Journal: Nature Communications

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

doi: 10.1038/s41467-024-55727-4

Figure Lengend Snippet: 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.

Article Snippet: The antibody against LC3 (catalog number: NB100-2220; clone number: N/A; lot number: D155067; 1:1000 dilution) was from Novus (USA).

Techniques: Pull Down Assay, Binding Assay, Incubation, Control, Immunofluorescence, Flow Cytometry, Transfection, Plasmid Preparation, Staining, Labeling, Western Blot, Activity Assay, Fluorescence, Positive Control

Journal: PLOS ONE

Article Title: St-N, a novel alkaline derivative of stevioside, reverses docetaxel resistance by targeting lysosomes in vitro and in vivo

doi: 10.1371/journal.pone.0316268

Figure Lengend Snippet: (A) Confocal microscopic analysis of lysosomes stained by LysoTracker Red in PC3/Doc cells exposed to 1 μM St-N or St-C for the indicated times. (B) Flow cytometric analysis of lysosomes stained with LysoTracker, and the relative ratio of geometric LysoTracker Red fluorescence. * P < 0.05, ** P < 0.01. (C) Electron micrographs showing lysosomal swelling induced by St-N. (D) ASM activities in lysates from the vehicle or St-N-treated PC3/Doc cells. * P < 0.05. (E) Western blotting analysis of LAMP1, LAMP2, CTSB, CTSD, p62, and LC3B in PC3/Doc cells after St-N treatment for different times. (F) Heatmap of the relative mRNA levels [logarithm value (base2)] of various cathepsins, LAMPs, and LC3B in St-N-treated PC3/Doc cells as determined by RT-qPCR. GAPDH served as an internal control. (G) Western blotting analysis of the active CTSB of lysosomal and cytosolic fractions in PC3/Doc cells treated with St-N. LAMP1 served as a lysosomal marker. (H) CTSB activity of lysosomal and cytosolic fractions was measured after subcellular fractionation of St-N-treated PC3/Doc cells. * P < 0.05.

Article Snippet: The primary antibodies used were as follows: cathepsin B (CTSB; cat. no. 12216-1-AP; Proteintech, Wuhan, China), Microtubule-associated protein 1 light chain 3 beta (LC3B; cat. no. NB100-2220; Novus Biologicals, Littleton, CO, USA), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP; Proteintech), lysosome-associated membrane protein 2 (LAMP2; cat. no. 66301-1-Ig; Proteintech), p62/SQSTM1 (cat. no. sc-28359; Santa Cruz Biotechnology, Dallas, TX, USA), tubulin alpha 1b (TUBA1B; cat. No. 11224-1-AP; Proteintech), cathepsin D (CTSD; cat. No. 21327-1-AP; Proteintech), poly (ADP-ribose) polymerase 1 (PARP1; cat. No. 80174-1-RR; Proteintech), β-actin (ACTB; cat. No. 60008-1-Ig; Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. sc-365062; Santa Cruz Biotechnology).

Techniques: Staining, Fluorescence, Western Blot, Quantitative RT-PCR, Control, Marker, Activity Assay, Fractionation

Journal: PLOS ONE

Article Title: St-N, a novel alkaline derivative of stevioside, reverses docetaxel resistance by targeting lysosomes in vitro and in vivo

doi: 10.1371/journal.pone.0316268

Figure Lengend Snippet: (A–D) Body weights (A, B) and tumor sizes (C, D) were measured every 2 days after the indicated treatment including 5 mg/kg Doc, 30 mg/kg St-N, or a combination of 30 mg/kg St-N and 5 mg/kg Doc. * P < 0.05, ** P < 0.01 and *** P < 0.001. (E) Tumor weights were determined at the time of sacrifice. * P < 0.05, ** P < 0.01 and *** P < 0.001. (F) Tumors from different groups are shown. (G) Ki67 staining of the indicated treated groups. Scale bar: 50 μm. (H) Effect of St-N on CTSB, p62, LC3B, and LAMP2 expression in RM-1/Doc homografts determined by western blotting. Representative patterns and the corresponding histograms were displayed. * P < 0.05, ** P < 0.01 compared with the vehicle control. (I) Effect of St-N on ASM activities in lysates from RM-1/Doc homografts. *** P < 0.001.

Article Snippet: The primary antibodies used were as follows: cathepsin B (CTSB; cat. no. 12216-1-AP; Proteintech, Wuhan, China), Microtubule-associated protein 1 light chain 3 beta (LC3B; cat. no. NB100-2220; Novus Biologicals, Littleton, CO, USA), lysosome-associated membrane protein 1 (LAMP1; cat. no. 21997-1-AP; Proteintech), lysosome-associated membrane protein 2 (LAMP2; cat. no. 66301-1-Ig; Proteintech), p62/SQSTM1 (cat. no. sc-28359; Santa Cruz Biotechnology, Dallas, TX, USA), tubulin alpha 1b (TUBA1B; cat. No. 11224-1-AP; Proteintech), cathepsin D (CTSD; cat. No. 21327-1-AP; Proteintech), poly (ADP-ribose) polymerase 1 (PARP1; cat. No. 80174-1-RR; Proteintech), β-actin (ACTB; cat. No. 60008-1-Ig; Proteintech), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; cat. no. sc-365062; Santa Cruz Biotechnology).

Techniques: Staining, Expressing, Western Blot, Control