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Autophagy Regulates Formation of Primary Cilia in Mefloquine-Treated Cells.

Shin JH, Bae DJ, Kim ES, Kim HB, Park SJ, Jo YK, Jo DS, Jo DG, Kim SY, Cho DH - Biomol Ther (Seoul) (2015)

Bottom Line: In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux.Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells.Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701.

ABSTRACT
Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

No MeSH data available.


Related in: MedlinePlus

Mefloquine induces autophagy in htRPE cells. (A) htRPE/GFP-LC3 cells were treated with Meflo (10 μM) and imaged by confocal microscopy. (B) htRPE/GFP-LC3 cells treated with Meflo (5, 10 μM) or Cyto D (50 nM) does and time dependent manner were fixed, and cells with autophagic punctate structures were counted under a fluorescence microscopy. (C) htRPE cells were treated with Meflo with different concentration and protein expression was detected by Western blotting with indicated antibodies. (D) htRPE cells were treated with Meflo (10 μM) in the presence or absence of bafilomycin A1 (Baf). The conversion of LC3 protein was detected by Western blotting. (E) htRPE cells treated with 3-methyladenine (3MA) were further incubated with Meflo (10 μM); the conversion of LC3 protein was detected by western blotting. (F) htRPE cells were transfected with scrambled siRNA (Sc) or a specific siRNA against ATG5 (siATG5). Three days later, the cells were incubated with or without Meflo (10 μM) for an additional 24 h. LC3 and ATG5 expression levels were examined by western blot analysis. Data were obtained from at least three independent experiments, and values are presented as the means ± S.E.M. (n>3, *p<0.05).
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f3-bt-23-327: Mefloquine induces autophagy in htRPE cells. (A) htRPE/GFP-LC3 cells were treated with Meflo (10 μM) and imaged by confocal microscopy. (B) htRPE/GFP-LC3 cells treated with Meflo (5, 10 μM) or Cyto D (50 nM) does and time dependent manner were fixed, and cells with autophagic punctate structures were counted under a fluorescence microscopy. (C) htRPE cells were treated with Meflo with different concentration and protein expression was detected by Western blotting with indicated antibodies. (D) htRPE cells were treated with Meflo (10 μM) in the presence or absence of bafilomycin A1 (Baf). The conversion of LC3 protein was detected by Western blotting. (E) htRPE cells treated with 3-methyladenine (3MA) were further incubated with Meflo (10 μM); the conversion of LC3 protein was detected by western blotting. (F) htRPE cells were transfected with scrambled siRNA (Sc) or a specific siRNA against ATG5 (siATG5). Three days later, the cells were incubated with or without Meflo (10 μM) for an additional 24 h. LC3 and ATG5 expression levels were examined by western blot analysis. Data were obtained from at least three independent experiments, and values are presented as the means ± S.E.M. (n>3, *p<0.05).

Mentions: We previously demonstrated that mefloquine induces autophagy and autophagy-associated cell death in a neuroblastoma cells. To examine whether mefloquine induces autophagy in htRPE cells, we generated the cell line htRPE/GFP-LC3, which stably expresses LC3, a molecular marker for activation of autophagy as a fusion protein with GFP. In accordance with previous results, treatment of mefloquine in htRPE cells significantly increased the punctate structure of GFP-LC3 in a dose- and time-dependent manner (Fig. 3A, B). However, Cyto D did not increase autophagy suggesting that induction of ciliogenesis does not directly activate autophagy in htRPE cells (Fig. 3B). In addition, autophagy related gene such as Beclin-1/ATG6 was not notably increased by in htRPE cells treated with mefloquine (Fig. 3C). We further examined autophagy flux by mefloquine treatment in htRPE cells. Combined treatment of mefloquine with bafilomycin A1, a lysosomal inhibitor, enhanced the level of LC3 II than that of Mefloquine alone (Fig. 3D). Next we investigated the effect of autophagy inhibition in mefloquine-treated cells. 3-methyladenine (3MA) can inhibits autophagy by blocking autophagosome formation via the inhibition of class 3 PI3K. Therefore, 3MA has been applied to block the autophagy activation (Klionsky et al., 2012). As shown in Fig. 3E and 3F, both chemical and genetic inhibition of autophagy by treatment with 3MA or ATG5 knock-down efficiently reduced mefloquine-mediated autophagy in htRPE cells treated with Mefloquine (Fig. 3E, 3F). These results suggested that mefloquine also strongly induces autophagy in htRPE cells.


Autophagy Regulates Formation of Primary Cilia in Mefloquine-Treated Cells.

Shin JH, Bae DJ, Kim ES, Kim HB, Park SJ, Jo YK, Jo DS, Jo DG, Kim SY, Cho DH - Biomol Ther (Seoul) (2015)

Mefloquine induces autophagy in htRPE cells. (A) htRPE/GFP-LC3 cells were treated with Meflo (10 μM) and imaged by confocal microscopy. (B) htRPE/GFP-LC3 cells treated with Meflo (5, 10 μM) or Cyto D (50 nM) does and time dependent manner were fixed, and cells with autophagic punctate structures were counted under a fluorescence microscopy. (C) htRPE cells were treated with Meflo with different concentration and protein expression was detected by Western blotting with indicated antibodies. (D) htRPE cells were treated with Meflo (10 μM) in the presence or absence of bafilomycin A1 (Baf). The conversion of LC3 protein was detected by Western blotting. (E) htRPE cells treated with 3-methyladenine (3MA) were further incubated with Meflo (10 μM); the conversion of LC3 protein was detected by western blotting. (F) htRPE cells were transfected with scrambled siRNA (Sc) or a specific siRNA against ATG5 (siATG5). Three days later, the cells were incubated with or without Meflo (10 μM) for an additional 24 h. LC3 and ATG5 expression levels were examined by western blot analysis. Data were obtained from at least three independent experiments, and values are presented as the means ± S.E.M. (n>3, *p<0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4489826&req=5

f3-bt-23-327: Mefloquine induces autophagy in htRPE cells. (A) htRPE/GFP-LC3 cells were treated with Meflo (10 μM) and imaged by confocal microscopy. (B) htRPE/GFP-LC3 cells treated with Meflo (5, 10 μM) or Cyto D (50 nM) does and time dependent manner were fixed, and cells with autophagic punctate structures were counted under a fluorescence microscopy. (C) htRPE cells were treated with Meflo with different concentration and protein expression was detected by Western blotting with indicated antibodies. (D) htRPE cells were treated with Meflo (10 μM) in the presence or absence of bafilomycin A1 (Baf). The conversion of LC3 protein was detected by Western blotting. (E) htRPE cells treated with 3-methyladenine (3MA) were further incubated with Meflo (10 μM); the conversion of LC3 protein was detected by western blotting. (F) htRPE cells were transfected with scrambled siRNA (Sc) or a specific siRNA against ATG5 (siATG5). Three days later, the cells were incubated with or without Meflo (10 μM) for an additional 24 h. LC3 and ATG5 expression levels were examined by western blot analysis. Data were obtained from at least three independent experiments, and values are presented as the means ± S.E.M. (n>3, *p<0.05).
Mentions: We previously demonstrated that mefloquine induces autophagy and autophagy-associated cell death in a neuroblastoma cells. To examine whether mefloquine induces autophagy in htRPE cells, we generated the cell line htRPE/GFP-LC3, which stably expresses LC3, a molecular marker for activation of autophagy as a fusion protein with GFP. In accordance with previous results, treatment of mefloquine in htRPE cells significantly increased the punctate structure of GFP-LC3 in a dose- and time-dependent manner (Fig. 3A, B). However, Cyto D did not increase autophagy suggesting that induction of ciliogenesis does not directly activate autophagy in htRPE cells (Fig. 3B). In addition, autophagy related gene such as Beclin-1/ATG6 was not notably increased by in htRPE cells treated with mefloquine (Fig. 3C). We further examined autophagy flux by mefloquine treatment in htRPE cells. Combined treatment of mefloquine with bafilomycin A1, a lysosomal inhibitor, enhanced the level of LC3 II than that of Mefloquine alone (Fig. 3D). Next we investigated the effect of autophagy inhibition in mefloquine-treated cells. 3-methyladenine (3MA) can inhibits autophagy by blocking autophagosome formation via the inhibition of class 3 PI3K. Therefore, 3MA has been applied to block the autophagy activation (Klionsky et al., 2012). As shown in Fig. 3E and 3F, both chemical and genetic inhibition of autophagy by treatment with 3MA or ATG5 knock-down efficiently reduced mefloquine-mediated autophagy in htRPE cells treated with Mefloquine (Fig. 3E, 3F). These results suggested that mefloquine also strongly induces autophagy in htRPE cells.

Bottom Line: In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux.Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells.Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of East-West Medical Science, Kyung Hee University, Yongin 446-701.

ABSTRACT
Primary cilia have critical roles in coordinating multiple cellular signaling pathways. Dysregulation of primary cilia is implicated in various ciliopathies. To identify specific regulators of autophagy, we screened chemical libraries and identified mefloquine, an anti-malaria medicine, as a potent regulator of primary cilia in human retinal pigmented epithelial (RPE) cells. Not only ciliated cells but also primary cilium length was increased in mefloquine-treated RPE cells. Treatment with mefloquine strongly induced the elongation of primary cilia by blocking disassembly of primary cilium. In addition, we found that autophagy was increased in mefloquine-treated cells by enhancing autophagic flux. Both chemical and genetic inhibition of autophagy suppressed ciliogenesis in mefloquine-treated RPE cells. Taken together, these results suggest that autophagy induced by mefloquine positively regulates the elongation of primary cilia in RPE cells.

No MeSH data available.


Related in: MedlinePlus