Trehalose, sucrose and raffinose are novel activators of autophagy in human. keratinocytes through an mtor-independent pathway
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1 Title page Trehalose, sucrose and raffinose are novel activators of autophagy in human keratinocytes through an mtor-independent pathway Xu Chen 1*, Min Li 1*, Li Li 1, Song Xu 1, Dan Huang 1, Mei Ju 1, Ju Huang 2, Kun Chen 1#, Heng Gu 1# 1. Institute of Dermatology, Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Chinese Academy of Medical Science & Peking Union Medical College, Nanjing, , China 2. Ontario Cancer Institute, University Health Network, Toronto, Ontario, M5G 2M1, Canada #Corresponding authors: Heng Gu Tel: Kun Chen Tel: Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Institute of Dermatology, Chinese Academy of Medical Science & Peking Union Medical College, 12 Jiangwangmiao St., Nanjing , China Fax: *: These authors contributed equally to this study.
2 Supplemental Information Figure.S1 legend (A) Total cell extracts from HeLa cells untreated or treated with 50 m chloroquine was subjected to western blot, together with two cell lysate samples from human primary keratinocytes with normal culture. LC3 protein level was determined and served as a loading control. (B) Total cell extracts from MCF-7 cells untreated or treated with insulin. The protein levels of mtor, p70 S6 Kinase, S6 Ribosomal protein and 4E-BP1, as well as their phosphorylation levels were detected. This experiment was performed to identify the sensitivity of primary antibodies. -actin served as a loading control. HaCaT cells (C) or primary keratinocytes (D) treated with or without 20 or 80 nm rapamycin. (E) Primary keratinocytes was treated with or without E64d and pepstatin. Then, the protein levels of LC3 and p62 were determined. served as a loading control. The cells treated with served as a vehicle control. Figure.S2 legend (A) The transgenes of GFP-LC3B or GFP-LC3 (G120A) were transfected into primary keratinocytes through the Premo Autophagy Sensor LC3B-GFP BacMam 2.0 system according to the instructions of manufacturer. Then the cells transfected with GFP-LC3B or GFP-LC3 (G120A) were treated with 50 m chloroquine. LC3B (G120A)-GFP served as a negative control. The cells were scanned to monitor GFP-LC3B puncta using a laser scanning confocal microscope. (B) Primary keratinocytes were treated with or without rapamycin or EBSS in the presence or absence of wortmannin. After treatment, the cells were incubated with AO, and then scanned using a laser scanning confocal microscope. In the AO G pictures, nucleus and cytoplasm showed deep green and slight green fluorescence respectively. In the AO R pictures, acidic vesicular organelles were marked with red fluorescence. Bars = 20 m. Figure.S3 legend
3 Primary keratinocytes were treated with or without rapamycin (an autophagy inducer) or Earle's balanced salt solution (EBSS, starvation-induced autophagy) in the presence or absence of wortmannin (an autophagy inhibitor). The protein levels of LC3 and Beclin-1 were determined, and served as a loading control. We did not observe change of Beclin-1 expression in human primary keratinocytes after treatment of rapamycin or EBSS incubation in the presence or absence of wortmannin, while autophagy can be activated or inhibited. These findings indicated that Beclin-1 was not involved in canonical autophagy regulation of keratinocytes through modifying its protein expression.
4 14kDa Figure S1 Figure S1a HeLa cells Primary keratinocytes 50 μm chloroquine NT 1 2 LC3(A/B)-I LC3(A/B)-II Figure S1c hours p.i. 4 hours Rapamycin (nm in ) HaCaT cells 62kDa 14kDa p62 Figure S1b NT Insulin 289kDa 289kDa 289kDa Phospho-mTOR (Ser2448) Phospho-mTOR (Ser2481) mtor Figure S1d hours p.i. 4 hours Rapamycin (nm in ) Primary keratinocytes 62kDa 14kDa p62 85kDa 70kDa 85kDa 70kDa 85kDa 70kDa 32kDa Phospho-p70 S6 Kinase (Thr371) Phospho-p70 S6 Kinase (Thr389) p70 S6 Kinase Phospho-S6 Ribosomal Protein (Ser240/244) Figure S1e E64pepstatin(in ) 62kDa 12 hours p62 32kDa 20kDa 15kDa 20kDa 15kDa S6 Ribosomal Protein Phospho-4E-BP1 Thr37/46 4E-BP1 Primary keratinocytes 14kDa 45kDa β-actin
5 Figure S2 Figure S2a chloroquine (50µM) - LC3B-GFP LC3B-GFP control LC3B(G120A)-GFP Bars=20µm Figure S2b NT 20nM Rapa EBSS GFP-LC3B AO G AO R AO Merge Bars=20µm 20nM Rapa 1µM wortmannin EBSS 1µM wortmannin
6 Figure S3 Beclin-1 protein in human keratinocytes NT 20nM Rapamycin 80nM Rapamycin EBSS 1µM wortmannin EBSS1µM wortmannin 20nM Rapamycin1µM wortmannin 80nM Rapamycin1µM wortmannin Primary keratinocytes 60kDa 14kDa Beclin-1
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