Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

SEM and TEM images of TNAP-positive cells induced to differentiate into osteocyte-like cells.

<p>After isolation by FACS, TNAP-positive and -negative cells were cultured in OBM for 120 days. (a) SEM images of TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. (b) Images of toluidine blue-stained semi-thin sections of TNAP-positive cells are shown at low (left) and high (right) magnification with light microscopy. (c) TEM image of TNAP-positive cells. Arrowheads indicate cytoplasmic processes.</p

Characterization of gene expression in TNAP-positive iPSCs.

<p>(a) Comparison of expression of ES cell markers in TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. qRT-PCR analysis of <i>OCT3/4</i>, <i>SOX2</i>, <i>NANOG</i>, <i>REX1</i>, <i>ESG1</i>, and <i>TERT</i> was performed in isolated cells using a FACSAria cell sorter. Parental iPSCs (hiPS) was used as a positive control. (b) Comparison of the expression of markers of osteoblast differentiation in TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells. qRT-PCR analysis was performed in cells isolated by FACS. Parental iPSCs (hiPS) were used as a negative control and osteogenic-differentiated HPDLCs (ODH) as a positive control. (c) qRT-PCR analysis was performed with cells grown in OBM for 40 days. (d) After isolation by FACS, TNAP-positive cells were treated with vehicle (cont), 10 nM active vitamin D3 (VD10), or 50 nM active vitamin D3 (VD50) for 6 days. Abbreviations: RUNX2, runt-related transcription factor 2; TNAP, tissue-nonspecific alkaline phosphatase; COL1A1, type I collagen; OSX, osterix; BSP, bone sialoprotein; OCN, osteocalcin. The expression of these genes was analyzed by qRT-PCR, and the mRNA levels of the genes were normalized to that of <i>GAPDH</i> or 18S rRNA. The experiments were performed in triplicate. Values represent mean ± S.D. (<i>n</i>  =  4). *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.005.</p

Characterization of ALP-positive cells derived from human iPSCs.

<p>(a) Single cells from hEBs were cultured with various cytokines for 2 weeks and stained for ALP activity. The cells were cultured in α-MEM containing 10% FBS (α-MEM); α-MEM containing 10% FBS, ascorbic acid, and β-glycerophosphate (β-GP) (OBM); OBM with FGF-2 and TGF-β1 (FGF2 + TGF); OBM with FGF-2, TGF-β1, and IGF-1 (FGF2 + TGF + IGF); OBM with FGF-2 and BMP-2/-7 (FGF2 + BMP); or OBM with FGF-2, BMP-2/-7, and IGF-1 (FGF2 + BMP + IGF). The percentages shown indicate the frequency of ALP-positive cells determined by FACS analysis. (b) FACS analysis for the isolation of ALP-positive cells (right) and isotype control (left). (c) Expression of ALP isoenzymes: germ cell-specific ALP (G), placenta-specific ALP (P), intestine-specific ALP (I), tissue-nonspecific ALP (T), and β-actin (B) in parental hiPSCs, isolated ALP-positive cells, and isolated ALP-negative cells. (d) FACS analysis of CD90 and E-cadherin in the TNAP-positive population. (e) Morphology of TNAP-positive (TNAP+) and TNAP-negative (TNAP−) cells.</p

TNAP-positive cells express various osteocyte markers.

<p>(a) Osteogenic differentiation was confirmed by Alizarin Red staining after 40 days in OBM. The upper panels are whole-well images and lower panels are magnified images. (b) Phase-contrast images of TNAP-negative and -positive cells derived from hiPSCs at day 40 of culture in OBM (upper panel). The black box in the upper images represents the region shown in the middle and lower images. (c) Comparison of the expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by qRT-PCR. Osteogenic-differentiated HPDLCs (ODH) were used as a control. (d) TNAP-positive cells were treated with vehicle (white bars), 10 nM vitamin D3 (gray bars), or 50 nM vitamin D3 (black bars) for 6 days. (e) Comparison of expression of osteocyte marker genes between TNAP-negative (TNAP−) and TNAP-positive (TNAP+) cells by RT-PCR. Abbreviations: SOST, sclerostin; RELN, reelin; NPY, neuropeptide Y. Expression of these genes was analyzed by qRT-PCR, and mRNA levels of the genes were normalized to that of <i>GAPDH</i>. The experiments were performed in triplicate. Values represent mean ± S.D. (<i>n</i>  =  4). *<i>p</i><0.05, **<i>p</i><0.01.</p

Schematic representation of the protocol for differentiation of hiPSCs into osteoblast-like cells.

<p>EBs were prepared by culturing on low-attachment Petri dishes for 6 days and dissociated in 0.5 mg/ml collagenase type IV and 0.05% trypsin–EDTA. The trypsinized EBs were cultured in OBM on cell culture dishes. Next day, various cytokines were added to the dishes (day 0) and the OBM containing cytokines was changed every 3 days. After 14 days, the cells were analyzed and isolated by FACS.</p

The C-terminal region of IKKε contains two functional domains required for IFNβ promoter activity.

<p>(A) Schematic structure of IKKε and C-terminal truncated mutants. The putative functional domains (641–657 and 686–705) are shown. (B) 293T cells were transfected with the IFNβ promoter-luciferase reporter along with the FLAG-tagged full length IKKε (1-716), kinase defective mutant (K38A) or C-terminal deletion mutants presented in (A). Cells were lysed at 24 h post-transfection and luciferase activities were quantified by normalization with renilla luciferase activity. The values represent the average of three samples +/− SD. Cell lysates were also subjected to SDS-PAGE and Western-blotted with the antibodies indicated on the left. (C) 293T cells were transfected with the IFNβ promoter-luciferase reporter along with plasmid for wt or mutant forms of TKB1 and IKKε. Luciferase activities were measured as shown in (B).</p

The C-terminal region of IKKε, but not TBK1, is required for IFNβ promoter activity.

<p>(A) 293T cells were transfected with an IFNβ promoter-luciferase reporter along with an increasing amount of FLAG-tagged full length IKKε (1-716), TBK1 (1-729), a kinase defective mutant (K38A) or C-terminal deletion mutants, as shown on the left. Cells were lysed 24 h post-transfection and luciferase activities were quantified by normalization with renilla luciferase activity. The values represent the average of three samples +/− SD. (B) L cells were transfected with indicated plasmid and total RNA were prepared at 24 h post-transfection. Relative amount of IFNβ mRNA were quantified by using qRT-PCR by normalization with HPRT mRNA. The values represent the average of three samples +/− SD.</p

Improvement of Rice Biomass Yield through QTL-Based Selection

<div><p>Biomass yield of rice (<i>Oryza sativa</i> L.) is an important breeding target, yet it is not easy to improve because the trait is complex and phenotyping is laborious. Using progeny derived from a cross between two high-yielding Japanese cultivars, we evaluated whether quantitative trait locus (QTL)-based selection can improve biomass yield. As a measure of biomass yield, we used plant weight (aboveground parts only), which included grain weight and stem and leaf weight. We measured these and related traits in recombinant inbred lines. Phenotypic values for these traits showed a continuous distribution with transgressive segregation, suggesting that selection can affect plant weight in the progeny. Four significant QTLs were mapped for plant weight, three for grain weight, and five for stem and leaf weight (at α = 0.05); some of them overlapped. Multiple regression analysis showed that about 43% of the phenotypic variance of plant weight was significantly explained (<i>P</i> < 0.0001) by six of the QTLs. From F₂ plants derived from the same parental cross as the recombinant inbred lines, we divergently selected lines that carried alleles with positive or negative additive effects at these QTLs, and performed successive selfing. In the resulting F₆ lines and parents, plant weight significantly differed among the genotypes (at α = 0.05). These results demonstrate that QTL-based selection is effective in improving rice biomass yield.</p></div

C-terminal mutants of IKKε induce nuclear translocation of IRF3.

<p>293ET cells were transfected with FLAG-tagged wt IKKε or mutants as indicated on top and were fixed at 20 h post-transfection. Fixed cells were stained with anti-FLAG and anti-IRF3 antibodies and were observed by confocal microscopy. Green, red and blue fluorescence in merged figures (top panels) indicate the endogenous IRF3, FLAG-tagged IKKε and nucleus, respectively. Single channel images are also shown in lower panels.</p