analyzed by immunoblot with the indicated antibodies in four independent experiments. Positions for marker proteins are indicated in kilodalton. C and D, representative confocal images of Lgl-depleted HepG2 cells in 3D culture. Cells transfected with the indicated siRNAs were cultured for 24 h, followed by staining with the indicated antibodies and Hoechst. The scale bars represent 10 μm. E–G, quantification of apical lumen formation in 3D culture of Lgl-depleted HepG2 cells. After 3D cultured for 24 h, cell clusters (n ≥ 100 for each condition) were scored for lumen formation in three independent experiments, and values are presented as the mean ± SD. ∗p < 0.05 (Student's t test) (E); ∗∗∗p < 0.001 (one-way ANOVA followed by the Tukey–Kramer test) (F and G).

Fig 4: Smooth muscle-specific deletion of LGL1 promoted neointimal hyperplasia via STAT3. (A) Control (CTR) and LGL1SMKO mice underwent left CCA ligation (Ligated) to induce neointimal hyperplasia; right CCA was applied as control (Sham). Carotid arteries were stained with HE. The intimal area and intima/media ratio were calculated (n = 6). Scale bar: 50 μm. (B,C) Cyclin D1 (B) and PCNA (C) levels tested by immunohistochemistry (n = 3). Scale bar: 50 μm. (D) Protein levels of Cyclin D1 and PCNA detected by western blot analysis (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 vs. CTR + Sham. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. CTR + Ligated.

Fig 5: LGL1 overexpression inhibited the proliferation and migration of VSMCs. (A) VSMCs were infected with adenovirus-expressing GFP or LGL1, then treated with PDGF-BB (20 ng/ml) for 48 h. The protein levels of Cyclin D1 and PCNA were detected by western blot (n = 3). *p < 0.05, **p < 0.01 vs. GFP + Vehicle. # p < 0.05 vs. GFP + PDGF-BB. (B, C) The mRNA levels of Cyclin D1 (B) and PCNA (C) were tested by qRT-PCR (n = 4). *p < 0.05, ***p < 0.001 vs. GFP + Vehicle. ## p < 0.01, ### p < 0.001 vs. GFP + PDGF-BB. (D) Cell proliferation measured by CCK-8 assay (n = 3). **p < 0.01, ***p < 0.001 vs. GFP + Vehicle. ## p < 0.01 vs. GFP + PDGF-BB. (E) Cell migration evaluated by scratch wound healing assay (n = 9). Wound closure (%) represented migrative ability. Scale bar: 200 μm. ***p < 0.001 vs. GFP.