Journal: PLoS ONE

Article Title: Forkhead Box Protein O3 Transcription Factor Negatively Regulates Autophagy in Human Cancer Cells by Inhibiting Forkhead Box Protein O1 Expression and Cytosolic Accumulation

doi: 10.1371/journal.pone.0115087

Figure Lengend Snippet: (A) Immunoblot analysis of lysates from PC3 cells transfected with control siRNA (siLuc) or that targeting FoxO1 (siFoxO1), with or without rapamycin treatment. PC3 cells were transfected with the indicated siRNA for 48 h before subsequent treatment with DMSO, 20 nM or 100 nm rapamycin (Rap) for 24 h prior to harvest and processing. (B) PC3 cells were transfected with the indicated siRNA for 48 h prior to media change, whereupon the cells were subjected to the indicated growth conditions; these conditions are DMEM in the presence (Normal) or absence of 10% FBS (serum -), or in the absence of D-glucose (with 10% FBS), as indicated. Cells were harvested 6 h after exposure to these conditions, and processed for immunoblot analysis of the indicate proteins. (C) Knockdown of FoxO1 with two targeting siRNAs suppresses autophagy in PC3 cells. (D, E, F) illustrate similar study procedures as (A, B, C), but with siRNAs targeting FoxO3a in PC3 cells. All experiments have been performed three times with similar results.

Article Snippet: Flag-FoxO1 and Flag-FoxO3a expression plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Western Blot, Transfection

Journal: PLoS ONE

Article Title: Forkhead Box Protein O3 Transcription Factor Negatively Regulates Autophagy in Human Cancer Cells by Inhibiting Forkhead Box Protein O1 Expression and Cytosolic Accumulation

doi: 10.1371/journal.pone.0115087

Figure Lengend Snippet: (A) PC3 cells were transfected with siRNA for luciferase (siLuc), FoxO1 (siFoxO1), or FoxO3a (siFoxO3a), or combinations, as indicated. Cells were harvested 72 h after transfection, and cell lysates processed for immunoblot analysis of the indicated proteins. (B) FoxO3a knockdown with two FoxO3a targeting siRNA to illustrate the impact on FoxO1 protein level. (C) Real-time PCR analysis of FoxO1 and FoxO3a mRNA levels in control siRNA (gray) or FoxO3a siRNA (black) transfected cells. Cells were harvested and processed 48 h after transfection; 18S ribosomal RNA was analyzed as the normalization control. Data was presented as Mean ± S.D. (“**”, p <0.01). (D) PC3 cells were transfected with control siRNA or FoxO3a siRNA as indicated. Following 48 h of transfection, cells were treated with either DMSO control or 10 µg/ml cycloheximide for 24 h as indicated prior to being harvested for immunoblot analysis of the indicated proteins. The FoxO1/GAPDH ratio for each condition is as presented after band quantification by ImageJ. (E) Real-time PCR analysis of endogenous FoxO1 expression level in PC3 cells transfected with either control plasmid (vector) or that expressing FoxO3a(r); in each group of the plasmid transfected cells, either control siRNA or FoxO3a siRNA were concurrently introduced. The cells are harvested for RNA preparation and q-PCR analysis 72 h post transfection; FoxO1 transcript levels were analyzed, as described in Experimental Procedures. 18S was used as the normalization control. Data was presented as Mean ± S.D. (“**”, p <0.01). All experiments have been performed three times with similar results.

Article Snippet: Flag-FoxO1 and Flag-FoxO3a expression plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Transfection, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation

Journal: PLoS ONE

Article Title: Forkhead Box Protein O3 Transcription Factor Negatively Regulates Autophagy in Human Cancer Cells by Inhibiting Forkhead Box Protein O1 Expression and Cytosolic Accumulation

doi: 10.1371/journal.pone.0115087

Figure Lengend Snippet: (A) PC3 cells were transfected with siRNA for luciferase (-) or FoxO3a (siFoxO3a) as indicated, and harvested for immunoblot analysis of the indicated proteins 72 h after transfection; see Experimental Procedures for details. Histone H3 and GAPDH were used as loading control for nuclear and cytosolic proteins, respectively. The band quantity ratios, FoxO1/GAPDH and FoxO1/Histone H3, for each condition were obtained using ImageJ. (B) Real-time PCR analysis for the relative expression levels of the indicated autophagy-related genes 48 h after transfection of PC3 cells with control siRNA (black) or two different siRNAs targeting FoxO3a (light and dark grey, respectively). Data was presented as Mean ± S.D. (C, D) Over-expression of a transcription function inactive/cytosolic form of FoxO1, FoxO1-ΔDB, increases autophagy. The quantities of LC3 positive vesicle of both PC3 (panel C) and H1299 (panel D) cells were compared in the same analysis between Flag-FoxO1-ΔDB over-expressing cells and un-transfected cells. FITC and rhodamine tagged secondary antibodies were used for the detection of anti-LC3 or anti-Flag tag, respectively. The autophagy level in each cell population was quantified using MetaMorph software; the data were plotted on the right side of each panel. >50 cells were analyzed for each condition. Data was presented as Mean ± S.E.M. (“**”, p <0.01).

Article Snippet: Flag-FoxO1 and Flag-FoxO3a expression plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Transfection, Luciferase, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Over Expression, FLAG-tag, Software

Journal: PLoS ONE

Article Title: Forkhead Box Protein O3 Transcription Factor Negatively Regulates Autophagy in Human Cancer Cells by Inhibiting Forkhead Box Protein O1 Expression and Cytosolic Accumulation

doi: 10.1371/journal.pone.0115087

Figure Lengend Snippet: (A) PC3 cells were transfected with siRNAs targeting FoxO3a as indicated. Following 48 h of transfection, cells were subjected to rapamycin treatment for 24 h prior to processing for immunoblot analysis of the indicated proteins. (B) PC3 cells underwent similar study as described in (A), except siRNA targeting FoxO1 was used as indicated. All experiments have been performed three times with similar results.

Article Snippet: Flag-FoxO1 and Flag-FoxO3a expression plasmids were purchased from Addgene (Cambridge, MA).

Techniques: Transfection, Western Blot