Fig. 4: Exploration of mechanism for cytoskeletal remodeling by hybrid lipoplex. | Nature Communications

Fig. 4: Exploration of mechanism for cytoskeletal remodeling by hybrid lipoplex.

From: The hybrid lipoplex induces cytoskeletal rearrangement via autophagy/RhoA signaling pathway for enhanced anticancer gene therapy

Fig. 4

a Pull-down assay of GTP-RhoA in HepG2 cells using RHOTEKIN binding. Cells were incubated with different lipoplexes or free HS@Au for 24 h. β-actin was a loading control, while GTPγS and His-RhoA acted as high-affinity positive controls. b Immunofluorescence of RhoA in cells after treatment with various lipoplexes or free HS@Au for 24 h. n = 3 independent experimental cell lines with similar results. Green: RhoA, Blue: DAPI. c Representative EGFP images and d flow cytometry analysis of HepG2 cells transfected with different lipoplexes, with or without C3 transferase for 48 h. n = 3 independent experimental cell lines. e Mean count of yellow and red puncta after LC3-EGFP-mCherry plasmid transfection for 24 and 48 h. n = 14 cells from 3 independent experimental cell lines. The data are mean ± SD. f Representative confocal images of cells transfected with various lipoplexes containing LC3-EGFP-mCherry for 24 h, followed by LAMP1 immunofluorescence staining. Channels: Green (LC3-EGFP), Red (LC3-mCherry), Blue (LAMP1), Violet (DAPI-labeled nuclei), Yellow (EGFP merged mCherry), and White (yellow merged blue). n = 3 independent experimental cell lines with similar results. g Representative TEM image of autophagic flux induced by RLS/HS@Au or free HS@Au on HepG2 cells. The red, white, and yellow arrows indicated endosomes containing Au nanoparticles, autophagosomes, and autolysosomes, respectively. n = 25 cells from 3 independent experimental cell lines with similar results. h Western blot analysis of autophagy-related proteins in HepG2 cells treated with various lipoplexes with or without chloroquine for 24 h. β-actin was the loading control. i Lysosomal proteolytic activity analysis of HepG2 cells treated with various lipoplexes or HS@Au for 24 h. The activity was evaluated by the red fluorescence recovery of derivative-quenched bovine serum albumin (DQ-BSA). Serum-free MEM was the positive control. n = 3 independent experimental cell lines. j Illustration of the GTP-RhoA accumulation due to autophagic flux suppression by the HS@Au. Two-sided unpaired Student’s t test was used for the comparisons in (e). p values < 0.05 were considered statistically significant. Source data are provided as a Source Data file.

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