Conditioned spin and charge dynamics of a single electron quantum dot

In this article we describe the incoherent and coherent spin and charge dynamics of a single electron quantum dot. We use a stochastic master equation to model the state of the system, as inferred by an observer with access to only the measurement signal. Measurements obtained during an interval of time contribute, by a past quantum state analysis, to our knowledge about the system at any time $t$ within that interval. Such analysis permits precise estimation of physical parameters, and we propose and test a modification of the classical Baum-Welch parameter re-estimation method to systems driven by both coherent and incoherent processes.Comment: 9 pages, 9 figure

mycUP1 expression is stimulated by the depletion of nuclear of hnRNP A1 and A2.

<p><b>A</b>, Western analysis of A1, A2 and mycUP1 from four selected HCT116 clones engineered to express a transfected myc-tagged mouse UP1. Stable transformants were transfected with various siRNA mixtures and proteins were extracted 72 hours later. Only siRNA A1₂ also targets the mouse mycUP1. <b>B</b>, Cytoplasmic accumulation of hnRNP A1 and hnRNP A2 in HCT116 cells upon osmotic shock. HCT116 cells growing on glass coverslips were left untreated (0 mM) or were exposed to 600 mM sorbitol. After 1 hour, cells were fixed and immunostained with anti-hnRNP A1 and anti-hnRNP A2 antibodies. <b>C</b>, Sorbitol stimulates mycUP1 expression in a p38 kinase-dependent manner. The p38 kinase inhibitor SB203580 was added to H55-34 cells for 2 hours (8 μg/ml) before treating cells with 600 mM sorbitol for 1 hour. Cells were then washed with PBS and incubated in complete media for 24 hours. Endogenous A1/A2 and mycUP1 proteins were detected simultaneously using the anti-A1/A2 antibody.</p

Transcription elongation recovery assay.

<p>Change in the rate of transcription of different portions of <i>Kitlg</i> upon depletion of A1 and A2 by RNAi. Seventy-two hours after transfecting a control siRNA against <i>luciferase</i> (Luc) or siA1₆ + siA2₁ (A1/A2) in HCT116 cells (H55-34 clone), DRB was applied, washed out, and the recovery of transcription was assessed at different positions. Each graph illustrates the position analyzed and the level of RNA at each time after the DRB block was released. The graphs represent averages of three independent experiments and standard deviations are provided. The positions analyzed on <i>Kitlg</i> are shown schematically in the map provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.g003" target="_blank">Fig 3C</a>.</p

The depletion of nuclear hnRNP A1 and A2 stimulates the transcription of mycUP1.

<p><b>A</b>, Steady-state levels of mycUP1 transcripts measured by semi-quantitative or real-time quantitative RT-PCR (sqRT-PCR or qRT-PCR). At least 5 independent depletion experiments (A1₆+A2₁ vs Luc) and sorbitol treatments (0 or 600 mM) were analyzed by sqRT-PCR (examples are shown in Fig B in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.s001" target="_blank">S1 File</a>). Fold increases are represented by white diamonds over an averaged logarithmic histogram. Numbers below the histograms indicate averaged fold stimulation in mycUP1 transcripts. <b>B</b>, Actinomycin D (ActD) abrogates the increase in mycUP1 protein and RNA expression following osmotic shock. The top panel represents a western with the A1/A2 antibody and the results of sqRT-PCR for mycUP1 and actin (ACTB) are shown below. <b>C</b>, ActD abrogates the increase in mycUP1 protein expression associated with the knockdown of A1 and A2. Western analysis of mycUP1 protein expression is shown at different times after siRNA treatment with or without ActD for the last 24 hours of each period. <b>D</b>, HCT116 cells expressing mycUP1 were treated with 1 or 10 μg/ml of cycloheximide 1 hour prior to sorbitol treatment. The mycUP1 protein and RNA levels are shown. Duplicate experiments with quantitated mycUP1 transcript levels are provided in Fig C in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126654#pone.0126654.s001" target="_blank">S1 File</a>.</p

The hnRNP A1 Protein Stimulates Large-Intron Splicing

<div><p>(A) Sequestering hnRNP A/B proteins affects large-intron splicing. The large-intron substrates 7-AdB lacking ABS (−.−) or containing ABS (a.a) were co-incubated with the small-intron 7B-Ad pre-mRNA (80 pM for the 7-AdB substrates and 16 pM for the control 7B-Ad pre-mRNA) in a HeLa extract for 90 min in the presence of increasing amounts of the telomeric oligonucleotide TS10 (0, 80, 160, 320, and 640 nM, respectively).</p> <p>(B) Splicing mixtures were incubated with increasing amounts of recombinant GST-A1 protein (0, 0.8, 1.6, and 3.2 μM). The 7-AdB pre-mRNA carrying inverted repeats (→.←) was also used.</p> <p>(C) Splicing mixtures containing the 7-AdB and the small-intron 7B-Ad were supplemented with His-A1 and GST-A1 (0.5 μM each).</p> <p>(D) The histogram depicts a compilation of three independent experiments performed with the indicated concentrations of recombinant proteins. In each case, individual values obtained for the splicing of 7-AdA (−.−) or 7-AdA (a.a) pre-mRNAs were normalized with the splicing efficiency of the small intron 7B-Ad pre-mRNA. Error bars indicate standard deviations.</p></div

Role of hnRNP F/H Proteins in the Activity of FBS

<div><p>(A) hnRNP H binds FBS specifically. The binding of hnRNP H and hnRNP A1 to FBS and ABS was monitored using a gel-shift assay. The sequence of the FBS RNA corresponds to the sequence shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-g004" target="_blank">Figure 4</a>A, while 2XABS is a 2′<i>O</i>-Me oligonucleotide that contains two U AGAGU elements (CCUUU AGAGUAGU AGAGU AGAAUAAG-CCUUGCAUAAAUGG). Binding conditions were as described previously [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-b043" target="_blank">43</a>] and used 1.25, 2.5, and 3 μM of hnRNP H or A1 proteins. </p> <p>(B) hnRNP F/H are required for the activity of FBS on 5′ splice-site selection. Nuclear extracts were prepared from HeLa cells that were treated with siRNAs against hnRNP F/H [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-b034" target="_blank">34</a>]. Pre-mRNA substrates lacking or containing FBS were assayed in extracts prepared from mock-treated and siF/H-treated cells. The siF/H extract was also supplemented with recombinant His-tagged hnRNP H protein prepared from baculovirus-infected cells (0.15 μM). The ratio of the products resulting from the use of the distal or proximal 5′ splice site is indicated below the lane number. The right panel shows a Western blot analysis of the content of hnRNP F and H proteins in extracts prepared from mock-treated and siF/H-treated cells. In addition to the anti-F or anti-H antibody, an anti-A1 antibody [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040021#pbio-0040021-b045" target="_blank">45</a>] was co-incubated to reveal A1 and monitor total protein loading.</p> <p>(C) Splicing assays using the 553 and the 553ff pre-mRNAs were performed in triplicate in extracts prepared from mock-treated and siF/H-treated HeLa cells. The ratio of the amplified products corresponding to the proximal and distal 5′ splice-site usage was calculated and plotted in a graph that displays error bars.</p> <p>(D) Oligonucleotide-mediated RNase H protection assays to monitor U1 snRNP occupancy on the competing 5′ splice sites. Pre-mRNAs lacking or containing FBS or ABS were incubated at 0 °C in mock-treated and U1 snRNP-inactivated extracts (ΔU1). Oligonucleotides complementary to the 5′ splice sites were added along with RNase H. The position of the fully protected pre-mRNAs and cleaved molecules is shown.</p></div