Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Structural identification of isoviolanthin. ( A ) The HPLC chromatographic analysis and ( B ) MS and MS 2 spectra of isoviolanthin.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Effects of isoviolanthin (isovio) on the viability of hepatocellular carcinoma (HCC) and human LO2 normal liver cells and on the clonogenic potential of HCC cells. ( A ) The cell viability of LO2; ( B ) HepG2, and Bel-7402 cells treated with isoviolanthin (2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM, 80 µM, and 100 μM) for 24 h and 48 h was determined by MTT [3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay; ( C ) HepG2 and Bel-7402 cells were incubated with 10 ng/mL transforming growth factor (TGF)-β1 and isoviolanthin (2.5 µM, 5 µM, and 10 µM) for two weeks, and the clonogenic potential of each group was analyzed by colony formation assay. Representative images and bar graphs (mean ± SD) are shown, n = 3. ** p < 0.01 versus the control group; ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Incubation, Colony Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Effects of isoviolanthin on TGF-β1-induced migration and invasion of HCC cells. ( A ) HepG2 and ( B ) Bel-7402 cells were pretreated with 10 ng/mL TGF-β1 for 48 h, and then treated with isoviolanthin (2.5 µM, 5 µM, and 10 µM) for the last 24 h. The migration and invasion of HepG2 and Bel-7402 cells were measured by wound healing and transwell assays. Scale bars: 100 µm. Representative images and bar graphs (mean ± SD) are shown, n = 3. The magnification is ×100. ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Migration, Control

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Effects of isoviolanthin on TGF-β1-induced matrix metalloproteinase (MMP)-2 and -9 in HCC cells. ( A ) HepG2 and Bel-7402 cells were treated with 10 ng/mL TGF-β1 for 48 h, during which isoviolanthin (2.5 µM, 5 µM, and 10 µM) was added for the last 24 h. The secretion of MMP-2 and -9 in HCC cell supernatants was detected by ELISA; ( B ) The protein levels of MMP-2 and -9 in HCC cells were examined by Western blot analysis. GAPDH was used as a loading control. Representative images and bar graphs (mean ± SD) are shown, n = 3. ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Effects of isoviolanthin on TGF-β1-induced epithelial–mesenchymal transition (EMT) in HCC cells. ( A ) HepG2 and Bel-7402 cells were treated with 10 ng/mL TGF-β1 for 48 h, during which the indicated concentrations of isoviolanthin were added for the last 24 h. The expression of the EMT markers E-cadherin, ZO-1, N-cadherin, vimentin, Snail, and Slug was assessed by Western blotting. GAPDH was used as a loading control; ( B ) E-cadherin and vimentin expression in HCC cells was determined by confocal microscopy. Green fluorescence indicates E-cadherin- and vimentin-positive expression, and blue fluorescence indicates 4’,6-diamidino-2-phenylindole (DAPI)-labeled nuclei. Scale bars: 20 µm. Representative images and bar graphs (mean ± SD) are shown, n = 3. ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Expressing, Western Blot, Control, Confocal Microscopy, Fluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Effects of isoviolanthin on the TGF-β/Smad and PI3K/Akt/mTOR pathways in HCC cells. ( A ) HepG2 and Bel-7402 cells were treated with 10 ng/mL TGF-β1 for 48 h, during which the indicated concentrations of isoviolanthin were added for the last 24 h. The expression of TGF-β/Smad pathway-related proteins, such as Smad2, p-Smad2, Smad3, and p-Smad3, was examined by Western blot analysis; ( B ) The expression of PI3K/Akt/mTOR-pathway-related proteins, including Akt, p-Akt, mTOR, p-mTOR, P70S6K, and p-P70S6K, was examined by Western blot analysis. GAPDH was used as a loading control. Representative images and bar graphs (mean ± SD) are shown, n = 3. ** p < 0.01 versus the control group; ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Verification that isoviolanthin affects TGF-β1-induced EMT, migration, and invasion in HCC cells by regulating the TGF-β/Smad and PI3K/Akt/mTOR pathways. ( A ) HepG2 and Bel-7402 cells were pretreated with 10 ng/mL TGF-β1 for 48 h and then treated with 10 µM isoviolanthin, 20 μM SB431542 (SB, TGF-β/Smad inhibitor), or 20 µM LY294002 (LY, PI3K/Akt inhibitor) for the last 24 h. HCC cell migration and invasion were measured by wound healing and transwell assays. Scale bars: 100 µm; ( B ) E-cadherin, N-cadherin, Snail, and MMP-2 expression in HCC cells was examined by Western blot analysis. GAPDH was used as a loading control. Representative images and bar graphs (mean ± SD) are shown, n = 3. ** p < 0.01 versus the control group; ## p < 0.01 versus the TGF-β1 group.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Migration, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Isoviolanthin Extracted from Dendrobium officinale Reverses TGF-β1-Mediated Epithelial–Mesenchymal Transition in Hepatocellular Carcinoma Cells via Deactivating the TGF-β/Smad and PI3K/Akt/mTOR Signaling Pathways

doi: 10.3390/ijms19061556

Figure Lengend Snippet: Schematic representation of the effect of isoviolanthin on TGF-β1-mediated EMT in HCC cells. Isoviolanthin inhibited the TGF-β/Smad and PI3K/Akt/mTOR pathways and finally suppressed HCC cell migration and invasion. “↓” indicates promotion; “⊥” indicates inhibition.

Article Snippet: HepG2 and Bel-7402 cells (2.5 × 10 5 cells/well) were pretreated with 10 ng/mL TGF-β1 for 48 h in 100 mm tissue culture dishes, followed by treatment with isoviolanthin (2.5 µM, 5 µM, and 10 µM), LY294002 (20 µM, MedChem Express, NJ, USA), or SB431542 (20 µM, MedChem Express) for the last 24 h. Total protein was isolated with radioimmunoprecipitation assay buffer (RIPA buffer) mixed 1% PMSF (Phenylmethanesulfony fluoride) as we described previously [ ], and 30 micrograms of protein was separated by 8–12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Techniques: Migration, Inhibition