Fig 2: NEDD4 regulates actin polymerisation.a Representative time-lapse spinning disc confocal images of F-actin and stress fibre recovery after cytochalasin D washout in WT and Nedd4-/- NCCs expressing EGFP–Actin (green). Acquisitions started 30 min prior and immediately after washout. Images were acquired with the interval of 30 min after the washout. Scale bar = 10 µm. Images are representative of n = 3 biologically independent samples. b Representative images of F-actin (red) and stress fibre recovery after 180 min of cytochalasin D washout in WT and Nedd4-/- NCCs treated with control or Pfn1 siRNAs. Scale bar = 10 µm. Nuclei were visualised with DAPI (blue). c Quantitation of average F-actin level in WT and Nedd4-/- NCCs after 180 min cytochalasin D washout. N = 3 independent replicates. Data are presented as mean +/- SEM. *P = 0.0025 for WT v Nedd4-/- NCCs and P = 0.016 for Nedd4-/- NCCs v Nedd4-/- NCCs treated with Pfn1 siRNAs as determined by type two Student’s t test. Source data are provided as a Source data file. d Abundance of Pfn1 protein detected with anti-Pfn1 antibodies in lysates of WT and Nedd4-/- NCCs treated with varying amounts of Pfn1 siRNAs (mM). Blots are representative of 3 repeats. Uncropped images are provided as a Source data file. e Representative western blot of F- and G-actin from WT and Nedd4-/- NCC protein lysates. Total load controls of known actin levels are shown. Quantitation of the F/G actin ratio is shown for n = 3 independent experiments. Data are presented as mean +/- SEM. *P = 0.03 as determined by type two Student’s t test. Source data are provided as a Source data file. f Representative western blot of F- and G-actin from WT and Nedd4-/- NCC protein lysates at 0 min and 180 min after cytochalasin D washout with and without Pfn1 knockdown. Total load controls of known actin levels are shown. Quantitation of the F/G actin ratio is shown for n = 3 independent experiments. Data are presented as mean +/- SEM. *P = 0.012 as determined by type two Student’s t test. Source data are provided as a Source data file.

Fig 3: PFN1 expression levels and fraction of PFN1 positive cells in advanced HCC predict metastasis-free survivalPFN1 expression level (A) and PFN1 protein positive cells (B) by tumor stage in HCC and adjacent tissue, respectively. Kaplan-Meier curves of survival by PFN1 expression in HCC tissue (C) and in adjacent tissue (D). The TMA contains 90 individual paired HCC and adjacent tissues, in which 86 were included in the analysis and the remaining four were unscorable due to tissue quality.

Fig 4: Restoration of PFN1 inhibits HCC cell metastasis(A) PFN1 protein expression in HepG2-Control, HepG2-PFN1, and L-02 cells by Western blotting. (B) After injection with HepG2-Control, HepG2-PFN1, and L-02 cells (1 × 106 cells per mouse) via tail veins of BALB/c nude mice for 28 days. Upper panel: the lungs were fixed in Bouin's buffer and photographed. Lower panel: the lungs were fixed in 4% PFA, and sectioned for H&E staining. Arrow points to the metastasis nodules. a: Control (No HepG2 cells injected); b: HepG2-Control; c: HepG2-PFN1; d: L-02. Scale bar = 1 mm. (C) Statistics of lung metastasis. (D) The body weight was recorded every three days. Data are shown as mean ± SEM; ###P < 0.001 vs. Control; ***P < 0.001 vs. HepG2-Control. n = 8 per group.

Fig 5: NEDD4 ubiquitinates PFN1.a In vivo ubiquitination assay with 293T cells transfected with HA-Ub, GFP-Pfn1 and Flag-Nedd4 or Flag-Nedd4-CS. Protein lysates were immunoprecipitated (IP) with GFP-trap beads. Ubiquitinated PFN1 (Ub-Pfn1) was probed with anti-HA antibody. Quantitation of the ubiquitinated species was performed from 4 independent experiments. Arrow indicates monoubuiqitinanted PFN1. Data are presented as mean +/- SEM. *P = 0.035 as determined by type two Student’s t test. Uncropped images are provided as a Source data file. b 293T cells were transfected with various combinations of Flag-Nedd4, Flag-Nedd4-CS and GFP-Pfn1. NEDD4 and NEDD4-CS were precipitated from protein lysates with anti-Flag antibodies and compared to mouse IgG. Corresponding antibodies were used to recognize co-immunoprecipitated proteins. Uncropped images are provided as a Source data file. Blots are representative of 3 repeats. c Single-guide RNA (sgRNA) sequence designed to target exon 2 of Nedd4 for gene knockout by CRISPR. Western blot of NCU10K NCC clones derived from single cells confirms complete KO of NEDD4 and altered abundance of PFN1. Quantitation of PFN1 levels from n = 3 biologically independent replicates. Data are presented as mean +/- SEM. *P = 0.038 as determined by type-one Student’s t test. Uncropped images are provided as a Source data file. d In vivo ubiquitination assay with WT and Nedd4-/- NCCs transfected with GFP-Pfn1. Protein lysates were immunoprecipitated with GFP-trap beads and ubiquitination detected with anti-Ub antibodies. Quantitation of ubiquitin levels on immunoprecipitated GFP-PFN1 from 3 WT and 3 Nedd4-/- NCC lines. Data are presented as mean +/- SEM. *P = 0.048 as determined by type-two Student’s t test. Source data are provided as a Source data file. e Cycloheximide (CHX) chase analysis of Pfn1 degradation in WT and Nedd4-/- NCCs. Blots are representative of 3 separate experiments. CQ, chloroquine. f Quantitation of cycloheximide chase assay from n = 3 biologically independent replicates. Data are presented as mean +/- SEM. *P = 0.043 as determined by type one Student’s t test. Source data are provided as a Source data file.