Flow cytometer analysis of the different cell populations in the absence or presence of 1 μg/ml doxycycline for 5 days.

<p>A. Representative examples of histograms based on DNA content showing the decrease of the proportion of the S+G2/M population upon induction of shRNA or PIT271, with the concurrent increase in the sub-G1 population in the latter case. B. Quantitative analysis of the different sub-populations. Results (mean ± SEM) are pooled from four independent experiments, with duplicate measures in each. +: p<0,05, ++: p<0.01, relative to the same population without doxycycline, *: p<0,05, **: p<0,01.</p

Effects of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation and survival of GH4C1 cells.

<p>A. Expression of mRNA's for PIT-1 or PIT271 (upper panel), as well as for PRL (lower panel), in the different cell lines in the absence or presence of 1 μg/ml doxycycline for 5 days. The cells used were from the same experiment as those grown on parallel plates to measure proliferation rate (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g003" target="_blank">Fig. 3B</a>). Results for 'Pit endo' and ‘Pit SIR’ have been obtained by normalizing the values obtained in qPCR to those obtained with known quantities of external controls, and can thus be compared. Values correspond to mean ± SEM (n = 3). The significance of the effect of docycycline-induction on the expression of endogenous PIT-1, of the transgene or PRL was evaluated by the Student's t-test. +: p<0,05; ++: p<0,01; +++: p<0,001. B. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation of GH4C1 or GH4C1-sh1 cells as well as on CV1 cells. Cells were counted on triplicate wells. Induction of the expression of the shRNA (GH4C1-sh1 cells) and of the transcription factors was started on day 0 and continued throughout the experiment. 'Ctrl' represent GH4C1, GH4C1-sh1 or CV1 cells not infected with pInducer-20 vector expressing a transcription factor. Counting of CV1 cells was discontinued after 5 days, as cells became too confluent. Values correspond to mean ± SEM (n = 3). C. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the proliferation of GH4C1 or GH4C1-sh1 cells. Data represent the pooled results of the four experiments (with triplicate wells in each) of which one is illustrated on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g003" target="_blank">Fig. 3B</a>, and correspond to the cell numbers found in the absence or the presence of doxycycline five day (D5) after the start of the induction. Mean ±SEM, +: p<0,05, ++: p<0,01, relative to the same population without doxycycline, *: p<0,05. D. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on DNA fragmentation as evaluated by Tunel assay following 4 days exposure to doxycycline. Labeled and unlabeled cells were counted from 6 micrographs taken at random from duplicate wells and the experiment was repeated twice. Mean ±SEM, +: p<0,05, relative to the same population without doxycycline. E. Effect of the expression of PITWT, PIT271 and/or shRNA for PIT-1 on the division of GH4C1 or GH4C1-sh1 cells using EDU as cell division marker. EDU was present for 15–17h between day 3 and 4 of the induction by doxycycline. Labeled and unlabeled cells were counted from 6 micrographs taken at random from duplicate wells and experiments were repeated three times. Numbers given within the columns correspond to the population doubling time in hours as calculated from the labeling percentages. Mean ±SEM, +: p<0,05, ++: p<0,01, relative to the same population without doxycycline, **: p<0,01.</p

Constructs used in this study.

<p>A. Oligonucleotides used for the construction of the shRNA expressing lentiviral vectors. In red the target sequence, in blue the antisense (guide) strand, in green the sequence corresponding to the miR-30 'head'. The sequence of the Ambion siRNA on which a given shRNA construct was based is indicated in italics, underlined is the base that has been modified in order to introduce a bulge into the miRNA produced. B. Scheme of the pInducer lentiviral vectors used [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.ref015" target="_blank">15</a>]. Expression of the red fluorescent protein (RFP) and the coupled miR30 construct containing the inserted shRNA sequence (pInducer-10) or of the inserted CDS (pInducer-20) is driven by the synthetic TRE2 promoter regulated in a doxycycline-dependent manner by the rtTA3 transcription factor expressed constitutively by the Ubc promoter. The vectors express resistance markers, allowing selection of infected cells by puromycin or G418. C. Modifications introduced in the sequences targeted by the shRNA's in the coding sequence of PIT-1. The CDS with these modifications have been used to construct the pInducer-20 based expression vectors. The wt sequence is given in the upper lines, the modified sequence with the introduced changes indicated in capital letter in the middle lines. The lower line indicated the amino acids corresponding to the codons. The numbers in parentheses indicate the ordinal number of the first codon.</p

Dose-dependence of the actions of shRNA, PITWT and PIT271.

<p>A. Dose-responses curves of mRNA's for PIT-1 and PRL, in the different cell lines grown in presence of different doses (0, 20, 40, 100 or 1000 ng/ml) of doxycycline for 5 days. Results for 'PIT endo' and ‘PIT SIR’ have been obtained by normalizing the values obtained in qPCR to those obtained with known quantities of external controls, and can thus be compared. The values at 'Ctrl' correspond to the levels measured in cells exposed to 0 ng/ml doxycycline. The results correspond to the mean ± SEM of three independent measurements. B. Western blots for cells included in the experiment depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g005" target="_blank">Fig. 5A</a>. C. Representative examples of histograms based on the fluorescence of RFP as evaluated by flow cytometry in GHC1/PITWT cells grown in presence of different doses of doxycycline for 5 days. D. Quantitation of Western blots of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g005" target="_blank">Fig. 5B</a>. Bands of the protein of interest were normalized relative to actin and expressed as percentage of control cells grown in the absence of doxycycline. Legends are the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g005" target="_blank">Fig. 5A</a>. E. Dose-response curves for the proportions of the sub-G1 and the S+G2M populations in the different cell lines grown in presence of different doses (0, 20, 40, 100 or 1000 ng/ml) of doxycycline for 5 days. The values at 'Ctrl' correspond to the levels measured in cells exposed to 0 ng/ml doxycycline. The results are given as mean ± SEM (n = 3), significant correlations (**: p<0.001) are indicated beside the corresponding curves. Legends are the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g005" target="_blank">Fig. 5A</a>. Pearson coefficient of correlations were for Sub-G1 results: GH4C1/PIT271: r = 0,9449 (df = 10), GH4C1/PITWT: r = 0,8144 (df = 13), GHC1/sh1/PIT271: r = 0,9668 (df = 13) and for M+G2M results: GHC1/PIT271: r = -0,8044 (df = 13), GH4C1/sh1: r = -0,9615 (df = 13), GH4C1/sh1/PIT271: r = -0,9802 (df = 10).</p

Development of GH4C1 cells clones with inducible expression of shRNA and/or PITWT and PIT271.

<p>A. Pit-1 mRNA levels in GH4C1 cells 48 hours after transfection of siRNA's (Ambion) specific for rat Pit-1. Results are expressed relative to levels found in Control ('Ctrl') cells corresponding to non-transfected cells and correspond to the mean ± SEM of two experiments. 'Mock' corresponds to GH4C1 cells transfected with an unrelated non-specific control siRNA. B. Time-course of PIT-1 mRNA levels following induction of shRNA expression in GH4C1 cells (polyclonal population) infected with shRNA expressing conditional lentiviral vectors. Results are the mean ± SEM of two experiments and are expressed relative to levels found in Control ('Ctrl') cells corresponding to GH4C1 cells infected with the sh1 expressing vector and not exposed to the inducer doxycycline. Induction was achieved by exposure to 1μg/ml doxycycline for 1 to 7 days. C. PIT-1 mRNA levels in individual clones of GH4C1 cells harboring the shRNA expressing conditional lentiviral vectors, 6 days after induction of shRNA expression by 1 μg/ml doxycycline. Results are expressed relative to values found for GH4C1 control cells. D. Western blots of control GH4C1 cells and GH4C1/sh1 A2 clone. Induction by 1 μg/ml doxycycline was maintained for 3 or 7 days. Molecular weight ladder (Thermo Page Ruler) on the right. E. Expression of mRNA's for PIT-1 or PIT271 in the different cell lines in the absence or presence of 1 μg/ml doxycycline for 6 days. 'Pit endo' corresponds to wt endogenous PIT-1, ‘Pit SIR’ (si-resistant) to PITWT or PIT271 expressed from the conditional expression vectors. The results for 'Pit endo' and ‘Pit SIR’ have been obtained by normalizing the values obtained in qPCR to those obtained with known quantities of external controls, and are thus comparable. Results are mean ± SEM of three measures. F. Expression, in the same cells as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120010#pone.0120010.g002" target="_blank">Fig. 2E, of</a> PRL mRNA's. Results are expressed relative to values found for GH4C1 control cells.</p

Effects of hPITWT on the viability of primary human pituitary tumor cells.

<p>A. Expression of mRNA's for PIT-1 in individual tumor populations following infection with the control, eGFP-expressing lentiviral vector or with the hPITWT expressing lentiviral vector. The global increase in the expression of PIT-1 mRNA following the infection was statistically significant (paired t-test, p<0,005). B. Viability of individual tumor populations following infection with the hPITWT expressing lentiviral vector, expressed relative to the viability following infection with the control, eGFP-expressing lentiviral vector, as evaluated by the Cell Titer-Glo assay. Mean ± SEM (n = 3), +: p < 0,05, ++: p < 0,01.</p

Effect of antagonists of different types of cell death on the PITWT or PIT271 induced cell death.

<p>The proportion of sub-G1 population was evaluated by flow cytometry. Drugs were Z-VAD-FMK (25 μM), a general caspase inhibitor; chloroquine (10 μM), an inhibitor of autophagy; IM-54 (10 μM) and necrostatin-1 (10 μM), inhibitors of necrosis. Flow cytometry was performed 4 days after the addition of the drugs together with 1 μg/ml doxycycline. Results (mean ±SEM) are pooled from two independent experiments, with triplicate measures in each. +: p < 0,05, ++: p < 0,01 relative to the same population without doxycycline; *: p < 0,05, **: p < 0,01 relative to the same condition in the absence of drug.</p

Live Stimulated Raman Histology for the Near-Instant Assessment of Central Nervous System Samples

Central nervous system tumors encompass many heterogeneous neoplasms with different outcomes and treatment strategies. The current classification of these tumors is based on molecular parameters in addition to histopathology to define tumor entities. This genomic characterization of tumors is also becoming increasingly essential for physicians to identify targeted therapy options. The deployment of such genomic profiling relies on an efficient surgical sampling. To perform an appropriate tumor resection and a correct sampling of the tumor, the neurosurgeon may request an intraoperative pathological consultation. Stimulated Raman histology (SRH), an emerging nondestructive imaging technology, can address this challenge. SRH allows for a rapid and label-free microscopic examination of unprocessed tissues samples in near-perfect concordance with standard histology. In this study we showed that SRH enabled the near-instant microscopic examination of various central nervous system samples without any tissue processing such as labeling, freezing nor sectioning. Since SRH imaging is a nondestructive approach, we demonstrated that the tissue could be readily recovered after SRH imaging and reintroduced into the conventional pathology workflow including immunohistochemistry and genomic profiling to establish a definitive diagnosis

Clinical features of exertional heat stroke, 2008–2010 cohort with questionnaire.

<p>Clinical features of exertional heat stroke, 2008–2010 cohort with questionnaire.</p

<i>In vitro</i> contracture test status, 2004–2010 cohort.

<p>Legend: MHN, malignant hyperthermia normal response; MHS, malignant hyperthermia susceptible.</p><p>^aIncluding MHS for caffeine and MHS for halothane patients.</p><p><i>In vitro</i> contracture test status, 2004–2010 cohort.</p