Journal: International Journal of Molecular Sciences

Article Title: Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells

doi: 10.3390/ijms23063113

Figure Lengend Snippet: Exosomes from HCC97H cells enhance proliferation and invasion of recipient HCC97L cells in a CPE-dependent manner. ( A , B ) Bar graph showing the absorbance values obtained in the MTT cell proliferation assay on day 5 of HCC97L cells treated with exosomes (corresponding to 75 µg of exosomal protein) from HCC97H cells (ExoHCCH, A ) or with exosomes isolated 48 h after lipofectamine- mediated transfection of HCC97H cells with either 25 nM of CPE targeting shRNA or control shRNA, (ExoHCCH-CPE-shRNA/ExoHCCH-CTRL-shRNA, B ) (N = 2, n = 3). ExoHCCH increase the proliferation of HCC97L cells, however downregulation of CPE expression in HCC97H cells before exosome isolation abolishes this effect. Data represents mean ± SD of 2 independent experiments. ( C ) Bar graph showing the fold change in knockdown of CPE mRNA levels in HCC97L cells treated with ExoHCCH-CPE-shRNA relative to cells treated with ExoHCCH-CTRL-shRNA (N = 2). Data represents mean ± SD of 2 independent experiments. The 2−ΔΔCt method was used for gene expression analysis and 18s rRNA was the internal control. ( D , E ) Bar graph and representative images of wells showing the number of HCC97L cells that invaded through matrigel after treatment with ExoHCCH ( D ) (N = 2, n = 2), or with either ExoHCCH-CPE-shRNA or ExoHCCH-CTRL-shRNA ( E ) (N = 1, n = 2). Data represents mean ± SD of 2 independent experiments ( D ) and mean ± SD of technical replicates ( E ). HCCH97L cells treated with ExoHCCH exhibit enhanced invasion through matrigel, and this effect is abolished if HCC97H cells are transfected with CPE-shRNA before exosome isolation. Scale bar = 100µm. Statistical analysis for all panels was performed by Student’s t -test: *, p < 0.05; ***, p < 0.001.

Article Snippet: To perform exosome transfer experiments using HCC97H-derived exosomes, HCC97H cells were seeded in a 60 mm dish and transfected with either 25 nM CPE-shRNA, which is a pool of three target-specific lentiviral vector constructs (each encoding 19–25 nt shRNAs) or control shRNA plasmids (Santa Cruz Biotechnology Inc, Cat#sc-45378-SH, sc-108060, Dallas, TX, USA) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: MTT Cell Proliferation, Isolation, Transfection, shRNA, Control, Expressing, Knockdown, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells

doi: 10.3390/ijms23063113

Figure Lengend Snippet: Characterization of exosomes loaded with CPE-shRNA. ( A ) Schematic showing the strategy of loading and transfer of CPE-shRNA via exosomes. Exosomes were isolated from supernatant media of HEK293T cells (ExoHEK) infected with adenovirus encoding either CPE-shRNA or CTRL-shRNA, fused to GFP. HCC97H cells treated with these modified exosomes exhibited green fluorescence, validating the transfer of CPE-shRNA through the exosomes. Representative images showing GFP fluorescence in target HCC97H cells, treated with either ExoHEK-CPE-shRNA or ExoHEK-CTRL-shRNA are included. Scale bar = 100 µm. ( B ) Graph showing the concentration and size distribution of ExoHEK-CPE-shRNA and ExoHEK-CTRL-shRNA, as determined by NanoSight analysis.

Article Snippet: To perform exosome transfer experiments using HCC97H-derived exosomes, HCC97H cells were seeded in a 60 mm dish and transfected with either 25 nM CPE-shRNA, which is a pool of three target-specific lentiviral vector constructs (each encoding 19–25 nt shRNAs) or control shRNA plasmids (Santa Cruz Biotechnology Inc, Cat#sc-45378-SH, sc-108060, Dallas, TX, USA) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: shRNA, Isolation, Infection, Modification, Fluorescence, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells

doi: 10.3390/ijms23063113

Figure Lengend Snippet: CPE-shRNA-loaded exosomes inhibit proliferation of malignant HCC cells. ( A ) Bar graph showing the fold change in downregulation of CPE mRNA levels in HCC97H cells treated for 48h with ExoHEK-CPE-shRNA in comparison to cells treated with ExoHEK-CTRL-shRNA (N = 2). The 2−ΔΔCt method was used for CPE mRNA expression analysis and 18s rRNA was used as the reference. Data represents mean ± SD of 2 independent experiments. ( B ) Western blot showing suppressed secreted CPE levels (70.91% ± 0.003 [SD] decrease) in the media of HCC97H cells treated with ExoHEK-CPE-shRNA relative to the media of cells treated with ExoHEK-CTRL-shRNA (N = 2). ns: non-specific. ( C ) Representative line graph showing the absorbance values obtained in the MTT cell proliferation assay from D1- D7/8 of HCC97H cells treated with HEK293T exosomes loaded with either CPE-shRNA or Control shRNA. CPE-shRNA loaded exosomes inhibit the proliferation of HCC97H cells (N = 3, n = 3). Data represents mean ± SD of the triplicate wells of the representative experiment. Statistical analysis was performed by Two-way ANOVA with Sidak’s multiple comparisons test. ****, p < 0.0001. ( D , E ) Representative images and bar graph showing the number of colonies formed by HCC97H cells treated with ExoHEK-CPE-shRNA or ExoHEK-CTRL shRNA. Exosomes loaded with CPE-shRNA significantly decreased the colony formation ability of HCC97H cells (N = 2, n = 3). Data represents mean ± SD of 2 independent experiments. Error bars denote SD ( F ) Bar graph showing the downregulation of Cyclin D1 mRNA expression in HCC97H cells incubated with ExoHEK-CPE-shRNA compared to the control (N = 3). Relative qRT-PCR was performed for Cyclin D1 mRNA quantification by 2−ΔΔCt method, and 18s rRNA was used as the reference. Data represents mean ± SD of 3 independent experiments. ( G ) Representative western blot showing reduced levels of Cyclin D1 (23.17% ± 0.022 [SD] decrease) in HCC97H cells treated with ExoHEK-CPE-shRNA compared to cells treated with ExoHEK-CTRL-shRNA (N = 2). ( H ) Bar graph showing the suppression of c-MYC mRNA levels in HCC97H cells after treating with ExoHEK-CPE-shRNA relative to cells treated with ExoHEK-CTRL-shRNA (N = 3). Relative qRT-PCR was performed for c-MYC mRNA quantification by 2−ΔΔCt method, and 18s rRNA was used as the reference. Data represents mean ± SD of 3 independent experiments. Statistical analysis for E, F and G panels was performed by Student’s t -test: **, p < 0.01, ***, p < 0.001.

Article Snippet: To perform exosome transfer experiments using HCC97H-derived exosomes, HCC97H cells were seeded in a 60 mm dish and transfected with either 25 nM CPE-shRNA, which is a pool of three target-specific lentiviral vector constructs (each encoding 19–25 nt shRNAs) or control shRNA plasmids (Santa Cruz Biotechnology Inc, Cat#sc-45378-SH, sc-108060, Dallas, TX, USA) using Lipofectamine 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: shRNA, Comparison, Expressing, Western Blot, MTT Cell Proliferation, Control, Incubation, Quantitative RT-PCR