ΔNp73 interacts directly with SBEs in vivo.

<p>Chromatin Immunoprecipitation (ChIP) of ΔNp73 binding with SBE. The relative amount of ΔNp73 associated DNA as pulled down with an antibody directed against HA, is represented as a fold enrichment compared to pull-down with IgG (background). Gene enrichment was quantified by qPCR using primers specific for the promoter regions of A) PAI-1, B) Col1a1 and C) p21^WAF within the SBEs. Primers specific for PTEN (D) were used as a positive control. Pulldown antibody is shown on the x-axis, with y-axis showing fold enrichment ± SEM. * represents p-value>0.05.</p

ΔNp73 and Smads form a complex with SBEs in vitro.

<p>A) DNA Affinity Precipitation assay (DNAP) of SBE DNA duplexes with extracts of cells left untreated or treated with TGF-β1 after triple transfection with YFP-tagged Smad4, V5-tagged Smad3 and either empty vector control (c), HA-tagged ΔNp73 (ΔN) or HA-tagged TAp73α (TA). B) Relative binding of V5Smad3 to SBE oligos, quantification and normalization for input values of the V5Smad3 results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050815#pone-0050815-g003" target="_blank">Figure 3D</a>. C) Relative binding of YFPSmad4 binding to SBE oligos, quantification and normalization for input values of the YFPSmad4 results from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050815#pone-0050815-g003" target="_blank">Figure 3D</a>. D) Pull-down of SBE oligo incubated with a cell lysate containing HA-ΔNp73 (extract 1) or with a cell-lysate containing V5Smad3 and YFPSmad4 (extract 2) or incubated first with extract 1, washed and incubated with extract 2 (1+2). E) Immunoprecipitation (IP) with α-HA of HA-ΔNp73 induced cells (tetracycline) detecting endogenous Smad3.</p

ΔNp73 enhances promoter activity via Smad Binding Elements in Hep3B cells that lack expression of endogenous p73 and p53 a) Induction of PAI-1-luc by p53 or p73 variants and/or TGF-β1 after transfection.

<p>control = empty vector. b) Induction of p21-luc by TAp73α and its inhibition by co-expression of increasing amounts of ΔNp73. c) Induction of PAI-1-luc by TAp73α and its enhancement by co-expression of increasing amounts of ΔNp73. d) Activation of the PAI-1-luc promoter with a mutated p53 Binding Element: induction of promoter activity by p53 and TAp73 depends on an intact p53 binding element in the promoter, whilst ΔNp73 shows activity even if the p53 binding element is lacking. Transfected cells were cultured in the presence of 1 ng/ml TGF-β1 for 24 hours where indicated (right). e) Induction of Smad Binding Elements by p53 or p73 variants and/or TGF-β. Only ΔNp73 shows activity. Transfected cells were cultured in the presence of 1 ng/ml TGF-β1 for 24 hours. control = empty vector. TAp73γ and δ forms are shown only in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050815#pone-0050815-g001" target="_blank">Figure 1a</a> and were omitted in the rest of the figures for simplicity; they always showed similar results as TAp73.</p

ΔNp73 and Smads cooperate in TGF-β signaling.

<p>A) Activation of the SBE-luc promoter by a Smad2+Smad4 combination (pink edged bars) and/or ΔNp73 (Black bars) and/or TGF-β1 (right panel). c = empty vector control. B) Activation of the SBE-luc promoter by a Smad3+Smad4 combination (red edged bars) and/or ΔNp73 (black bars) and/or TGF-β1 (right panel). C) Luciferase assay of cells transfected with SBE-luc reporter and ΔNp73 in combination with increasing amounts of (inhibitory) Smad7. Cells in the right panel were also treated with 1 ng/ml TGF-β1.</p

Stimulation of TGF-β signaling by ΔNp73 in Hep3B, Hek293 and MDA-MB-468 cells.

<p>a) Luciferase assays comparing the effect of ΔNp73 on Hep3B cells versus Hek293 cells using SBE-luc as reporter, cotransfected with either empty vector or 10 ng/well ΔNp73, and/or treated with 1 ng/ml TGF-β1. b) Luciferase assay of MDA-MB-468 cells (deficient for Smad4) transfected with 400 ng/well PAI1-luc, 100 ng/well empty vector (to compensate for Smad4 plasmid in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050815#pone-0050815-g002" target="_blank">Figure 2c</a>) and either 10 ng/well empty vector (control), TAp73 or ΔNp73. Cells were grown with or without TGF-β1. c) Luciferase assay of MDA-MB-468 cells (deficient for Smad4) transfected with 400 ng/well SBE-luc, 100 ng/well Smad4 and 10 ng/well of either empty vector (control) or ΔNp73. Cells were grown in the presence of TGF-β1 where indicated. ** p<0.05 and *p<0.10 in a two-tailed T-test. d) Immunoblot analysis using a PAN-p73 specific antibody using lysates of Hek293 cells transfected with either empty pSuper vector as control (C1 or C2) or pSuper vectors expressing shRNA directed to the ΔN specific part of ΔNp73, a lysate of Hek293 cells transfected with a plasmid expressing HA-tagged ΔNp73α was used to serve as marker for the height of ΔNp73α. The bands corresponding to this height are shown additionally with a light balance appropriate for this band. Two ΔN p73 targeting sequences were used: ΔN1 and ΔN2. The PAN-p73 antibody detected multiple bands including the ΔNp73α variant. However, only a few specific bands, including a band with the height of ΔNp73α, were reduced whereas other bands were not affected, indicating that ΔNp73 variants were specifically downregulated. e) Luciferase assays of Hek293 cells transfected with SBE-luc and either pSuper empty vector control or pSuper ΔN1 and ΔN2 (combination), showing that (partial) ΔNp73 specific downregulation significantly decreases TGF-β signaling. ** p<0.05 in a two-tailed T-test. f) QPCR analysis of PAI-1 mRNA in tetracycline regulated ΔNp73 expressing cells, left untreated or after induction of ΔNp73. g) QPCR analysis of Col1a1 mRNA in tetracycline regulated ΔNp73 expressing cells left untreated or after induction of ΔNp73. ** p<0.05 in a two-tailed T-test.</p

Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion

<p>Macroautophagy/autophagy is a conserved transport pathway where targeted structures are sequestered by phagophores, which mature into autophagosomes, and then delivered into lysosomes for degradation. Autophagy is involved in the pathophysiology of numerous diseases and its modulation is beneficial for the outcome of numerous specific diseases. Several lysosomal inhibitors such as bafilomycin A₁ (BafA₁), protease inhibitors and chloroquine (CQ), have been used interchangeably to block autophagy in <i>in vitro</i> experiments assuming that they all primarily block lysosomal degradation. Among them, only CQ and its derivate hydroxychloroquine (HCQ) are FDA-approved drugs and are thus currently the principal compounds used in clinical trials aimed to treat tumors through autophagy inhibition. However, the precise mechanism of how CQ blocks autophagy remains to be firmly demonstrated. In this study, we focus on how CQ inhibits autophagy and directly compare its effects to those of BafA₁. We show that CQ mainly inhibits autophagy by impairing autophagosome fusion with lysosomes rather than by affecting the acidity and/or degradative activity of this organelle. Furthermore, CQ induces an autophagy-independent severe disorganization of the Golgi and endo-lysosomal systems, which might contribute to the fusion impairment. Strikingly, HCQ-treated mice also show a Golgi disorganization in kidney and intestinal tissues. Altogether, our data reveal that CQ and HCQ are not <i>bona fide</i> surrogates for other types of late stage lysosomal inhibitors for <i>in vivo</i> experiments. Moreover, the multiple cellular alterations caused by CQ and HCQ call for caution when interpreting results obtained by blocking autophagy with this drug.</p