第16回千葉カルシウム代謝研究会

Gene ontology term enrichments for RNA-Seq data from differentiated TSC2 deletion cell lines and microarray data of patient SEGAs (related to Fig. 2f). (XLSX 27.7 kb

Macrophages do not contribute to erythroid inhibition induced by hemozoin.

<p><b>A</b>, Cytokine levels in supernatants from erythroid cultures in the absence or presence of a high dose of hemozoin (25µg/ml) are shown for days 7 and 14. Empty bars represent levels detected in control cultures with media alone for each time point. The average of 3 or more independent experiments is shown with SEMs. <b>B</b>, Induction of MCP-1 by hemozoin is compared between erythroid cultures depleted of CD14⁺ macrophages on day 0 (−MØ) and cultures in which macrophages are present (+MØ). A representative experiment of 2 is shown with error bars as standard deviation of measurements in triplicate. <b>C</b>, The maturation of erythroblasts on day 14 to CD71^loCD235a⁺ precursors in cultures depleted of CD14⁺ on day 0 (−MØ) is reduced compared with the same cultures that have not had macrophages removed (+MØ). The effect of macrophage loss on erythroid development was taken into account by normalizing the yield of erythroblasts with hemozoin to the yield obtained in macrophage-depleted control cultures, where media but no hemozoin was added. The average of 2 independent experiments is shown where error bars are SEMs. **p = 0.003. <b>D</b>, O-dianisidine staining of cytospin preparations to show changes in morphology and hemoglobinization (brown staining) of cells cultured without macrophages and cultures with macrophages that are found associated with larger clusters of hemozoin. Images are representative of 3 or more experiments and were taken at ×40 magnification.</p

Assessment of reactive oxygen species (ROS) in erythroid cells following incubation with hemozoin.

<p><b>A</b>, Relative levels of ROS were determined using the fluorescent dye 2, 7-dichlorofluoroscein diacetate. The mean fluorescence intensity relative to media controls at each time point is shown. <b>B</b>, Viable erythroid cells in the same cultures with controls (MEDIA) or with 6µg/ml hemozoin (HZ). <b>C</b>, The proportion of late erythroblasts (CD71^loCD235a⁺) and <b>D</b>, the total number of CD235a⁺ erythroid cells in culture on day 14 when treated with the anti-oxidant vitamin E. Cells were incubated with 6µg/ml hemozoin on day 0 or with the same dose of hemozoin after pre incubation with 30µM vitamin E and normalized to media controls in the absence or presence of vitamin E respectively. The averages of 3 independent experiments with SEMs are shown. <b>A</b> * p = 0.021, <b>B</b> *** p = 0.0003 <b>C</b> * p = 0.0278 and <b>D</b> * p = 0.0236.</p

Erythroid Expansion <i>in vitro</i>.

<p><b>A</b>, Cytospins of cells stained with 1% dianisidine/10% Giemsa are shown to illustrate the morphological changes within this culture as erythroblasts differentiate from larger basophilic cells on day 7 to smaller hemoglobin positive cells on day 10 and with pyknotic nuclei on day 14. The brown stain is indicative of hemoglobinization. <b>B</b>, The generation of erythroid precursors was monitored by labelling cells with fluorescent antibodies specific to the cell surface markers CD71 (transferrin receptor) and CD235a (glycophorin A) and analysed by flow cytometry. <b>C</b>, The changes in FSC and SSC of gated cells are shown in B to indicate the reduced size of erythroid precursors. Representative plots and cytospins viewed at ×60 magnification from 3 or more independent experiments are shown.</p

Macrophage content required to reduce inhibition by hemozoin.

<p>Increasing proportions of isogenic CD14⁺ cells (MØ) isolated from the same culture were added on day 0 together with hemozoin. <b>A</b>, Absolute numbers of erythroblasts on day 14 and <b>B</b>, normalization of the same cell counts to media controls with each concentration of CD14⁺ cells to allow assessment of rescue by CD14⁺ cells. A representative experiment of 3 is shown where error bars are SDs of measurements in triplicate. ** p = 0.007.</p

Activation of Caspases in erythroid cells.

<p><b>A</b>, Induction of cleaved caspase 3 and cytochrome C in basophilic erythroblasts purified from day 6 cultures 24 hours after incubation with media (controls), anti-CD95 or 6µg/ml hemozoin. <b>B</b>, Western blots of lysates taken from purified basophilic erythroblasts incubated with hemozoin for 4 hours demonstrate increased levels of cleaved caspase 8 and activation of caspase 3 with slight induction of cleaved Bid (tBid). <b>C</b>, Detection of cleaved caspase 9 after 4 and 24 hours incubation with hemozoin. Each band was normalised to that for alpha tubulin. Fold changes in normalized levels of pro-apoptotic proteins relative to those in lysates from media controls are shown below each band.</p

Neutralization of TNF-α does not reverse inhibition induced by hemozoin.

<p>Erythroid cultures were pre-incubated with 25µg/ml of the neutralising antibody to TNF-α, InfliximAb (Centocor, Horsham, USA) on day 0 of the erythropoietin dependent stage of culture. After 1 hour hemozoin (Hz) and TNF-α were added at 6µg/ml and 10ng/ml respectively. The number of live erythroid CD235a positive events acquired (out of a total of 10, 000) by flow cytometry on day 14 is shown. Data from a representative experiment of 3 is shown.</p

Additional file 9: Table S6. of Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

Gene ontology term enrichments for ribosome profiling data (related to Fig. 3f). (XLSX 11 kb

Additional file 11: Table S8. of Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

Gene sets and associated genes that show after mTOR treatment a reversal of the change in expression detected in untreated TSC2 deletion cells (related to Fig. 4b). (XLSX 25.8 kb

Additional file 2: Figure S2. of Genomic analysis of the molecular neuropathology of tuberous sclerosis using a human stem cell model

Loss of TSC2 triggers expression changes related to inflammatory response, metabolism, and neuronal function. (PDF 1.15 mb