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11'-Deoxyverticillin A (C42) promotes autophagy through K-Ras/GSK3 signaling pathway in HCT116 cells.

Niu S, Yuan D, Jiang X, Che Y - Protein Cell (2014)

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.

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Baf A1 addition resulted in further accumulation of LC3-II in the cells tested (Fig... S1B), confirming the aforementioned results that C42 activates autophagic process... We use specific siRNA to down-regulate the mRNA level of H- or K-Ras in HCT116 cells (Fig.  2B)... Interestingly, K-Ras silencing remarkably decreased LC3-II accumulation upon stimulation with C42, whereas the compound was found to increase LC3-II levels in the H-Ras-depleted cells (Fig.  2C and 2D)... Notably, the deprivation of K-Ras, but not H-Ras, blocked the C42-induced phosphorylation of GSK3 at Tyr279/216 (Fig.  2F), suggesting that C42 enhanced autophagy via K-Ras/GSK3 signaling pathway (Fig.  2G)... In the present study, we clearly showed that K-Ras, not H-Ras, plays an essential role in mediating the C42-induced autophagy... Interestingly, we observed that GSK3 also functioned as a downstream signaling molecular of K-Ras to regulate the C42-induced autophagy... In one work, autophagy was activated through the GSK3-Tip60-ULK1 signaling pathway (Lin et al., )... Moreover, cadmium was found to promote autophagy through the ROS-GSK3β signaling pathway (Wang et al., )... Consistent with aforementioned studies, we found that GSK3 was further activated at Tyr279/216 in the C42-treated cells accompanied by accumulation of LC3-II, indicating that C42 also induce autophagy through the GSK3 dependent pathway... The new finding in the present study is that K-Ras, not H-Ras, acts upstream of GSK3 to mediate the C42-induced autophagy... Shubin Niu, Dongdong Yuan, Xuejun Jiang and Yongsheng Che declare that they have no conflict of interest... This article does not contain any studies with human or animal subjects performed by the any of the authors.

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The C42 enhances autophagy via GSK3 signaling pathway. (A) Electron microscopy was performed on vehicle (ctrl) and the C42-treated (0.5 μmol/L, 3 h) HCT116 cells as described in Materials and Methods. The right picture of the lower row indicated the high-contrast image of the cell region marked by the box. (B and E) HCT116 cells were transfected with a plasmid expressing GFP-LC3. After 12 h, the cells were treated with the presence or absence of LiCl for 3 h at 37°C in RPMI-1640 medium with 1/1000 DMSO (Ctrl), and C42 (0.5 μmol/L). Following fixation, the cells were stained with DAPI and visualized immediately by fluorescence microscopy. The number of punctuate GFP-LC3 in each cell was counted, and at least ten cells were included in each group. The data were normally distributed and were statistically analyzed. The asterisks denote a significant difference between the groups (P < 0.01). (C) HCT116 cells were treated with increasing concentrations of C42 (0.1–1.0 μmol/L) for 1 h, harvested, lysed, and immunoblotted for indicated proteins. The levels of p-p70S6 K (S6K1, Thr389) and p-GSK (Y216/279) were detected by Western blot analysis. (D) HCT116 cells were treated with C42 (0.5 μmol/L) in the presence or absence of LiCl and chloroquine (CQ) for up to 4 h before analysis by immunoblotting with the indicated antibodies. The lysates were analyzed by Western blot with the antibodies indicated. Densitometry was performed for quantification. The adjusted ratios of LC3-II to actin were calculated and presented below the blots. The ratios represent the results of three independent experiments
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Fig1: The C42 enhances autophagy via GSK3 signaling pathway. (A) Electron microscopy was performed on vehicle (ctrl) and the C42-treated (0.5 μmol/L, 3 h) HCT116 cells as described in Materials and Methods. The right picture of the lower row indicated the high-contrast image of the cell region marked by the box. (B and E) HCT116 cells were transfected with a plasmid expressing GFP-LC3. After 12 h, the cells were treated with the presence or absence of LiCl for 3 h at 37°C in RPMI-1640 medium with 1/1000 DMSO (Ctrl), and C42 (0.5 μmol/L). Following fixation, the cells were stained with DAPI and visualized immediately by fluorescence microscopy. The number of punctuate GFP-LC3 in each cell was counted, and at least ten cells were included in each group. The data were normally distributed and were statistically analyzed. The asterisks denote a significant difference between the groups (P < 0.01). (C) HCT116 cells were treated with increasing concentrations of C42 (0.1–1.0 μmol/L) for 1 h, harvested, lysed, and immunoblotted for indicated proteins. The levels of p-p70S6 K (S6K1, Thr389) and p-GSK (Y216/279) were detected by Western blot analysis. (D) HCT116 cells were treated with C42 (0.5 μmol/L) in the presence or absence of LiCl and chloroquine (CQ) for up to 4 h before analysis by immunoblotting with the indicated antibodies. The lysates were analyzed by Western blot with the antibodies indicated. Densitometry was performed for quantification. The adjusted ratios of LC3-II to actin were calculated and presented below the blots. The ratios represent the results of three independent experiments

Mentions: Electron microscopy, which is believed to be one of the most convincing instruments to detect autophagy, was used to investigate the autophagy induced by C42 (Klionsky et al., 2012). Compared to the control, an obvious accumulation of membrane vacuoles was found in the C42-treated HCT116 cells and cytosolic components or organelles were sequestered in some of the vacuoles. Autophagosome-like vacuoles with double-membrane structures (high magnification) and a segment of the double-membrane between a vacuole and mitochondrion were also observed (Fig. 1A).Figure. 1


11'-Deoxyverticillin A (C42) promotes autophagy through K-Ras/GSK3 signaling pathway in HCT116 cells.

Niu S, Yuan D, Jiang X, Che Y - Protein Cell (2014)

The C42 enhances autophagy via GSK3 signaling pathway. (A) Electron microscopy was performed on vehicle (ctrl) and the C42-treated (0.5 μmol/L, 3 h) HCT116 cells as described in Materials and Methods. The right picture of the lower row indicated the high-contrast image of the cell region marked by the box. (B and E) HCT116 cells were transfected with a plasmid expressing GFP-LC3. After 12 h, the cells were treated with the presence or absence of LiCl for 3 h at 37°C in RPMI-1640 medium with 1/1000 DMSO (Ctrl), and C42 (0.5 μmol/L). Following fixation, the cells were stained with DAPI and visualized immediately by fluorescence microscopy. The number of punctuate GFP-LC3 in each cell was counted, and at least ten cells were included in each group. The data were normally distributed and were statistically analyzed. The asterisks denote a significant difference between the groups (P < 0.01). (C) HCT116 cells were treated with increasing concentrations of C42 (0.1–1.0 μmol/L) for 1 h, harvested, lysed, and immunoblotted for indicated proteins. The levels of p-p70S6 K (S6K1, Thr389) and p-GSK (Y216/279) were detected by Western blot analysis. (D) HCT116 cells were treated with C42 (0.5 μmol/L) in the presence or absence of LiCl and chloroquine (CQ) for up to 4 h before analysis by immunoblotting with the indicated antibodies. The lysates were analyzed by Western blot with the antibodies indicated. Densitometry was performed for quantification. The adjusted ratios of LC3-II to actin were calculated and presented below the blots. The ratios represent the results of three independent experiments
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Fig1: The C42 enhances autophagy via GSK3 signaling pathway. (A) Electron microscopy was performed on vehicle (ctrl) and the C42-treated (0.5 μmol/L, 3 h) HCT116 cells as described in Materials and Methods. The right picture of the lower row indicated the high-contrast image of the cell region marked by the box. (B and E) HCT116 cells were transfected with a plasmid expressing GFP-LC3. After 12 h, the cells were treated with the presence or absence of LiCl for 3 h at 37°C in RPMI-1640 medium with 1/1000 DMSO (Ctrl), and C42 (0.5 μmol/L). Following fixation, the cells were stained with DAPI and visualized immediately by fluorescence microscopy. The number of punctuate GFP-LC3 in each cell was counted, and at least ten cells were included in each group. The data were normally distributed and were statistically analyzed. The asterisks denote a significant difference between the groups (P < 0.01). (C) HCT116 cells were treated with increasing concentrations of C42 (0.1–1.0 μmol/L) for 1 h, harvested, lysed, and immunoblotted for indicated proteins. The levels of p-p70S6 K (S6K1, Thr389) and p-GSK (Y216/279) were detected by Western blot analysis. (D) HCT116 cells were treated with C42 (0.5 μmol/L) in the presence or absence of LiCl and chloroquine (CQ) for up to 4 h before analysis by immunoblotting with the indicated antibodies. The lysates were analyzed by Western blot with the antibodies indicated. Densitometry was performed for quantification. The adjusted ratios of LC3-II to actin were calculated and presented below the blots. The ratios represent the results of three independent experiments
Mentions: Electron microscopy, which is believed to be one of the most convincing instruments to detect autophagy, was used to investigate the autophagy induced by C42 (Klionsky et al., 2012). Compared to the control, an obvious accumulation of membrane vacuoles was found in the C42-treated HCT116 cells and cytosolic components or organelles were sequestered in some of the vacuoles. Autophagosome-like vacuoles with double-membrane structures (high magnification) and a segment of the double-membrane between a vacuole and mitochondrion were also observed (Fig. 1A).Figure. 1

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Baf A1 addition resulted in further accumulation of LC3-II in the cells tested (Fig... S1B), confirming the aforementioned results that C42 activates autophagic process... We use specific siRNA to down-regulate the mRNA level of H- or K-Ras in HCT116 cells (Fig.  2B)... Interestingly, K-Ras silencing remarkably decreased LC3-II accumulation upon stimulation with C42, whereas the compound was found to increase LC3-II levels in the H-Ras-depleted cells (Fig.  2C and 2D)... Notably, the deprivation of K-Ras, but not H-Ras, blocked the C42-induced phosphorylation of GSK3 at Tyr279/216 (Fig.  2F), suggesting that C42 enhanced autophagy via K-Ras/GSK3 signaling pathway (Fig.  2G)... In the present study, we clearly showed that K-Ras, not H-Ras, plays an essential role in mediating the C42-induced autophagy... Interestingly, we observed that GSK3 also functioned as a downstream signaling molecular of K-Ras to regulate the C42-induced autophagy... In one work, autophagy was activated through the GSK3-Tip60-ULK1 signaling pathway (Lin et al., )... Moreover, cadmium was found to promote autophagy through the ROS-GSK3β signaling pathway (Wang et al., )... Consistent with aforementioned studies, we found that GSK3 was further activated at Tyr279/216 in the C42-treated cells accompanied by accumulation of LC3-II, indicating that C42 also induce autophagy through the GSK3 dependent pathway... The new finding in the present study is that K-Ras, not H-Ras, acts upstream of GSK3 to mediate the C42-induced autophagy... Shubin Niu, Dongdong Yuan, Xuejun Jiang and Yongsheng Che declare that they have no conflict of interest... This article does not contain any studies with human or animal subjects performed by the any of the authors.

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Related in: MedlinePlus