*** 0

*** 0.001 (paired Student’s = 3). of Stx17 function. Depletion of MAP1B\LC1 causes Stx17\dependent autophagosome accumulation even under nutrient\rich conditions, whereas its overexpression blocks starvation\induced autophagosome formation. MAP1B\LC1 links microtubules and Stx17 in fed cells, and starvation causes the dephosphorylation of MAP1B\LC1 at Thr217, allowing Stx17 to dissociate from MAP1B\LC1 and bind to Atg14L. Our results reveal the mechanism by which Stx17 changes its binding partners in response to nutrient status. = 3). *** 0.001 (paired Student’s = 3). ** 0.01 (paired Student’s = 3). ** 0.01 (paired Student’s = 3). B HeLa cells were treated as described in (A) and subjected to immunoelectron microscopic analysis using an antibody against LC3. Scale bar, 1 m. C, D HeLa cells or ones transiently expressing FLAG\DFCP1 or 3x\FLAG\Atg14L were mock\treated (Mock) or treated with siRNA LC1 #1 (MAP1B KD), siRNA for Stx17 (Stx17 KD), or both (MAP1B/Stx17 DKD) for 72 Duloxetine HCl h and immunostained for LC3 (C) or FLAG (D). Scale bar, 10 m. Data in the bar graphs are expressed as a fold change compared with mock\treated cells. Error bars indicate SD (30 cells/condition (C) and 40 cells/condition (D), = 3). *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). HeLa cells were transfected with a plasmid encoding the FLAG\MAP1B\LC1 wild Rabbit polyclonal to MGC58753 type, the T217A mutant, or the T217E mutant. After 24 h, the cells were transfected with siRNA HC #1, incubated for 72 h and double\immunostained using antibodies against FLAG and LC3. Of note is that siRNA HC #1 does not target the MAP1B\LC1 coding region. Scale bar, 10 m. The bar graph shows the ratio of the LC3 staining intensity of FLAG\expressing to that of non\expressing cells. Error bars indicate SD (20 cells/condition, = 3). *** 0.001 (paired Student’s = 3) normalized to that before starvation. HeLa cells were mock\transfected (Mock) or transfected with siRNA LC1 #1 (MAP1B KD). At 48 h after transfection, the cells were transfected with a plasmid encoding mRFP\GFP\LC3. At 24 h after the second transfection, the cells were not subjected to starvation treatment (Fed) or starved for 2 h (Starved) in the absence or presence of 0.1 M Baf. Scale bar, 10 m. The bar graph shows the quantification of the merged (both GFP and mRFP) images shown in the left upper two rows. Only, the cytoplasmic puncta were analyzed, and the nuclear staining was ignored. Error bars indicate SD (30 cells/condition, = 3). ** 0.01 (paired Student’s = 3). ** 0.01 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). * 0.05, ** 0.01 (paired Student’s = 3). *** 0.001 (paired Duloxetine HCl Student’s = 3). *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s = 3). ** 0.01, *** 0.001 (paired Student’s 0.05, ** 0.01; *** 0.001. Author contributions This study was conceived and designed by HN, KA, and MT. HN, KA, YK, HK, NN, and ND performed the experiments; AY performed the electron microscopic experiments; SY contributed with reagents; YW and HI contributed with expertise in experiments. Conflict of interest The authors declare that they have no conflict of interest. Supporting information Expanded View Figures PDF Click here for additional data file.(1.8M, pdf) Source Data for Expanded View Click here for additional data file.(72K, zip) Review Process File Click here for additional data file.(379K, pdf) Source Data for Figure 1 Click here for additional data file.(36K, xlsx) Source Data for Figure 2 Click here for additional data file.(32K, xlsx) Source Data for Figure 3 Click here for additional data file.(28K, xlsx) Source Data for Figure 4 Click here for additional data file.(29K, xlsx) Source Data for Figure 5 Click here for additional data file.(28K, xlsx) Source Data for Figure 6 Click here for additional data file.(29K, xlsx) Acknowledgements We thank Dr. N. Mizushima, Dr. N. Ishihara, and Dr. F. Propst for the gifts of plasmids. This paper is dedicated to the memory of Dr. Akitsugu Yamamoto who prematurely passed away on September 28, 2016. This work was supported in part by Grants\in\Aid for Scientific Research, #25291029 and. Duloxetine HCl

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