vbw area  (Worthington Biochemical)


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  • 91
    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    ls002004
    Price:
    33
    Size:
    5 mg
    Source:
    Bovine Pancreas
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical vbw area
    <t>TAGLN</t> protein expression in the ventral midline during the closure process. Transverse sections are shown of thoracic (A-C) and abdominal (D-G) <t>VBW</t> from Tagln -Cre:Rosa26-tdTom embryos stained with TAGLN antibody. (A) E12.5, showing the left primary body wall. Complete overlap between Tagln -Cre (tdTom) and TAGLN signals is seen. (B) At E14.5 there is still near complete overlap between the tdTom and TAGLN signals. A magnified view of the closing midline (boxed area in B) is shown to the right. (C) At E16.5 the thoracic midline has completely closed. The tdTom signal is still seen as a narrow line in the midline, but TAGLN signal cannot be identified. The magnified view of the midline (boxed area in C) shows the fine line of tdTom + cells that have now become negative for TAGLN. (D) The abdominal ventral midline at E12.5, Tagln -Cre and TAGLN signals show complete overlap. (E) At E13.5 the Tagln -Cre-derived cells (tdTom + ) of the primary body wall still express TAGLN. (F) At E14.5 the TAGLN signal area in the primary ventral midline is restricted compared with the tdTom signal area of the Tagln -Cre cells. (G) The ventral midline, labelled by tdTom, at E15.5 (same level as in F) has largely downregulated TAGLN. The magnified view of the midline (boxed area in G) shows tdTom + cells of the midline that have now become negative for TAGLN. H, heart; LV, liver; IN, intestine. Scale bars: 200 µm, except 25 µm in higher magnification images in C,G.
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/vbw area/product/Worthington Biochemical
    Average 91 stars, based on 593 article reviews
    Price from $9.99 to $1999.99
    vbw area - by Bioz Stars, 2020-10
    91/100 stars

    Images

    1) Product Images from "Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling"

    Article Title: Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.152843

    TAGLN protein expression in the ventral midline during the closure process. Transverse sections are shown of thoracic (A-C) and abdominal (D-G) VBW from Tagln -Cre:Rosa26-tdTom embryos stained with TAGLN antibody. (A) E12.5, showing the left primary body wall. Complete overlap between Tagln -Cre (tdTom) and TAGLN signals is seen. (B) At E14.5 there is still near complete overlap between the tdTom and TAGLN signals. A magnified view of the closing midline (boxed area in B) is shown to the right. (C) At E16.5 the thoracic midline has completely closed. The tdTom signal is still seen as a narrow line in the midline, but TAGLN signal cannot be identified. The magnified view of the midline (boxed area in C) shows the fine line of tdTom + cells that have now become negative for TAGLN. (D) The abdominal ventral midline at E12.5, Tagln -Cre and TAGLN signals show complete overlap. (E) At E13.5 the Tagln -Cre-derived cells (tdTom + ) of the primary body wall still express TAGLN. (F) At E14.5 the TAGLN signal area in the primary ventral midline is restricted compared with the tdTom signal area of the Tagln -Cre cells. (G) The ventral midline, labelled by tdTom, at E15.5 (same level as in F) has largely downregulated TAGLN. The magnified view of the midline (boxed area in G) shows tdTom + cells of the midline that have now become negative for TAGLN. H, heart; LV, liver; IN, intestine. Scale bars: 200 µm, except 25 µm in higher magnification images in C,G.
    Figure Legend Snippet: TAGLN protein expression in the ventral midline during the closure process. Transverse sections are shown of thoracic (A-C) and abdominal (D-G) VBW from Tagln -Cre:Rosa26-tdTom embryos stained with TAGLN antibody. (A) E12.5, showing the left primary body wall. Complete overlap between Tagln -Cre (tdTom) and TAGLN signals is seen. (B) At E14.5 there is still near complete overlap between the tdTom and TAGLN signals. A magnified view of the closing midline (boxed area in B) is shown to the right. (C) At E16.5 the thoracic midline has completely closed. The tdTom signal is still seen as a narrow line in the midline, but TAGLN signal cannot be identified. The magnified view of the midline (boxed area in C) shows the fine line of tdTom + cells that have now become negative for TAGLN. (D) The abdominal ventral midline at E12.5, Tagln -Cre and TAGLN signals show complete overlap. (E) At E13.5 the Tagln -Cre-derived cells (tdTom + ) of the primary body wall still express TAGLN. (F) At E14.5 the TAGLN signal area in the primary ventral midline is restricted compared with the tdTom signal area of the Tagln -Cre cells. (G) The ventral midline, labelled by tdTom, at E15.5 (same level as in F) has largely downregulated TAGLN. The magnified view of the midline (boxed area in G) shows tdTom + cells of the midline that have now become negative for TAGLN. H, heart; LV, liver; IN, intestine. Scale bars: 200 µm, except 25 µm in higher magnification images in C,G.

    Techniques Used: Expressing, Staining, Derivative Assay

    Directional migration of TAGLN + cells towards the ventral midline. Still images from 9 h time-lapse (time shown bottom right) of ex vivo body wall explant culture. The VBW is located at the righthand side of each panel and dorsally located tdTom + cells (white arrows) are in the left top corner. (A) Time zero, showing location of tdTom + cells. (B) Time zero, with added tracks and migration paths. Each tdTom + cell centre is labelled with a grey square and the path and time course of the journey are marked with a colour-coded line. (C) At 3 h VBW cells show directional migration towards the ventral midline, whereas dorsal cells show little change in position. (D,E) At 6 and 9 h, respectively, midline directional migration continues in VBW cells. (F) Trajectories and journey length in the analysed cells. Grey arrows indicate the direction and length of each migration path. VBW cells show consistent directional migration towards the midline, whereas dorsal cells show little change in position.
    Figure Legend Snippet: Directional migration of TAGLN + cells towards the ventral midline. Still images from 9 h time-lapse (time shown bottom right) of ex vivo body wall explant culture. The VBW is located at the righthand side of each panel and dorsally located tdTom + cells (white arrows) are in the left top corner. (A) Time zero, showing location of tdTom + cells. (B) Time zero, with added tracks and migration paths. Each tdTom + cell centre is labelled with a grey square and the path and time course of the journey are marked with a colour-coded line. (C) At 3 h VBW cells show directional migration towards the ventral midline, whereas dorsal cells show little change in position. (D,E) At 6 and 9 h, respectively, midline directional migration continues in VBW cells. (F) Trajectories and journey length in the analysed cells. Grey arrows indicate the direction and length of each migration path. VBW cells show consistent directional migration towards the midline, whereas dorsal cells show little change in position.

    Techniques Used: Migration, Ex Vivo

    Characterisation of ventral midline cells in Tagln -Cre:Rosa26-tdTom during VBW closure. Expression of smooth muscle contractile proteins (A-D,H) in the primary wall is more evident at early stages of midline closure. (A) αSMA and vimentin are expressed in the thoracic primary body wall at E12.5 and correlate with tdTom signal. Insets are magnified views (at cellular level) of the boxed areas. (B) At E14.5 primary body wall cells labelled by tdTom are still strongly positive for vimentin and express the smooth muscle intermediate filament protein desmin. (C) E15.5 midline cells are immunopositive for the fibroblast marker ER-TR7. Inset shows the ventral midline area (boxed) at higher magnification. (D) When the thoracic midline is fully closed at E16.5 the residual primary midline cells still labelled by tdTom have now downregulated αSMA. As shown in the higher magnification inset, only a small number of cells (arrow) of the midline show expression of αSMA. (E) Numbered lines indicate the level of transverse sections shown in (1) A-D,F,G and (2) H-J. (F,G) Tendon markers are absent in the primary body wall. (F) Tendon marker tenascin-C is expressed at E13.5 around the rib primordium and just lateral to primary elements (bottom box), and sporadic low-level expression is seen in the primary body wall (top box). (G) At E14.5 no tenascin-C expression is seen in the primary body wall in the midline. Sternal primordium cells express tenascin-C and are seen encircling the primary body wall cells. (H-J) Abdominal primary body wall is made of myofibroblasts. (H) In the abdominal midline at E14.5, primary body wall cells express vimentin and desmin. (I) At E15.5 the cells of the abdominal midline are immunopositive for the fibroblast marker ER-TR7. (J) At E16.5 the ventral midline has fully closed and resident tdTom + cells are seen in the midline. Tenascin-C expression can be detected in the edges of the falciform ligament, but not at the midline. Scale bars: 100 µm.
    Figure Legend Snippet: Characterisation of ventral midline cells in Tagln -Cre:Rosa26-tdTom during VBW closure. Expression of smooth muscle contractile proteins (A-D,H) in the primary wall is more evident at early stages of midline closure. (A) αSMA and vimentin are expressed in the thoracic primary body wall at E12.5 and correlate with tdTom signal. Insets are magnified views (at cellular level) of the boxed areas. (B) At E14.5 primary body wall cells labelled by tdTom are still strongly positive for vimentin and express the smooth muscle intermediate filament protein desmin. (C) E15.5 midline cells are immunopositive for the fibroblast marker ER-TR7. Inset shows the ventral midline area (boxed) at higher magnification. (D) When the thoracic midline is fully closed at E16.5 the residual primary midline cells still labelled by tdTom have now downregulated αSMA. As shown in the higher magnification inset, only a small number of cells (arrow) of the midline show expression of αSMA. (E) Numbered lines indicate the level of transverse sections shown in (1) A-D,F,G and (2) H-J. (F,G) Tendon markers are absent in the primary body wall. (F) Tendon marker tenascin-C is expressed at E13.5 around the rib primordium and just lateral to primary elements (bottom box), and sporadic low-level expression is seen in the primary body wall (top box). (G) At E14.5 no tenascin-C expression is seen in the primary body wall in the midline. Sternal primordium cells express tenascin-C and are seen encircling the primary body wall cells. (H-J) Abdominal primary body wall is made of myofibroblasts. (H) In the abdominal midline at E14.5, primary body wall cells express vimentin and desmin. (I) At E15.5 the cells of the abdominal midline are immunopositive for the fibroblast marker ER-TR7. (J) At E16.5 the ventral midline has fully closed and resident tdTom + cells are seen in the midline. Tenascin-C expression can be detected in the edges of the falciform ligament, but not at the midline. Scale bars: 100 µm.

    Techniques Used: Expressing, Marker

    Tagln- Cre: Tgfbr2 flx/flx embryos develop VBW closure defects. (A,B) Morphological comparison between Tagln- Cre: Tgfbr2 flx/flx and Tagln- Cre: Tgfbr2 flx/wt mouse embryos. (A) E13.5 Tagln- Cre: Tgfbr2 flx/flx embryos show a translucent ventral midline, a more lateral limit to the secondary body wall (arrow) and absence of midline raphe (arrowhead) when compared with Tagln -Cre: Tgfbr2 flx/wt . (B) The ventral midline closure defect in Tagln- Cre: Tgfbr2 flx/flx . A thin membrane covers the VBW cavities, as compared with the nearly closed thoracic midline in the WT (arrow) and the embryos show a large exomphalos compared with the physiological umbilical hernia in the WT (arrowhead). (C) Transverse section in mid-thorax at E14.5 in WT (left) and Tagln -Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. The VBW is composed of a thin sac in the mutant, whereas the two lateral sternebrae are nearly meeting in the midline in the WT. (D) Transverse section at level of the umbilical hernia at E14.5 in WT (left) and Tagln- Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. In the WT only a small physiological umbilical hernia is present and the small intestine is returning to the abdominal cavity, whereas the mutant shows a large exomphalos defect and very few bowel loops are present in the abdominal cavity. (E-H) Characterisation of cell type in Tagln- Cre: Tgfbr2 flx/flx thoracic (right) and abdominal (left) body wall by immunohistochemistry. (E) E13.5 mutant embryos show normal lateral body wall muscles (MF20 + ) and ribs (SOX9 + ), whereas the ventral midline is made of a thin sac. Condensations of SOX9 + and MF20 + cells (arrow) are seen just lateral to the VBW in the thoracic and abdominal areas, respectively. (F) E14.5 mutant embryo shows very little progression in secondary element migration, and the condensation of chondrocyte and myocyte (arrow) is still seen lateral to the VBW in the thoracic and abdominal compartments, respectively. (G) The VBW of Tagln- Cre: Tgfbr2 flx/flx still expresses TAGLN. (H) The skin covering the premature VBW in Tagln- Cre: Tgfbr2 flx/flx is made of a single layer of squamous epithelial cells (insets P), while in the secondary elements multilayered cuboid epithelium covers the lateral body wall (insets S). Bottom row of insets shows E-cadherin channel. H, heart; L, lungs; LV, liver; IN, intestine; P, primary body wall; S, secondary body wall; TA, transverses abdominis; IO, internal oblique; EO, external oblique; PC, panniculus carnosus; IC, intercostal muscles; R, rib. Scale bars: 1000 µm in A,B; 500 µm in C-G; 200 µm in H, 50 µm in insets.
    Figure Legend Snippet: Tagln- Cre: Tgfbr2 flx/flx embryos develop VBW closure defects. (A,B) Morphological comparison between Tagln- Cre: Tgfbr2 flx/flx and Tagln- Cre: Tgfbr2 flx/wt mouse embryos. (A) E13.5 Tagln- Cre: Tgfbr2 flx/flx embryos show a translucent ventral midline, a more lateral limit to the secondary body wall (arrow) and absence of midline raphe (arrowhead) when compared with Tagln -Cre: Tgfbr2 flx/wt . (B) The ventral midline closure defect in Tagln- Cre: Tgfbr2 flx/flx . A thin membrane covers the VBW cavities, as compared with the nearly closed thoracic midline in the WT (arrow) and the embryos show a large exomphalos compared with the physiological umbilical hernia in the WT (arrowhead). (C) Transverse section in mid-thorax at E14.5 in WT (left) and Tagln -Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. The VBW is composed of a thin sac in the mutant, whereas the two lateral sternebrae are nearly meeting in the midline in the WT. (D) Transverse section at level of the umbilical hernia at E14.5 in WT (left) and Tagln- Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. In the WT only a small physiological umbilical hernia is present and the small intestine is returning to the abdominal cavity, whereas the mutant shows a large exomphalos defect and very few bowel loops are present in the abdominal cavity. (E-H) Characterisation of cell type in Tagln- Cre: Tgfbr2 flx/flx thoracic (right) and abdominal (left) body wall by immunohistochemistry. (E) E13.5 mutant embryos show normal lateral body wall muscles (MF20 + ) and ribs (SOX9 + ), whereas the ventral midline is made of a thin sac. Condensations of SOX9 + and MF20 + cells (arrow) are seen just lateral to the VBW in the thoracic and abdominal areas, respectively. (F) E14.5 mutant embryo shows very little progression in secondary element migration, and the condensation of chondrocyte and myocyte (arrow) is still seen lateral to the VBW in the thoracic and abdominal compartments, respectively. (G) The VBW of Tagln- Cre: Tgfbr2 flx/flx still expresses TAGLN. (H) The skin covering the premature VBW in Tagln- Cre: Tgfbr2 flx/flx is made of a single layer of squamous epithelial cells (insets P), while in the secondary elements multilayered cuboid epithelium covers the lateral body wall (insets S). Bottom row of insets shows E-cadherin channel. H, heart; L, lungs; LV, liver; IN, intestine; P, primary body wall; S, secondary body wall; TA, transverses abdominis; IO, internal oblique; EO, external oblique; PC, panniculus carnosus; IC, intercostal muscles; R, rib. Scale bars: 1000 µm in A,B; 500 µm in C-G; 200 µm in H, 50 µm in insets.

    Techniques Used: Staining, Mutagenesis, Immunohistochemistry, Migration

    Tagln -Cre expression in the ventral midline and mitotic activity of TAGLN + cells. (A) Transverse section at a thoracic level in an E12.5 wild-type (WT) mouse embryo stained for TAGLN, showing expression in the primary VBW (area between arrows). (B) Transverse section at an abdominal level in an E13.5 WT embryo stained for TAGLN showing expression in the primary abdominal wall (area between arrows) that is encircling the umbilical hernia. (C) Whole-mount β-galactosidase staining in Tagln -Cre:Rosa26-NGZ at three embryonic stages. The expression of TAGLN is evident in the somite at E11.5 and localises to the midline area when VBW closure is complete. Dotted lines delineate forelimb and hindlimb. (D) (Left) Numbered lines indicate the level of transverse sections shown in (1) A,E,H, (2) B,F and (3) G. (Right) Schematic of midline (red) and para-midline (grey) areas presented in the KI67 analysis in H,I. (E,F) Expression of Tagln -Cre:Rosa26-tdTom in the thoracic (E) and abdominal (F) ventral midline over a 4 day time window during the closure process and at postnatal day (P) 20. TAGLN expression becomes restricted to the midline area with advanced gestation and this expression is maintained postnatally. Inset in E15.5 shows high magnification of the midline. Arrowheads indicate internal mammary/superior epigastric vessels and asterisk indicates the xiphisternum. (G) TUNEL assay for apoptosis in the ventral midline at E15.5. There is no obvious pattern of apoptosis in TAGLN + -derived cells in the midline. Boxes show examples of individual TUNEL + cells in the midline and para-midline areas. (H) KI67 staining of the ventral midline at E14.5. Primary body wall remnant at this stage shows limited mitotic activity, which is evident in the KI67 channel. (I) Comparison of KI67 expression between midline (ML) primary VBW cells (tdTom + ) and para-midline (PML) secondary body wall cells (tdTom − ) in the thoracic and abdominal regions. Comparison was made on 200 cells from three different sections at each level; data presented as mean±s.e.m. ** P
    Figure Legend Snippet: Tagln -Cre expression in the ventral midline and mitotic activity of TAGLN + cells. (A) Transverse section at a thoracic level in an E12.5 wild-type (WT) mouse embryo stained for TAGLN, showing expression in the primary VBW (area between arrows). (B) Transverse section at an abdominal level in an E13.5 WT embryo stained for TAGLN showing expression in the primary abdominal wall (area between arrows) that is encircling the umbilical hernia. (C) Whole-mount β-galactosidase staining in Tagln -Cre:Rosa26-NGZ at three embryonic stages. The expression of TAGLN is evident in the somite at E11.5 and localises to the midline area when VBW closure is complete. Dotted lines delineate forelimb and hindlimb. (D) (Left) Numbered lines indicate the level of transverse sections shown in (1) A,E,H, (2) B,F and (3) G. (Right) Schematic of midline (red) and para-midline (grey) areas presented in the KI67 analysis in H,I. (E,F) Expression of Tagln -Cre:Rosa26-tdTom in the thoracic (E) and abdominal (F) ventral midline over a 4 day time window during the closure process and at postnatal day (P) 20. TAGLN expression becomes restricted to the midline area with advanced gestation and this expression is maintained postnatally. Inset in E15.5 shows high magnification of the midline. Arrowheads indicate internal mammary/superior epigastric vessels and asterisk indicates the xiphisternum. (G) TUNEL assay for apoptosis in the ventral midline at E15.5. There is no obvious pattern of apoptosis in TAGLN + -derived cells in the midline. Boxes show examples of individual TUNEL + cells in the midline and para-midline areas. (H) KI67 staining of the ventral midline at E14.5. Primary body wall remnant at this stage shows limited mitotic activity, which is evident in the KI67 channel. (I) Comparison of KI67 expression between midline (ML) primary VBW cells (tdTom + ) and para-midline (PML) secondary body wall cells (tdTom − ) in the thoracic and abdominal regions. Comparison was made on 200 cells from three different sections at each level; data presented as mean±s.e.m. ** P

    Techniques Used: Expressing, Activity Assay, Staining, TUNEL Assay, Derivative Assay

    TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P
    Figure Legend Snippet: TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P

    Techniques Used: Expressing, Staining, Dissection, Quantitative RT-PCR, FACS, Derivative Assay

    2) Product Images from "Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling"

    Article Title: Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.152843

    TAGLN protein expression in the ventral midline during the closure process. Transverse sections are shown of thoracic (A-C) and abdominal (D-G) VBW from Tagln -Cre:Rosa26-tdTom embryos stained with TAGLN antibody. (A) E12.5, showing the left primary body wall. Complete overlap between Tagln -Cre (tdTom) and TAGLN signals is seen. (B) At E14.5 there is still near complete overlap between the tdTom and TAGLN signals. A magnified view of the closing midline (boxed area in B) is shown to the right. (C) At E16.5 the thoracic midline has completely closed. The tdTom signal is still seen as a narrow line in the midline, but TAGLN signal cannot be identified. The magnified view of the midline (boxed area in C) shows the fine line of tdTom + cells that have now become negative for TAGLN. (D) The abdominal ventral midline at E12.5, Tagln -Cre and TAGLN signals show complete overlap. (E) At E13.5 the Tagln -Cre-derived cells (tdTom + ) of the primary body wall still express TAGLN. (F) At E14.5 the TAGLN signal area in the primary ventral midline is restricted compared with the tdTom signal area of the Tagln -Cre cells. (G) The ventral midline, labelled by tdTom, at E15.5 (same level as in F) has largely downregulated TAGLN. The magnified view of the midline (boxed area in G) shows tdTom + cells of the midline that have now become negative for TAGLN. H, heart; LV, liver; IN, intestine. Scale bars: 200 µm, except 25 µm in higher magnification images in C,G.
    Figure Legend Snippet: TAGLN protein expression in the ventral midline during the closure process. Transverse sections are shown of thoracic (A-C) and abdominal (D-G) VBW from Tagln -Cre:Rosa26-tdTom embryos stained with TAGLN antibody. (A) E12.5, showing the left primary body wall. Complete overlap between Tagln -Cre (tdTom) and TAGLN signals is seen. (B) At E14.5 there is still near complete overlap between the tdTom and TAGLN signals. A magnified view of the closing midline (boxed area in B) is shown to the right. (C) At E16.5 the thoracic midline has completely closed. The tdTom signal is still seen as a narrow line in the midline, but TAGLN signal cannot be identified. The magnified view of the midline (boxed area in C) shows the fine line of tdTom + cells that have now become negative for TAGLN. (D) The abdominal ventral midline at E12.5, Tagln -Cre and TAGLN signals show complete overlap. (E) At E13.5 the Tagln -Cre-derived cells (tdTom + ) of the primary body wall still express TAGLN. (F) At E14.5 the TAGLN signal area in the primary ventral midline is restricted compared with the tdTom signal area of the Tagln -Cre cells. (G) The ventral midline, labelled by tdTom, at E15.5 (same level as in F) has largely downregulated TAGLN. The magnified view of the midline (boxed area in G) shows tdTom + cells of the midline that have now become negative for TAGLN. H, heart; LV, liver; IN, intestine. Scale bars: 200 µm, except 25 µm in higher magnification images in C,G.

    Techniques Used: Expressing, Staining, Derivative Assay

    Directional migration of TAGLN + cells towards the ventral midline. Still images from 9 h time-lapse (time shown bottom right) of ex vivo body wall explant culture. The VBW is located at the righthand side of each panel and dorsally located tdTom + cells (white arrows) are in the left top corner. (A) Time zero, showing location of tdTom + cells. (B) Time zero, with added tracks and migration paths. Each tdTom + cell centre is labelled with a grey square and the path and time course of the journey are marked with a colour-coded line. (C) At 3 h VBW cells show directional migration towards the ventral midline, whereas dorsal cells show little change in position. (D,E) At 6 and 9 h, respectively, midline directional migration continues in VBW cells. (F) Trajectories and journey length in the analysed cells. Grey arrows indicate the direction and length of each migration path. VBW cells show consistent directional migration towards the midline, whereas dorsal cells show little change in position.
    Figure Legend Snippet: Directional migration of TAGLN + cells towards the ventral midline. Still images from 9 h time-lapse (time shown bottom right) of ex vivo body wall explant culture. The VBW is located at the righthand side of each panel and dorsally located tdTom + cells (white arrows) are in the left top corner. (A) Time zero, showing location of tdTom + cells. (B) Time zero, with added tracks and migration paths. Each tdTom + cell centre is labelled with a grey square and the path and time course of the journey are marked with a colour-coded line. (C) At 3 h VBW cells show directional migration towards the ventral midline, whereas dorsal cells show little change in position. (D,E) At 6 and 9 h, respectively, midline directional migration continues in VBW cells. (F) Trajectories and journey length in the analysed cells. Grey arrows indicate the direction and length of each migration path. VBW cells show consistent directional migration towards the midline, whereas dorsal cells show little change in position.

    Techniques Used: Migration, Ex Vivo

    Characterisation of ventral midline cells in Tagln -Cre:Rosa26-tdTom during VBW closure. Expression of smooth muscle contractile proteins (A-D,H) in the primary wall is more evident at early stages of midline closure. (A) αSMA and vimentin are expressed in the thoracic primary body wall at E12.5 and correlate with tdTom signal. Insets are magnified views (at cellular level) of the boxed areas. (B) At E14.5 primary body wall cells labelled by tdTom are still strongly positive for vimentin and express the smooth muscle intermediate filament protein desmin. (C) E15.5 midline cells are immunopositive for the fibroblast marker ER-TR7. Inset shows the ventral midline area (boxed) at higher magnification. (D) When the thoracic midline is fully closed at E16.5 the residual primary midline cells still labelled by tdTom have now downregulated αSMA. As shown in the higher magnification inset, only a small number of cells (arrow) of the midline show expression of αSMA. (E) Numbered lines indicate the level of transverse sections shown in (1) A-D,F,G and (2) H-J. (F,G) Tendon markers are absent in the primary body wall. (F) Tendon marker tenascin-C is expressed at E13.5 around the rib primordium and just lateral to primary elements (bottom box), and sporadic low-level expression is seen in the primary body wall (top box). (G) At E14.5 no tenascin-C expression is seen in the primary body wall in the midline. Sternal primordium cells express tenascin-C and are seen encircling the primary body wall cells. (H-J) Abdominal primary body wall is made of myofibroblasts. (H) In the abdominal midline at E14.5, primary body wall cells express vimentin and desmin. (I) At E15.5 the cells of the abdominal midline are immunopositive for the fibroblast marker ER-TR7. (J) At E16.5 the ventral midline has fully closed and resident tdTom + cells are seen in the midline. Tenascin-C expression can be detected in the edges of the falciform ligament, but not at the midline. Scale bars: 100 µm.
    Figure Legend Snippet: Characterisation of ventral midline cells in Tagln -Cre:Rosa26-tdTom during VBW closure. Expression of smooth muscle contractile proteins (A-D,H) in the primary wall is more evident at early stages of midline closure. (A) αSMA and vimentin are expressed in the thoracic primary body wall at E12.5 and correlate with tdTom signal. Insets are magnified views (at cellular level) of the boxed areas. (B) At E14.5 primary body wall cells labelled by tdTom are still strongly positive for vimentin and express the smooth muscle intermediate filament protein desmin. (C) E15.5 midline cells are immunopositive for the fibroblast marker ER-TR7. Inset shows the ventral midline area (boxed) at higher magnification. (D) When the thoracic midline is fully closed at E16.5 the residual primary midline cells still labelled by tdTom have now downregulated αSMA. As shown in the higher magnification inset, only a small number of cells (arrow) of the midline show expression of αSMA. (E) Numbered lines indicate the level of transverse sections shown in (1) A-D,F,G and (2) H-J. (F,G) Tendon markers are absent in the primary body wall. (F) Tendon marker tenascin-C is expressed at E13.5 around the rib primordium and just lateral to primary elements (bottom box), and sporadic low-level expression is seen in the primary body wall (top box). (G) At E14.5 no tenascin-C expression is seen in the primary body wall in the midline. Sternal primordium cells express tenascin-C and are seen encircling the primary body wall cells. (H-J) Abdominal primary body wall is made of myofibroblasts. (H) In the abdominal midline at E14.5, primary body wall cells express vimentin and desmin. (I) At E15.5 the cells of the abdominal midline are immunopositive for the fibroblast marker ER-TR7. (J) At E16.5 the ventral midline has fully closed and resident tdTom + cells are seen in the midline. Tenascin-C expression can be detected in the edges of the falciform ligament, but not at the midline. Scale bars: 100 µm.

    Techniques Used: Expressing, Marker

    Tagln -Cre expression in the ventral midline and mitotic activity of TAGLN + cells. (A) Transverse section at a thoracic level in an E12.5 wild-type (WT) mouse embryo stained for TAGLN, showing expression in the primary VBW (area between arrows). (B) Transverse section at an abdominal level in an E13.5 WT embryo stained for TAGLN showing expression in the primary abdominal wall (area between arrows) that is encircling the umbilical hernia. (C) Whole-mount β-galactosidase staining in Tagln -Cre:Rosa26-NGZ at three embryonic stages. The expression of TAGLN is evident in the somite at E11.5 and localises to the midline area when VBW closure is complete. Dotted lines delineate forelimb and hindlimb. (D) (Left) Numbered lines indicate the level of transverse sections shown in (1) A,E,H, (2) B,F and (3) G. (Right) Schematic of midline (red) and para-midline (grey) areas presented in the KI67 analysis in H,I. (E,F) Expression of Tagln -Cre:Rosa26-tdTom in the thoracic (E) and abdominal (F) ventral midline over a 4 day time window during the closure process and at postnatal day (P) 20. TAGLN expression becomes restricted to the midline area with advanced gestation and this expression is maintained postnatally. Inset in E15.5 shows high magnification of the midline. Arrowheads indicate internal mammary/superior epigastric vessels and asterisk indicates the xiphisternum. (G) TUNEL assay for apoptosis in the ventral midline at E15.5. There is no obvious pattern of apoptosis in TAGLN + -derived cells in the midline. Boxes show examples of individual TUNEL + cells in the midline and para-midline areas. (H) KI67 staining of the ventral midline at E14.5. Primary body wall remnant at this stage shows limited mitotic activity, which is evident in the KI67 channel. (I) Comparison of KI67 expression between midline (ML) primary VBW cells (tdTom + ) and para-midline (PML) secondary body wall cells (tdTom − ) in the thoracic and abdominal regions. Comparison was made on 200 cells from three different sections at each level; data presented as mean±s.e.m. ** P
    Figure Legend Snippet: Tagln -Cre expression in the ventral midline and mitotic activity of TAGLN + cells. (A) Transverse section at a thoracic level in an E12.5 wild-type (WT) mouse embryo stained for TAGLN, showing expression in the primary VBW (area between arrows). (B) Transverse section at an abdominal level in an E13.5 WT embryo stained for TAGLN showing expression in the primary abdominal wall (area between arrows) that is encircling the umbilical hernia. (C) Whole-mount β-galactosidase staining in Tagln -Cre:Rosa26-NGZ at three embryonic stages. The expression of TAGLN is evident in the somite at E11.5 and localises to the midline area when VBW closure is complete. Dotted lines delineate forelimb and hindlimb. (D) (Left) Numbered lines indicate the level of transverse sections shown in (1) A,E,H, (2) B,F and (3) G. (Right) Schematic of midline (red) and para-midline (grey) areas presented in the KI67 analysis in H,I. (E,F) Expression of Tagln -Cre:Rosa26-tdTom in the thoracic (E) and abdominal (F) ventral midline over a 4 day time window during the closure process and at postnatal day (P) 20. TAGLN expression becomes restricted to the midline area with advanced gestation and this expression is maintained postnatally. Inset in E15.5 shows high magnification of the midline. Arrowheads indicate internal mammary/superior epigastric vessels and asterisk indicates the xiphisternum. (G) TUNEL assay for apoptosis in the ventral midline at E15.5. There is no obvious pattern of apoptosis in TAGLN + -derived cells in the midline. Boxes show examples of individual TUNEL + cells in the midline and para-midline areas. (H) KI67 staining of the ventral midline at E14.5. Primary body wall remnant at this stage shows limited mitotic activity, which is evident in the KI67 channel. (I) Comparison of KI67 expression between midline (ML) primary VBW cells (tdTom + ) and para-midline (PML) secondary body wall cells (tdTom − ) in the thoracic and abdominal regions. Comparison was made on 200 cells from three different sections at each level; data presented as mean±s.e.m. ** P

    Techniques Used: Expressing, Activity Assay, Staining, TUNEL Assay, Derivative Assay

    TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P
    Figure Legend Snippet: TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P

    Techniques Used: Expressing, Staining, Dissection, Quantitative RT-PCR, FACS, Derivative Assay

    3) Product Images from "FSP1-positive fibroblasts are adipogenic niche and regulate adipose homeostasis"

    Article Title: FSP1-positive fibroblasts are adipogenic niche and regulate adipose homeostasis

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001493

    Ablation of FSP1 + fibroblasts disturbs adipose homeostasis. (A) FACS analyses of tdTomato + cells in the SVF from Fsp1 - Cre ; tdTomato and Fsp1 - Cre ; Rosa26 - DTA ; tdTomato compound mice. (B) Body weight of male F-DTA compound mice and their littermates. n = 5 for male Ctrl mice; n = 5 for male F-DTA mice. (C) Percent body fat (NMR spectroscopy) of Ctrl and F-DTA littermates at 4 months of age. n = 9 for male Ctrl mice, and n = 8 for male F-DTA mice. (D, E) Representative pictures (panel D) and weight (panel E) of the adipose tissues of F-DTA mice and their littermates at 4 months of age. n = 12 for male male Ctrl mice, and n = 14 for male F-DTA mice. (F) GSEA data showing negative enrichment of adipogenesis signature in F-DTA SVF cells compared with the control SVF cells. (G, H) Control or F-DTA SVF cells were subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel G). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel H). (I) FACS analyses of CD34 + Sca1 + cells in the SVF of 4-month-old Ctrl and F-DTA mice ( n = 3). (J) RT-PCR analyses of PDGF expression in control and F-DTA SVF cells ( n = 3). (K) SVF cells isolated from F-DTA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days before FACS analyses of CD34 + Sca1 + populations ( n = 3). (L, M) SVF cells isolated from F-DTA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days and subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel L). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel M). (N) GSEA of YAP signature in F-DTA SVF cells. (O) RT-PCR analyses of YAP and YAP target gene expression in F-DTA and control SVF cells ( n = 3). (P, Q) F-DTA SVF cells were pretreated with or without YAP inhibitor VP before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel P). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (Q). Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C, E, H, I, J, and O) or paired (panel K, M, and Q) student t test or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B). * p
    Figure Legend Snippet: Ablation of FSP1 + fibroblasts disturbs adipose homeostasis. (A) FACS analyses of tdTomato + cells in the SVF from Fsp1 - Cre ; tdTomato and Fsp1 - Cre ; Rosa26 - DTA ; tdTomato compound mice. (B) Body weight of male F-DTA compound mice and their littermates. n = 5 for male Ctrl mice; n = 5 for male F-DTA mice. (C) Percent body fat (NMR spectroscopy) of Ctrl and F-DTA littermates at 4 months of age. n = 9 for male Ctrl mice, and n = 8 for male F-DTA mice. (D, E) Representative pictures (panel D) and weight (panel E) of the adipose tissues of F-DTA mice and their littermates at 4 months of age. n = 12 for male male Ctrl mice, and n = 14 for male F-DTA mice. (F) GSEA data showing negative enrichment of adipogenesis signature in F-DTA SVF cells compared with the control SVF cells. (G, H) Control or F-DTA SVF cells were subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel G). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel H). (I) FACS analyses of CD34 + Sca1 + cells in the SVF of 4-month-old Ctrl and F-DTA mice ( n = 3). (J) RT-PCR analyses of PDGF expression in control and F-DTA SVF cells ( n = 3). (K) SVF cells isolated from F-DTA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days before FACS analyses of CD34 + Sca1 + populations ( n = 3). (L, M) SVF cells isolated from F-DTA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days and subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel L). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel M). (N) GSEA of YAP signature in F-DTA SVF cells. (O) RT-PCR analyses of YAP and YAP target gene expression in F-DTA and control SVF cells ( n = 3). (P, Q) F-DTA SVF cells were pretreated with or without YAP inhibitor VP before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel P). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (Q). Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C, E, H, I, J, and O) or paired (panel K, M, and Q) student t test or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B). * p

    Techniques Used: FACS, Mouse Assay, Nuclear Magnetic Resonance, Spectroscopy, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Two Tailed Test

    PDGF-BB is responsible for the maintenance of preadipocyte number. (A, B) Ctrl or F-BCA SVF cells were pretreated with CM harvested from Ctrl or F-BCA SVF cell culture before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel A). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (B). (C) RT-PCR analyses of PDGF expression in Ctrl and F-BCA SVF cells ( n = 3). (D) Immunostaining of phospho-PDGFR-β on I-WAT sections from Ctrl and F-BCA mice at 4 months of age. Scale bar: 200 μm. (E) SVF cells isolated from F-BCA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days before FACS analyses of CD34 + Sca1 + populations ( n = 4). (F, G) SVF cells isolated from F-BCA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days and subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel F). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 6) (G). (H) Western blot analyses of PPARγ and c/ebpα in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB and subjected to adipogenic induction. (I–K) Matrigel plugs containing vehicle or 10 ng PDGF-BB were implanted in contralateral I-WATs of F-BCA mice for 2 weeks. Isolated SVF cells were subjected to FACS analyses ( n = 4) (panel I) and adipogenic induction (panel J and K). Adipogenesis was assayed with Oil Red O staining (panel J). Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel K). Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C) or paired (panel E, G, I, and K) student t test or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B). * p
    Figure Legend Snippet: PDGF-BB is responsible for the maintenance of preadipocyte number. (A, B) Ctrl or F-BCA SVF cells were pretreated with CM harvested from Ctrl or F-BCA SVF cell culture before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel A). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (B). (C) RT-PCR analyses of PDGF expression in Ctrl and F-BCA SVF cells ( n = 3). (D) Immunostaining of phospho-PDGFR-β on I-WAT sections from Ctrl and F-BCA mice at 4 months of age. Scale bar: 200 μm. (E) SVF cells isolated from F-BCA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days before FACS analyses of CD34 + Sca1 + populations ( n = 4). (F, G) SVF cells isolated from F-BCA I-WAT were treated with 10 ng/mL PDGF-BB for 4 days and subjected to adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel F). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 6) (G). (H) Western blot analyses of PPARγ and c/ebpα in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB and subjected to adipogenic induction. (I–K) Matrigel plugs containing vehicle or 10 ng PDGF-BB were implanted in contralateral I-WATs of F-BCA mice for 2 weeks. Isolated SVF cells were subjected to FACS analyses ( n = 4) (panel I) and adipogenic induction (panel J and K). Adipogenesis was assayed with Oil Red O staining (panel J). Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel K). Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C) or paired (panel E, G, I, and K) student t test or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B). * p

    Techniques Used: BIA-KA, Cell Culture, Staining, Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Mouse Assay, Isolation, FACS, Western Blot, Two Tailed Test

    FSP1 + fibroblasts localize adjacent to preadipocytes without adipogenic potential. (A) FACS analyses of tdTomato + cells in SVF cells isolated from I-WAT and E-WAT of 4-month-old Rosa26-tdTomato (mT) and Fsp1 - Cre ; tdTomato (F-mT) mice. (B, C) SVF cells isolated from Rosa26-tdTomato (mT) and Fsp1 - Cre ; tdTomato (F-mT) I-WAT were adipogenically induced. Cells were stained with Bodipy 493/503 (panel C). Scale bar: 200 μm. (D) FACS analyses of CD34 + Sca1 + cells in I-WAT and E-WAT SVF of 4-month-old Fsp1 - Cre ; tdTomato mice. (E) Immunofluorescent staining of GFP and PPARγ on I-WAT sections of 4-month-old mTmG and Fsp1 - Cre ; mTmG (F-mTmG) mice. Scale bar: 200 μm. CD34, cluster of differentiation 34; E-WAT, epididymal white adipose tissue; FACS, fluorescence-activated cell sorting; FSP1, fibroblast-specific protein-1; GFP, green fluorescent protein; I-WAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator-activated receptor-γ; Sca1, stem cell antigen 1; SVF, stromal vascular fraction.
    Figure Legend Snippet: FSP1 + fibroblasts localize adjacent to preadipocytes without adipogenic potential. (A) FACS analyses of tdTomato + cells in SVF cells isolated from I-WAT and E-WAT of 4-month-old Rosa26-tdTomato (mT) and Fsp1 - Cre ; tdTomato (F-mT) mice. (B, C) SVF cells isolated from Rosa26-tdTomato (mT) and Fsp1 - Cre ; tdTomato (F-mT) I-WAT were adipogenically induced. Cells were stained with Bodipy 493/503 (panel C). Scale bar: 200 μm. (D) FACS analyses of CD34 + Sca1 + cells in I-WAT and E-WAT SVF of 4-month-old Fsp1 - Cre ; tdTomato mice. (E) Immunofluorescent staining of GFP and PPARγ on I-WAT sections of 4-month-old mTmG and Fsp1 - Cre ; mTmG (F-mTmG) mice. Scale bar: 200 μm. CD34, cluster of differentiation 34; E-WAT, epididymal white adipose tissue; FACS, fluorescence-activated cell sorting; FSP1, fibroblast-specific protein-1; GFP, green fluorescent protein; I-WAT, inguinal white adipose tissue; PPARγ, peroxisome proliferator-activated receptor-γ; Sca1, stem cell antigen 1; SVF, stromal vascular fraction.

    Techniques Used: FACS, Isolation, Mouse Assay, Staining, Fluorescence

    Activation of canonical Wnt signaling in FSP1 + fibroblasts modulates adipogenesis through extracellular matrix remodeling and YAP signaling. (A) Sirius red staining of WAT of 4-month-old F-BCA mice and their littermates. Scale bar: 200 μm. (B) Western blot analyses of Col I and YAP in control and F-BCA SVF cells. (C–E) RT-PCR analyses of expression of extracellular matrix proteins (panel C), MMPs (panel D), and TIMPs (panel E) in F-BCA and control SVF cells ( n = 3). (F) Gelatin zymography of conditioned medium of F-BCA and control SVF cells. (G) Gelatin zymography of conditioned medium of F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB. (H) Western blot analyses of Col I and YAP in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB. (I) GSEA of YAP signature in F-BCA SVF cells. (J) RT-PCR analyses of YAP and YAP target gene expression in F-BCA and control SVF cells ( n = 3). (K) RT-PCR analyses of YAP and YAP target gene expression in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB ( n = 3). (L, M) F-BCA SVF cells were pretreated with or without YAP inhibitor VP before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel L). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel M). (N) Western blot analyses of PPARγ, Col I, and YAP in F-BCA SVF cells treated with or without VP and subjected to adipogenic induction. Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C, D, E, and J) or paired (panel K and M) student t test. * p
    Figure Legend Snippet: Activation of canonical Wnt signaling in FSP1 + fibroblasts modulates adipogenesis through extracellular matrix remodeling and YAP signaling. (A) Sirius red staining of WAT of 4-month-old F-BCA mice and their littermates. Scale bar: 200 μm. (B) Western blot analyses of Col I and YAP in control and F-BCA SVF cells. (C–E) RT-PCR analyses of expression of extracellular matrix proteins (panel C), MMPs (panel D), and TIMPs (panel E) in F-BCA and control SVF cells ( n = 3). (F) Gelatin zymography of conditioned medium of F-BCA and control SVF cells. (G) Gelatin zymography of conditioned medium of F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB. (H) Western blot analyses of Col I and YAP in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB. (I) GSEA of YAP signature in F-BCA SVF cells. (J) RT-PCR analyses of YAP and YAP target gene expression in F-BCA and control SVF cells ( n = 3). (K) RT-PCR analyses of YAP and YAP target gene expression in F-BCA SVF cells treated with or without 10 ng/mL PDGF-BB ( n = 3). (L, M) F-BCA SVF cells were pretreated with or without YAP inhibitor VP before adipogenic induction. Adipogenesis was assayed with Oil Red O staining (panel L). Scale bar: 100 μm. Oil Red O staining was quantitated by isopropanol extraction ( n = 3) (panel M). (N) Western blot analyses of PPARγ, Col I, and YAP in F-BCA SVF cells treated with or without VP and subjected to adipogenic induction. Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired (panel C, D, E, and J) or paired (panel K and M) student t test. * p

    Techniques Used: Activation Assay, Staining, BIA-KA, Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Zymography, Two Tailed Test

    Activation of canonical Wnt signaling in FSP1 + fibroblasts disturbs adipose homeostasis. (A) Western blot analyses of β-catenin expression in adipocytes (left panels) and SVF cells (right panels) isolated from WAT from F-BCA compound mice and their littermates with WT, Fsp1 - Cre , or Ctnnb1 exon 3 fl/+ genotypes (Ctrl). TTFs were used as a positive Ctrl for activated form β-catenin expression. (B) Body weight of male F-BCA mice and their littermates on ND. n = 9 for male Ctrl mice, and n = 10 for male F-BCA mice. (C) Ventral view of subcutaneous and visceral adipose depots of male Ctrl and F-BCA littermates on ND at 4 months of age. Adipose depots are circled with dashed lines. (D) Percent body fat (NMR spectroscopy) of Ctrl and F-BCA littermates on ND at 4 months of age. n = 6 for male Ctrl and F-BCA mice. (E, F) Representative pictures and weight of the adipose tissues of F-BCA mice and their littermates on ND at 4 months of age. n = 8 for male Ctrl mice, and n = 14 for male F-BCA mice. (G) HE staining of WAT sections of 4-month-old F-BCA mice and their littermates on ND. Scale bar: 200 μm. (H) GTT of F-BCA mice and their littermates on ND at 4 months of age. n = 13 for male Ctrl mice, and n = 5 for male F-BCA mice. (I) Quantification of the AUC of the GTT relative to Ctrl group. (J) ITT of F-BCA mice and their littermates on ND at 4 months of age. n = 10 for male Ctrl mice, and n = 5 for male F-BCA mice. (K) Quantification of the AUC of the ITT relative to Ctrl group. Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired student t test (panel D, F, I, and K) or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B, H, and J). * p
    Figure Legend Snippet: Activation of canonical Wnt signaling in FSP1 + fibroblasts disturbs adipose homeostasis. (A) Western blot analyses of β-catenin expression in adipocytes (left panels) and SVF cells (right panels) isolated from WAT from F-BCA compound mice and their littermates with WT, Fsp1 - Cre , or Ctnnb1 exon 3 fl/+ genotypes (Ctrl). TTFs were used as a positive Ctrl for activated form β-catenin expression. (B) Body weight of male F-BCA mice and their littermates on ND. n = 9 for male Ctrl mice, and n = 10 for male F-BCA mice. (C) Ventral view of subcutaneous and visceral adipose depots of male Ctrl and F-BCA littermates on ND at 4 months of age. Adipose depots are circled with dashed lines. (D) Percent body fat (NMR spectroscopy) of Ctrl and F-BCA littermates on ND at 4 months of age. n = 6 for male Ctrl and F-BCA mice. (E, F) Representative pictures and weight of the adipose tissues of F-BCA mice and their littermates on ND at 4 months of age. n = 8 for male Ctrl mice, and n = 14 for male F-BCA mice. (G) HE staining of WAT sections of 4-month-old F-BCA mice and their littermates on ND. Scale bar: 200 μm. (H) GTT of F-BCA mice and their littermates on ND at 4 months of age. n = 13 for male Ctrl mice, and n = 5 for male F-BCA mice. (I) Quantification of the AUC of the GTT relative to Ctrl group. (J) ITT of F-BCA mice and their littermates on ND at 4 months of age. n = 10 for male Ctrl mice, and n = 5 for male F-BCA mice. (K) Quantification of the AUC of the ITT relative to Ctrl group. Data are presented as mean ± SEM. Statistical analyses were performed with two-tailed unpaired student t test (panel D, F, I, and K) or two-way ANOVA followed by Bonferroni's multiple comparison test (panel B, H, and J). * p

    Techniques Used: Activation Assay, Western Blot, Expressing, Isolation, BIA-KA, Mouse Assay, Nuclear Magnetic Resonance, Spectroscopy, Staining, Two Tailed Test

    4) Product Images from "CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle"

    Article Title: CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.104950

    Effects of CAS-1 on exchange of actin-bound nucleotides in the absence and presence of UNC-60B. ATP–G-actin (1 µM) was incubated with etheno–ATP in the presence of 0–0.5 µM MBP–CAS-1 ( A ,
    Figure Legend Snippet: Effects of CAS-1 on exchange of actin-bound nucleotides in the absence and presence of UNC-60B. ATP–G-actin (1 µM) was incubated with etheno–ATP in the presence of 0–0.5 µM MBP–CAS-1 ( A ,

    Techniques Used: Incubation

    5) Product Images from "Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment"

    Article Title: Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.03.064

    Tracking cell division with iH2B-GFP reporter identifies quiescent population in GBM organoids. a) Targeting strategy for iH2B-GFP reporter knock-in by CRISPR-assisted homologous recombination into AAVS1 locus (gene symbol PPP1R12C ). SA: splice acceptor; Neo: Neomycin resistance gene; pA: poly-adenylation signal; CAG: CAG promoter; rtTA: reverse tetracycline-controlled transactivator; H2B-GFP: histone2B-green fluorescent protein; tetO: tet operator. b) Principle of doxycyline (Dox)-inducible expression of H2B-GFP. c) Schematic depiction of divisional dilution of H2B-GFP label during -Dox chase period. Quiescent cells retain H2B-GFP label (GFP high ), while proliferative cells dilute the label (GFP low ). d) GBM cell line SD3-iH2B-GFP grown as proliferative culture on 2D laminin-coated dishes. In the presence of doxycycline (+Dox), nuclei are uniformly labeled with H2B-GFP. Cells dilute H2B-GFP label during -Dox chase periods (5, 10, and 20 days shown) by cell division. DAPI is used for nuclear counter staining. e) Flow cytometry analysis of SD3-iH2B-GFP cells grown on 2D laminin for the indicated -Dox chase periods. A small fraction of SD3-iH2B-GFP cells remained GFP-negative even in +Dox conditions (denoted as “[s]”), possibly due to sporadic silencing of transgene. Histograms are normalized on y-axis to modal scale (FlowJo). f) Experimental design for isolation of quiescent GBM cells from 3D GBM organoids. GBM organoids are generated by seeding cells in Matrigel droplets and expanding them as floating cultures. After growth for 2 weeks with +Dox pulse, organoids are chased for 2 or 4 weeks in -Dox conditions. Dissociated cells are separated into GFP high and GFP low populations by FACS. g) Images of GBM organoids in culture dishes, after 2 or 4 week -Dox chase periods. h) Fluorescence images of sections of 3D GBM organoids show a declining number of label-retaining GFP high cells during organoid expansion. i) Immunofluorescence images show absence of proliferation markers Ki67 and phospho-Vimentin (pVim) in GFP high cells (arrows), confirming slow dividing nature of GFP high cells. Notice debris from dead cells accumulates during organoid culture, which is more prominent after 4 week chase. j) Representative FACS results of GBM organoids analyzed after 2 or 4 week -Dox chase. After 2 week -Dox chase, 3·1% of cells remained GFP high ; after 4 week chase, only 0·4% of cells remained GFP high . Three independent experiments (10–12 pooled organoids per experiment) yielded similar results. X-axis in left histograms shows red auto-fluorescence of cells. Histograms are normalized on y-axis to modal scale (FlowJo). Scale bars: 50 μm (d), 10 mm (g), 200 μm (h), 20 μm (i).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
    Figure Legend Snippet: Tracking cell division with iH2B-GFP reporter identifies quiescent population in GBM organoids. a) Targeting strategy for iH2B-GFP reporter knock-in by CRISPR-assisted homologous recombination into AAVS1 locus (gene symbol PPP1R12C ). SA: splice acceptor; Neo: Neomycin resistance gene; pA: poly-adenylation signal; CAG: CAG promoter; rtTA: reverse tetracycline-controlled transactivator; H2B-GFP: histone2B-green fluorescent protein; tetO: tet operator. b) Principle of doxycyline (Dox)-inducible expression of H2B-GFP. c) Schematic depiction of divisional dilution of H2B-GFP label during -Dox chase period. Quiescent cells retain H2B-GFP label (GFP high ), while proliferative cells dilute the label (GFP low ). d) GBM cell line SD3-iH2B-GFP grown as proliferative culture on 2D laminin-coated dishes. In the presence of doxycycline (+Dox), nuclei are uniformly labeled with H2B-GFP. Cells dilute H2B-GFP label during -Dox chase periods (5, 10, and 20 days shown) by cell division. DAPI is used for nuclear counter staining. e) Flow cytometry analysis of SD3-iH2B-GFP cells grown on 2D laminin for the indicated -Dox chase periods. A small fraction of SD3-iH2B-GFP cells remained GFP-negative even in +Dox conditions (denoted as “[s]”), possibly due to sporadic silencing of transgene. Histograms are normalized on y-axis to modal scale (FlowJo). f) Experimental design for isolation of quiescent GBM cells from 3D GBM organoids. GBM organoids are generated by seeding cells in Matrigel droplets and expanding them as floating cultures. After growth for 2 weeks with +Dox pulse, organoids are chased for 2 or 4 weeks in -Dox conditions. Dissociated cells are separated into GFP high and GFP low populations by FACS. g) Images of GBM organoids in culture dishes, after 2 or 4 week -Dox chase periods. h) Fluorescence images of sections of 3D GBM organoids show a declining number of label-retaining GFP high cells during organoid expansion. i) Immunofluorescence images show absence of proliferation markers Ki67 and phospho-Vimentin (pVim) in GFP high cells (arrows), confirming slow dividing nature of GFP high cells. Notice debris from dead cells accumulates during organoid culture, which is more prominent after 4 week chase. j) Representative FACS results of GBM organoids analyzed after 2 or 4 week -Dox chase. After 2 week -Dox chase, 3·1% of cells remained GFP high ; after 4 week chase, only 0·4% of cells remained GFP high . Three independent experiments (10–12 pooled organoids per experiment) yielded similar results. X-axis in left histograms shows red auto-fluorescence of cells. Histograms are normalized on y-axis to modal scale (FlowJo). Scale bars: 50 μm (d), 10 mm (g), 200 μm (h), 20 μm (i).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

    Techniques Used: Knock-In, CRISPR, Homologous Recombination, Expressing, Labeling, Staining, Flow Cytometry, Cytometry, Isolation, Generated, FACS, Fluorescence, Immunofluorescence

    6) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    7) Product Images from "STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells"

    Article Title: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1302750

    The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites
    Figure Legend Snippet: The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites

    Techniques Used:

    8) Product Images from "Primary Culture of Mammalian Taste Epithelium"

    Article Title: Primary Culture of Mammalian Taste Epithelium

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-62703-125-7_7

    Attachment and morphology of cultured human fungiform taste cells. ( a ) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. ( b ) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. ( c and d ) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.
    Figure Legend Snippet: Attachment and morphology of cultured human fungiform taste cells. ( a ) Primary cell cultures grown on collagen type-1-coated plates were imaged after 2 days. ( b ) Cells from human fungiform papillae grew for up to 2–4 weeks under attached cell clusters. ( c and d ) Represent day 2 and 4 weeks after harvesting, respectively. During this period we did not observe growth of cells with the appearance of non-taste epithelial cells.

    Techniques Used: Cell Culture

    9) Product Images from "3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine"

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

    Journal: Nucleic Acids Research

    doi:

    MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.
    Figure Legend Snippet: MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Techniques Used: Modification, Sequencing

    10) Product Images from "Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae"

    Article Title: Rational Manipulation of mRNA Folding Free Energy Allows Rheostat Control of Pneumolysin Production by Streptococcus pneumoniae

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119823

    Differential PLY production by S . pneumoniae strains is directly related to PLY production. Lysis of primary human erythrocytes as assessed by hemoglobin release assay correlates with (A) ply 5’ mRNA ΔG and (B) PLY as measured by ELISA (R 2 = 0.95; outside lines represent 95% CI). *, P
    Figure Legend Snippet: Differential PLY production by S . pneumoniae strains is directly related to PLY production. Lysis of primary human erythrocytes as assessed by hemoglobin release assay correlates with (A) ply 5’ mRNA ΔG and (B) PLY as measured by ELISA (R 2 = 0.95; outside lines represent 95% CI). *, P

    Techniques Used: Lysis, Release Assay, Enzyme-linked Immunosorbent Assay

    11) Product Images from "E-Cadherin-dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1"

    Article Title: E-Cadherin-dependent Growth Suppression is Mediated by the Cyclin-dependent Kinase Inhibitor p27KIP1

    Journal: The Journal of Cell Biology

    doi:

    Increased p27 binding to cyclin E mediates reduced Cyclin E–associated kinase activity in aggregated cells. ( a ) Cyclin E–associated cdk2 kinase activity, as measured by phosphorylation of Histone H1 substrate in representative neomycin-resistant control transfectants ( neo1 , neo4 , neo8 ) or E-cadherin transfectants (Ecad1, Ecad9, Ecad18) grown in three-dimensional culture. Also included are Pc5-T cells grown in monolayer (2-D) culture, or in three-dimensional culture in either the presence or absence of hyaluronidase (Hy). ( b ) Increased levels of p27 bound to cyclin E in tightly adherent EMT/6 spheroids. After immunoprecipitating cyclin E from tightly- or loosely adherent aggregates, levels of cyclin E–bound p27 were detected by immunoblotting. Cell-free lysis buffer was used for the control sample. ( c ) Immunodepletion of p27 from aggregated E-cadherin transfectants. Cell extracts immunodepleted one (1×) or two (2×) times with antibodies against p27 were probed for p27 by immunoblotting. Controls ( Con ) represent extracts before immunodepletion ( d ). Cyclin E, cyclin E–associated cdk2, and p27 are markedly reduced in p27-immunodepleted ( ID ) supernatents from E-cadherin–aggregated cells. Cyclin E immunoprecipitates from lysates with or without prior immunodepletion with p27 antibodies were probed for cyclin E, cdk2, or p27 by immunoblotting. As a control, Ecad9 lysates were immunoprecipitated with nonspecific rabbit IgG antibodies instead of cyclin E antibodies. ( e ) Cyclin E–associated cdk2 kinase activity is increased after p27-immunodepletion ( ID ) of E-cadherin–arrested cells. The representative control in this experiment was Ecad7 lysate immunoprecipitated with nonspecific rabbit IgG antibodies instead of cyclin E antibodies. In each of these experiments cells were cultured for 48 h in three-dimensional culture.
    Figure Legend Snippet: Increased p27 binding to cyclin E mediates reduced Cyclin E–associated kinase activity in aggregated cells. ( a ) Cyclin E–associated cdk2 kinase activity, as measured by phosphorylation of Histone H1 substrate in representative neomycin-resistant control transfectants ( neo1 , neo4 , neo8 ) or E-cadherin transfectants (Ecad1, Ecad9, Ecad18) grown in three-dimensional culture. Also included are Pc5-T cells grown in monolayer (2-D) culture, or in three-dimensional culture in either the presence or absence of hyaluronidase (Hy). ( b ) Increased levels of p27 bound to cyclin E in tightly adherent EMT/6 spheroids. After immunoprecipitating cyclin E from tightly- or loosely adherent aggregates, levels of cyclin E–bound p27 were detected by immunoblotting. Cell-free lysis buffer was used for the control sample. ( c ) Immunodepletion of p27 from aggregated E-cadherin transfectants. Cell extracts immunodepleted one (1×) or two (2×) times with antibodies against p27 were probed for p27 by immunoblotting. Controls ( Con ) represent extracts before immunodepletion ( d ). Cyclin E, cyclin E–associated cdk2, and p27 are markedly reduced in p27-immunodepleted ( ID ) supernatents from E-cadherin–aggregated cells. Cyclin E immunoprecipitates from lysates with or without prior immunodepletion with p27 antibodies were probed for cyclin E, cdk2, or p27 by immunoblotting. As a control, Ecad9 lysates were immunoprecipitated with nonspecific rabbit IgG antibodies instead of cyclin E antibodies. ( e ) Cyclin E–associated cdk2 kinase activity is increased after p27-immunodepletion ( ID ) of E-cadherin–arrested cells. The representative control in this experiment was Ecad7 lysate immunoprecipitated with nonspecific rabbit IgG antibodies instead of cyclin E antibodies. In each of these experiments cells were cultured for 48 h in three-dimensional culture.

    Techniques Used: Binding Assay, Activity Assay, Lysis, Immunoprecipitation, Cell Culture

    E-cadherin suppresses growth of EMT/6 cells in three-dimensional culture. ( a ) Proliferation of EMT/6 cells transfected with E-cadherin ( Ecad 9) or neomycin vector alone ( neo1 ) as measured by BrdU incorporation into DNA. 24 h after plating in three-dimensional culture, cells were either untreated (−BrdU) or pulsed with BrdU (+BrdU) for a further 24 h. Cells were prepared as described in the Materials and Methods, and cell cycle profiles were analyzed by flow cytometry. Tightly adherent or hyaluronidase ( HYase ) dispersed Pc5-T cells were also included as an additional control. ( b ) Proliferation of neomycin-resistant control transfectants ( neo4 , neo7 , neo8 ) or E-cadherin transfectants ( Ecad3 , Ecad7 , Ecad9 , and Ecad18 ) as measured by [ 3 H]thymidine incorporation into DNA. Intercellular adhesion of E-cadherin transfectants was prevented by treatment with 2 μg/ml DECMA-1 antibody at the time of plating.
    Figure Legend Snippet: E-cadherin suppresses growth of EMT/6 cells in three-dimensional culture. ( a ) Proliferation of EMT/6 cells transfected with E-cadherin ( Ecad 9) or neomycin vector alone ( neo1 ) as measured by BrdU incorporation into DNA. 24 h after plating in three-dimensional culture, cells were either untreated (−BrdU) or pulsed with BrdU (+BrdU) for a further 24 h. Cells were prepared as described in the Materials and Methods, and cell cycle profiles were analyzed by flow cytometry. Tightly adherent or hyaluronidase ( HYase ) dispersed Pc5-T cells were also included as an additional control. ( b ) Proliferation of neomycin-resistant control transfectants ( neo4 , neo7 , neo8 ) or E-cadherin transfectants ( Ecad3 , Ecad7 , Ecad9 , and Ecad18 ) as measured by [ 3 H]thymidine incorporation into DNA. Intercellular adhesion of E-cadherin transfectants was prevented by treatment with 2 μg/ml DECMA-1 antibody at the time of plating.

    Techniques Used: Transfection, Plasmid Preparation, BrdU Incorporation Assay, Flow Cytometry, Cytometry

    Changes in cell cycle molecules in response to intercellular adhesion of EMT/6 cells as detected by immunoblotting. ( a ) Three-dimensional cultures of EMT/6 neomycin–resistant control transfectants ( neo1 , neo4 ), E-cadherin transfectants ( Ecad3 , Ecad7, and Ecad9 ), EMT/6 loosely-adherent variants ( Pc10-L and Pc11-L ) or HA-dependent tightly adherent variants ( Pc5-T , PcT-7 ) were examined. Immunoblots were probed for the cell cycle molecules indicated. E-cadherin- or HA-dependent intercellular adhesion was prevented by treatment with either DECMA-1 antibody ( Dec ) or hyaluronidase ( Hy ), respectively. Note changes in pRb phosphorylation and levels of cyclin D1 and p27 in cells allowed to aggregate in three-dimensional culture. ( b ) Time course demonstrating changes in p27 and cyclin D1 protein levels as cells aggregate in three-dimensional culture. Cells transfected with E-cadherin ( Ecad3 ) grown in the presence or absence of 2 μg/ml DECMA-1 antibody for the indicated times were probed for p27 and cyclin D1 by immunoblotting. At time = 0 cells were transferred from two- to three-dimensional culture.
    Figure Legend Snippet: Changes in cell cycle molecules in response to intercellular adhesion of EMT/6 cells as detected by immunoblotting. ( a ) Three-dimensional cultures of EMT/6 neomycin–resistant control transfectants ( neo1 , neo4 ), E-cadherin transfectants ( Ecad3 , Ecad7, and Ecad9 ), EMT/6 loosely-adherent variants ( Pc10-L and Pc11-L ) or HA-dependent tightly adherent variants ( Pc5-T , PcT-7 ) were examined. Immunoblots were probed for the cell cycle molecules indicated. E-cadherin- or HA-dependent intercellular adhesion was prevented by treatment with either DECMA-1 antibody ( Dec ) or hyaluronidase ( Hy ), respectively. Note changes in pRb phosphorylation and levels of cyclin D1 and p27 in cells allowed to aggregate in three-dimensional culture. ( b ) Time course demonstrating changes in p27 and cyclin D1 protein levels as cells aggregate in three-dimensional culture. Cells transfected with E-cadherin ( Ecad3 ) grown in the presence or absence of 2 μg/ml DECMA-1 antibody for the indicated times were probed for p27 and cyclin D1 by immunoblotting. At time = 0 cells were transferred from two- to three-dimensional culture.

    Techniques Used: Western Blot, Transfection

    Morphology of EMT/6 cells displaying either E-cadherin or HA-dependent intercellular adhesion in three-dimensional culture. ( a–c ) EMT/6 cells transfected with E-cadherin (Ecad9); ( d ) with neomycin vector alone (neo8), or ( e and f ) Pc5-T cells with HA-dependent adhesion were cultured with 1,000 U/ml hyaluronidase ( a–d, f ), 10 μg/ml DECMA-1 antibody ( c and e ), or 10 μg/ml of GoH3 anti-α6-integrin antibody ( b ). Cells were photographed after 24 h in three-dimensional culture. Bar, 50 μm.
    Figure Legend Snippet: Morphology of EMT/6 cells displaying either E-cadherin or HA-dependent intercellular adhesion in three-dimensional culture. ( a–c ) EMT/6 cells transfected with E-cadherin (Ecad9); ( d ) with neomycin vector alone (neo8), or ( e and f ) Pc5-T cells with HA-dependent adhesion were cultured with 1,000 U/ml hyaluronidase ( a–d, f ), 10 μg/ml DECMA-1 antibody ( c and e ), or 10 μg/ml of GoH3 anti-α6-integrin antibody ( b ). Cells were photographed after 24 h in three-dimensional culture. Bar, 50 μm.

    Techniques Used: Transfection, Plasmid Preparation, Cell Culture

    12) Product Images from "TNF is required for TLR ligand–mediated but not protease-mediated allergic airway inflammation"

    Article Title: TNF is required for TLR ligand–mediated but not protease-mediated allergic airway inflammation

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI90890

    TNF from CD11c + macrophages acts on radioresistant AECs to reprogram them and promote allergic sensitization. ( A and B ) TNF in BALF after instillation of LPS/OVA into WT and Myd88 –/– mice ( A ) or Myd88 fl/fl mice crossed to Sftpc-cre mice or Cd11c-cre mice to delete Myd88 in AECs or Cd11c -expressing cells, respectively ( B ) ( n = 8 mice per group). ( C ) TNF in medium alone (Med), or culture supernatants of alveolar (Alv) macrophages, interstitial (Int) macrophages, monocytes (Mon), and cDCs purified by FACS from lungs of LPS/OVA-treated mice ( n = 6 mice per group). ( D ) Cell numbers for the indicated leukocytes in airways of WT and TNFR1/2-DKO (KO) reciprocal bone marrow chimeric mice sensitized with OVA with or without LPS and subsequently challenged with aerosolized OVA ( n = 6 mice per group). ( A – D ) Data represent mean values ± SEM and were confirmed by at least 1 repeat experiment. * P
    Figure Legend Snippet: TNF from CD11c + macrophages acts on radioresistant AECs to reprogram them and promote allergic sensitization. ( A and B ) TNF in BALF after instillation of LPS/OVA into WT and Myd88 –/– mice ( A ) or Myd88 fl/fl mice crossed to Sftpc-cre mice or Cd11c-cre mice to delete Myd88 in AECs or Cd11c -expressing cells, respectively ( B ) ( n = 8 mice per group). ( C ) TNF in medium alone (Med), or culture supernatants of alveolar (Alv) macrophages, interstitial (Int) macrophages, monocytes (Mon), and cDCs purified by FACS from lungs of LPS/OVA-treated mice ( n = 6 mice per group). ( D ) Cell numbers for the indicated leukocytes in airways of WT and TNFR1/2-DKO (KO) reciprocal bone marrow chimeric mice sensitized with OVA with or without LPS and subsequently challenged with aerosolized OVA ( n = 6 mice per group). ( A – D ) Data represent mean values ± SEM and were confirmed by at least 1 repeat experiment. * P

    Techniques Used: Mouse Assay, Expressing, Purification, FACS

    TNF is required in a TLR ligand–mediated model of asthma. ( A – E and G ) WT and TNFR1/2-DKO mice were sensitized to OVA using the indicated adjuvants, and subsequently challenged with aerosolized OVA. Shown are cell numbers ( A ) and cytokine ( B ) and chemokine ( C ) concentrations in BALF, mucin-producing cells in airways (×100 magnification; scale bars: 500 μm) ( D ), and airway resistance (R) after allergen challenge ( E ). Resistance values were obtained at baseline (B) and after administration of the indicated doses of methacholine (MCH). ( F ) Cell numbers for the indicated leukocytes in BALF of WT and TNFR1- and TNFR2-single-KO mice following LPS/OVA sensitization and subsequent challenge with OVA. ( G ) Cell numbers for the indicated leukocytes in BALF after challenge. * P
    Figure Legend Snippet: TNF is required in a TLR ligand–mediated model of asthma. ( A – E and G ) WT and TNFR1/2-DKO mice were sensitized to OVA using the indicated adjuvants, and subsequently challenged with aerosolized OVA. Shown are cell numbers ( A ) and cytokine ( B ) and chemokine ( C ) concentrations in BALF, mucin-producing cells in airways (×100 magnification; scale bars: 500 μm) ( D ), and airway resistance (R) after allergen challenge ( E ). Resistance values were obtained at baseline (B) and after administration of the indicated doses of methacholine (MCH). ( F ) Cell numbers for the indicated leukocytes in BALF of WT and TNFR1- and TNFR2-single-KO mice following LPS/OVA sensitization and subsequent challenge with OVA. ( G ) Cell numbers for the indicated leukocytes in BALF after challenge. * P

    Techniques Used: Mouse Assay

    TNF signaling through TNFR1 promotes type 2, but not type 17, cell differentiation. ( A – C ) Mice receiving adoptive transfer of OVA-specific CD4 + T cells were sensitized to inhaled OVA using the indicated adjuvants. Lung-draining LNs were excised 4 days later, cells from them were restimulated ex vivo with OVA, and the indicated cytokines in the culture supernatants were measured. Shown are cytokines from LN cultures of WT and Tlr4 –/– mice sensitized to OVA using rmTNF as an adjuvant ( n = 5 mice per group) ( A ), WT and TNFR1/2-DKO mice sensitized to OVA using either LPS or ASP as an adjuvant ( n = 6 mice per group) ( B ), and WT and TNFR1- and TNFR2-single-KO mice sensitized to OVA using LPS as an adjuvant ( n = 12 mice per group) ( C ). Data represent mean ± SEM from a single experiment, representative of 2, except C , which shows the combined data of 2 experiments yielding similar results. * P
    Figure Legend Snippet: TNF signaling through TNFR1 promotes type 2, but not type 17, cell differentiation. ( A – C ) Mice receiving adoptive transfer of OVA-specific CD4 + T cells were sensitized to inhaled OVA using the indicated adjuvants. Lung-draining LNs were excised 4 days later, cells from them were restimulated ex vivo with OVA, and the indicated cytokines in the culture supernatants were measured. Shown are cytokines from LN cultures of WT and Tlr4 –/– mice sensitized to OVA using rmTNF as an adjuvant ( n = 5 mice per group) ( A ), WT and TNFR1/2-DKO mice sensitized to OVA using either LPS or ASP as an adjuvant ( n = 6 mice per group) ( B ), and WT and TNFR1- and TNFR2-single-KO mice sensitized to OVA using LPS as an adjuvant ( n = 12 mice per group) ( C ). Data represent mean ± SEM from a single experiment, representative of 2, except C , which shows the combined data of 2 experiments yielding similar results. * P

    Techniques Used: Cell Differentiation, Mouse Assay, Adoptive Transfer Assay, Ex Vivo

    13) Product Images from "G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases"

    Article Title: G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

    Journal: eLife

    doi: 10.7554/eLife.04871

    A ternary complex of DNase I, G-actin, PP1G and PPP1R15A retains its eIF2a P -directed phosphatase activity. ( A ) UV protein absorbance trace of a PPP1R15A (539–614)-PP1G(7–323)-G-actin and DNase I complex assembled from the bacterially-expressed binary complex, rabbit muscle G-actin and bovine pancreatic DNase I, resolved by size-exclusion chromatography. The indicated fractions from the chromatogram are presented in the Coomassie-stained SDS-PAGE below. The positions of G-actin, PP1, DNase I, and the PPP1R15A peptide are indicated. ( B ) Cartoon representation of a model of the PPP1R15B, PP1G, and G-actin ternary complex with DNase I placed by superimposing the actin and DNase I complex (PDB: 2A41) ( Chereau et al., 2005 ) onto the PPP1R15B, PP1G and G-actin ternary complex (PDB: 4V0U). Note that DNase I is bound to the backside of the ternary complex, facing away from the PP1 active site (arrow). ( C ) Images of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated and dephosphorylated eIF2a (eIF2a P and eIF2a 0 ) have been resolved. Escalating amounts of G-actin or a complex of G-actin and DNase I (final concentration, 10 nM–1 µM) were added to a reaction containing 25 nM PPP1R15B-MBP and PP1G complex (as in Figure 3A ) and 2 µM eIF2a P substrate for 20 min. DOI: http://dx.doi.org/10.7554/eLife.04871.022
    Figure Legend Snippet: A ternary complex of DNase I, G-actin, PP1G and PPP1R15A retains its eIF2a P -directed phosphatase activity. ( A ) UV protein absorbance trace of a PPP1R15A (539–614)-PP1G(7–323)-G-actin and DNase I complex assembled from the bacterially-expressed binary complex, rabbit muscle G-actin and bovine pancreatic DNase I, resolved by size-exclusion chromatography. The indicated fractions from the chromatogram are presented in the Coomassie-stained SDS-PAGE below. The positions of G-actin, PP1, DNase I, and the PPP1R15A peptide are indicated. ( B ) Cartoon representation of a model of the PPP1R15B, PP1G, and G-actin ternary complex with DNase I placed by superimposing the actin and DNase I complex (PDB: 2A41) ( Chereau et al., 2005 ) onto the PPP1R15B, PP1G and G-actin ternary complex (PDB: 4V0U). Note that DNase I is bound to the backside of the ternary complex, facing away from the PP1 active site (arrow). ( C ) Images of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated and dephosphorylated eIF2a (eIF2a P and eIF2a 0 ) have been resolved. Escalating amounts of G-actin or a complex of G-actin and DNase I (final concentration, 10 nM–1 µM) were added to a reaction containing 25 nM PPP1R15B-MBP and PP1G complex (as in Figure 3A ) and 2 µM eIF2a P substrate for 20 min. DOI: http://dx.doi.org/10.7554/eLife.04871.022

    Techniques Used: Activity Assay, Size-exclusion Chromatography, Staining, SDS Page, Concentration Assay

    14) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.
    Figure Legend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Techniques Used: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.
    Figure Legend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Techniques Used: Functional Assay, Preserving, Sequencing, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.
    Figure Legend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Techniques Used: Refining, Sequencing

    15) Product Images from "Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *"

    Article Title: Sequence, Distance, and Accessibility are Determinants of 5? End-Directed Cleavages by Retroviral RNases H *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M510504200

    Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.
    Figure Legend Snippet: Alignment of sequences flanking RNA 5′ end-directed cleavage sites recognized by HIV-1 RNase H ). In the center column, the sequence surrounding each cleavage site is given, with the location of the cleavage site represented as a gap. The right column gives the position of each cleavage site counting from the 5′ end of the RNA.

    Techniques Used: Sequencing

    Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.
    Figure Legend Snippet: Comparison of HIV-1 and M-MuLV RNase H 5′ end-directed cleavages in the sequences of RNAs Md1 - Md10 . The sequences of the 29-mer RNAs Md1 through Md10 are aligned by the RNA 5′ ends to compare the positions of 5′ end-directed cleavage sites. In each sequence, the extent of cleavage at a site is indicated as strong (large arrows) or medium (small arrows) for HIV-1 reverse transcriptase (above) or M-MuLV reverse transcriptase (below). As described in the Discussion, the range of the closest and furthest independent 5′ end-directed cleavage sites is indicated by the positions of the bordering nucleotides from the RNA 5′ end, the position of site G in substrates Md1 and Md7 is indicated, and the grey box highlights nucleotide positions +13 and +20 that include the range of distances where the 5′ end-directed cleavages occur.

    Techniques Used: Sequencing

    Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.
    Figure Legend Snippet: Extent of cleavage and optimal distances for cleavage at sites F, G, and H in RNAs Md1 through Md10 by HIV-1 and M-MuLV reverse transcriptases . The amount of product generated by cleavage (% of total) at sites F, G, and H in the indicated substrates was determined for HIV-1 (A) or M-MuLV (B) reverse transcriptase. Data from the 1 min time points in three (A) or four (B) independent experiments with 5′ end-labeled RNAs were used to determine the amount of product that resulted from the cleavages at site F (gray bars), site G (black bars), or site H (white bars) (± S.D.). These same data were also used to analyze the optimal distance for cleavage of each site relative to the 5′ RNA ends for HIV-1 (C) or M-MuLV (D) reverse transcriptase. The amount of product generated by cleavage (% of total) for sites F (gray squares), G (black circles), or H (open triangles) is plotted as a function of the cleavage site distance in nucleotides from the 5′ end of each substrate.

    Techniques Used: Generated, Labeling

    16) Product Images from "Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro"

    Article Title: Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw675

    Analysis of the products of enzymatic digestion of the PAR–DNA adducts. ( A ) Graphical representation of formation and chemical structure of poly(ADP-ribose) polymers. ( B ) Denaturing PAGE analysis of the products of PARG- and CIP-catalysed digestion of the 5′-[ 32 P]labelled PAR–DNA products. ( C ) Denaturing PAGE analysis of the products of CIP- and SVPDE1-catalysed digestion of the PARP1-generated 3′- 32 P-cordycepin-labelled PAR–DNA products. To generate the PAR–DNA products, 20 nM 3′dAM 32 P-ExoA•RexT rec was incubated with 50 nM PARP1 or PARP2 in the presence of 1 mM NAD + for 30 min at 37°C. After that, the samples were heated for 20 min at 80°C and then incubated with 50 pg/μl PARG (in PARP1 buffer), 0.1 U SVPDE1 (in SVPDE1 buffer) or 10 U CIP (in CIP buffer) for 60 min or 30 min at 37°C. Arrows depict HMW PAR–DNA products and free oligonucleotides.
    Figure Legend Snippet: Analysis of the products of enzymatic digestion of the PAR–DNA adducts. ( A ) Graphical representation of formation and chemical structure of poly(ADP-ribose) polymers. ( B ) Denaturing PAGE analysis of the products of PARG- and CIP-catalysed digestion of the 5′-[ 32 P]labelled PAR–DNA products. ( C ) Denaturing PAGE analysis of the products of CIP- and SVPDE1-catalysed digestion of the PARP1-generated 3′- 32 P-cordycepin-labelled PAR–DNA products. To generate the PAR–DNA products, 20 nM 3′dAM 32 P-ExoA•RexT rec was incubated with 50 nM PARP1 or PARP2 in the presence of 1 mM NAD + for 30 min at 37°C. After that, the samples were heated for 20 min at 80°C and then incubated with 50 pg/μl PARG (in PARP1 buffer), 0.1 U SVPDE1 (in SVPDE1 buffer) or 10 U CIP (in CIP buffer) for 60 min or 30 min at 37°C. Arrows depict HMW PAR–DNA products and free oligonucleotides.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Generated, Incubation

    Separation and characterisation of PAR–DNA mono-adducts by denaturing PAGE and TLC. ( A ) The long-run denaturing PAGE analysis of the products of SVPDE1-catalysed digestion of the 3′- 32 P-cordycepin- and 5′-[ 32 P]labelled PAR–DNA adducts, free DNA and the PAR polymer. The samples were desalted before loading on the gel. ( B ) As in panel A but short-run denaturing PAGE. The samples were not desalted before loading on the gel. ( C ) TLC separation of the products of SVPDE1-catalysed digestion of the 3′- 32 P-cordycepin- and 5′-[ 32 P]labelled PAR–DNA adducts, free DNA and the PAR polymer. The samples were not desalted before loading on the plate. ( D ) Graphical representation of chemical structures of the phosphoribosyl adenosine monophosphate adducts. The pRib-AMP adduct is generated by digestion of the [ 32 P]labelled PAR by SVPDE1, whereas the 2′-pRib-3′dAMP and 5′P-pRib-dG adducts are generated by digestion of the 3′- 32 P-cordycepin-labelled and 5′-[ 32 P]labelled PAR–DNA by SVPDE1, respectively. Arrow ‘X’ indicates the putative 2′-pRib-3′dAMP adduct, ‘Y’ points to traces of a putative 3′-terminal dAMP-3′dAMP dinucleotide, ‘Z’ indicates the putative 5′P-pRib-dG adduct containing a pRib moiety covalently attached to 5′P of dGMP, ‘AM 32 P’ means adenosine 5′-[ 32 P]monophosphate, ‘pRib-AM 32 P’ stands for pRib-AMP with adenosine 5′-[ 32 P]monophosphate, ‘ 32 P-NAD + ’ means [adenylate- 32 P]NAD + , ‘3′dAM 32 P’ is cordycepin 5′-[ 32 P]monophosphate, ‘dGM 32 P’ denotes 2′-deoxyguanosine 5′-[ 32 P]monophosphate, and 32 P means free phosphate.
    Figure Legend Snippet: Separation and characterisation of PAR–DNA mono-adducts by denaturing PAGE and TLC. ( A ) The long-run denaturing PAGE analysis of the products of SVPDE1-catalysed digestion of the 3′- 32 P-cordycepin- and 5′-[ 32 P]labelled PAR–DNA adducts, free DNA and the PAR polymer. The samples were desalted before loading on the gel. ( B ) As in panel A but short-run denaturing PAGE. The samples were not desalted before loading on the gel. ( C ) TLC separation of the products of SVPDE1-catalysed digestion of the 3′- 32 P-cordycepin- and 5′-[ 32 P]labelled PAR–DNA adducts, free DNA and the PAR polymer. The samples were not desalted before loading on the plate. ( D ) Graphical representation of chemical structures of the phosphoribosyl adenosine monophosphate adducts. The pRib-AMP adduct is generated by digestion of the [ 32 P]labelled PAR by SVPDE1, whereas the 2′-pRib-3′dAMP and 5′P-pRib-dG adducts are generated by digestion of the 3′- 32 P-cordycepin-labelled and 5′-[ 32 P]labelled PAR–DNA by SVPDE1, respectively. Arrow ‘X’ indicates the putative 2′-pRib-3′dAMP adduct, ‘Y’ points to traces of a putative 3′-terminal dAMP-3′dAMP dinucleotide, ‘Z’ indicates the putative 5′P-pRib-dG adduct containing a pRib moiety covalently attached to 5′P of dGMP, ‘AM 32 P’ means adenosine 5′-[ 32 P]monophosphate, ‘pRib-AM 32 P’ stands for pRib-AMP with adenosine 5′-[ 32 P]monophosphate, ‘ 32 P-NAD + ’ means [adenylate- 32 P]NAD + , ‘3′dAM 32 P’ is cordycepin 5′-[ 32 P]monophosphate, ‘dGM 32 P’ denotes 2′-deoxyguanosine 5′-[ 32 P]monophosphate, and 32 P means free phosphate.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Thin Layer Chromatography, Generated

    Analysis of the products of NUDT16-catalysed hydrolysis of PAR-DNA adducts generated by PARP1 and PARP2. The 10 nM 3′dAM 32 P-labelled and 3′-[ 32 P]labelled ExoA•RexT rec duplexes were incubated with 100 nM PARP1 and 1 mM NAD + , and the 40 nM 5′-[ 32 P]labelled ExoA•RexT nick duplex was incubated with 100 nM PARP2 and 1 mM NAD + at 37°C for 30 min. After incubation with PARPs, the samples were heated for 20 min at 80°C and the resulting [ 32 P]labelled HMW products were further incubated with 50 pg/μl PARG, 1 U SVPDE1, 10 U CIP or 2–20 μM NUDIX16. ( A ) Denaturing PAGE analysis of the products of NUDT16 and CIP catalysed hydrolysis of the PARP1-generated 3′-[ 32 P]labelled PAR-DNA adducts. To generate a DNA duplex containing a 3′-terminal 32 P residue, the 3′dAM 32 P-labelled ExoA•RexT rec duplex was treated with Tdp1. Lanes 1–8, 3′dAM 32 P-labelled ExoA•RexT rec duplex; lanes 9–17, 3′-[ 32 P]labelled ExoA•RexT rec duplex. ( B ) Denaturing PAGE analysis of the products of PARG-, SVPDE1-, CIP- and NUDT16-catalysed hydrolysis of the PARP2-generated 5′-[ 32 P]labelled PAR-DNA adducts (lanes 1–9). Arrows indicate phosphoribosylated (Rib-p), ribosylated (Rib) and native [ 32 P]labelled 21-mer and 22-mer oligonucleotides, ‘*p’ stands for a labelled phosphate residue. For more details, see Materials and Methods.
    Figure Legend Snippet: Analysis of the products of NUDT16-catalysed hydrolysis of PAR-DNA adducts generated by PARP1 and PARP2. The 10 nM 3′dAM 32 P-labelled and 3′-[ 32 P]labelled ExoA•RexT rec duplexes were incubated with 100 nM PARP1 and 1 mM NAD + , and the 40 nM 5′-[ 32 P]labelled ExoA•RexT nick duplex was incubated with 100 nM PARP2 and 1 mM NAD + at 37°C for 30 min. After incubation with PARPs, the samples were heated for 20 min at 80°C and the resulting [ 32 P]labelled HMW products were further incubated with 50 pg/μl PARG, 1 U SVPDE1, 10 U CIP or 2–20 μM NUDIX16. ( A ) Denaturing PAGE analysis of the products of NUDT16 and CIP catalysed hydrolysis of the PARP1-generated 3′-[ 32 P]labelled PAR-DNA adducts. To generate a DNA duplex containing a 3′-terminal 32 P residue, the 3′dAM 32 P-labelled ExoA•RexT rec duplex was treated with Tdp1. Lanes 1–8, 3′dAM 32 P-labelled ExoA•RexT rec duplex; lanes 9–17, 3′-[ 32 P]labelled ExoA•RexT rec duplex. ( B ) Denaturing PAGE analysis of the products of PARG-, SVPDE1-, CIP- and NUDT16-catalysed hydrolysis of the PARP2-generated 5′-[ 32 P]labelled PAR-DNA adducts (lanes 1–9). Arrows indicate phosphoribosylated (Rib-p), ribosylated (Rib) and native [ 32 P]labelled 21-mer and 22-mer oligonucleotides, ‘*p’ stands for a labelled phosphate residue. For more details, see Materials and Methods.

    Techniques Used: Generated, Incubation, Polyacrylamide Gel Electrophoresis

    17) Product Images from "Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis"

    Article Title: Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis

    Journal: Metabolism: clinical and experimental

    doi: 10.1016/j.metabol.2019.01.008

    Tissue-specific expression of mouse CEACAM1 (mCC1). (A) Primary hepatocytes from Alb – Cc1 +/+ (white bar), Alb + Cc1 +/+ (light grey bar), Alb – Cc1 fl/fl (dark grey bar) and Alb + Cc1 fl/fl (black bar) were isolated and analyzed by qRT-PCR in triplicate to assess mouse Ceacam1 mRNA level. Values are expressed as mean ± SEM; * P
    Figure Legend Snippet: Tissue-specific expression of mouse CEACAM1 (mCC1). (A) Primary hepatocytes from Alb – Cc1 +/+ (white bar), Alb + Cc1 +/+ (light grey bar), Alb – Cc1 fl/fl (dark grey bar) and Alb + Cc1 fl/fl (black bar) were isolated and analyzed by qRT-PCR in triplicate to assess mouse Ceacam1 mRNA level. Values are expressed as mean ± SEM; * P

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR

    18) Product Images from "Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept"

    Article Title: Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept

    Journal: AMB Express

    doi: 10.1186/s13568-014-0064-5

    Mean [±S.D] glucose, fructose and fructan concentrations in untreated GJ (open bars) and GJ + t fosEp (filled bars). a Fructan = polyfructose.
    Figure Legend Snippet: Mean [±S.D] glucose, fructose and fructan concentrations in untreated GJ (open bars) and GJ + t fosEp (filled bars). a Fructan = polyfructose.

    Techniques Used:

    19) Product Images from "Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling"

    Article Title: Transgelin-expressing myofibroblasts orchestrate ventral midline closure through TGFβ signalling

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.152843

    Tgfbr2 knockout in myogenic and chondrogenic cells does not affect midline closure. (A,B) Transverse sections in the thoracic and abdominal region of an E14.5 MyoD -Cre: Tgfbr2 flx/flx embryo. (A) Alcian Blue (AB) and Nuclear Fast Red (NFR) staining show normal developmental milestones, comparable to the WT (see Fig. 6 C,D). (B) Normal muscle (MF20 + ) and chondrocyte [NG2 (CSPG4) + ] development in the midline area of the mutant mouse. (C) Whole-mount MF20 staining of a 2-day-old pup, showing normal muscle development in the midline postnatally in the mutant. The umbilicus site is marked with a dotted circle. (D,E) Transverse sections in the thoracic and abdominal region of an E15.5 NG2- CreER™: Tgfbr2 flx/flx embryo. (D) Alcian Blue and Nuclear Fast Red staining show normal developmental milestones, comparable to WT. (E) Normal muscle (MF20 + ) and chondrocyte (NG2 + ) development in the midline area of the mutant mouse. (F) Whole-mount Alizarin Red and Alcian Blue showing normal rib cage development and fused sternum in the midline at the fetal stage in the mutant. Scale bars: 500 µm in A,B,D,E; 1000 µm in C,F.
    Figure Legend Snippet: Tgfbr2 knockout in myogenic and chondrogenic cells does not affect midline closure. (A,B) Transverse sections in the thoracic and abdominal region of an E14.5 MyoD -Cre: Tgfbr2 flx/flx embryo. (A) Alcian Blue (AB) and Nuclear Fast Red (NFR) staining show normal developmental milestones, comparable to the WT (see Fig. 6 C,D). (B) Normal muscle (MF20 + ) and chondrocyte [NG2 (CSPG4) + ] development in the midline area of the mutant mouse. (C) Whole-mount MF20 staining of a 2-day-old pup, showing normal muscle development in the midline postnatally in the mutant. The umbilicus site is marked with a dotted circle. (D,E) Transverse sections in the thoracic and abdominal region of an E15.5 NG2- CreER™: Tgfbr2 flx/flx embryo. (D) Alcian Blue and Nuclear Fast Red staining show normal developmental milestones, comparable to WT. (E) Normal muscle (MF20 + ) and chondrocyte (NG2 + ) development in the midline area of the mutant mouse. (F) Whole-mount Alizarin Red and Alcian Blue showing normal rib cage development and fused sternum in the midline at the fetal stage in the mutant. Scale bars: 500 µm in A,B,D,E; 1000 µm in C,F.

    Techniques Used: Knock-Out, Staining, Mutagenesis

    Tagln- Cre: Tgfbr2 flx/flx embryos develop VBW closure defects. (A,B) Morphological comparison between Tagln- Cre: Tgfbr2 flx/flx and Tagln- Cre: Tgfbr2 flx/wt mouse embryos. (A) E13.5 Tagln- Cre: Tgfbr2 flx/flx embryos show a translucent ventral midline, a more lateral limit to the secondary body wall (arrow) and absence of midline raphe (arrowhead) when compared with Tagln -Cre: Tgfbr2 flx/wt . (B) The ventral midline closure defect in Tagln- Cre: Tgfbr2 flx/flx . A thin membrane covers the VBW cavities, as compared with the nearly closed thoracic midline in the WT (arrow) and the embryos show a large exomphalos compared with the physiological umbilical hernia in the WT (arrowhead). (C) Transverse section in mid-thorax at E14.5 in WT (left) and Tagln -Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. The VBW is composed of a thin sac in the mutant, whereas the two lateral sternebrae are nearly meeting in the midline in the WT. (D) Transverse section at level of the umbilical hernia at E14.5 in WT (left) and Tagln- Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. In the WT only a small physiological umbilical hernia is present and the small intestine is returning to the abdominal cavity, whereas the mutant shows a large exomphalos defect and very few bowel loops are present in the abdominal cavity. (E-H) Characterisation of cell type in Tagln- Cre: Tgfbr2 flx/flx thoracic (right) and abdominal (left) body wall by immunohistochemistry. (E) E13.5 mutant embryos show normal lateral body wall muscles (MF20 + ) and ribs (SOX9 + ), whereas the ventral midline is made of a thin sac. Condensations of SOX9 + and MF20 + cells (arrow) are seen just lateral to the VBW in the thoracic and abdominal areas, respectively. (F) E14.5 mutant embryo shows very little progression in secondary element migration, and the condensation of chondrocyte and myocyte (arrow) is still seen lateral to the VBW in the thoracic and abdominal compartments, respectively. (G) The VBW of Tagln- Cre: Tgfbr2 flx/flx still expresses TAGLN. (H) The skin covering the premature VBW in Tagln- Cre: Tgfbr2 flx/flx is made of a single layer of squamous epithelial cells (insets P), while in the secondary elements multilayered cuboid epithelium covers the lateral body wall (insets S). Bottom row of insets shows E-cadherin channel. H, heart; L, lungs; LV, liver; IN, intestine; P, primary body wall; S, secondary body wall; TA, transverses abdominis; IO, internal oblique; EO, external oblique; PC, panniculus carnosus; IC, intercostal muscles; R, rib. Scale bars: 1000 µm in A,B; 500 µm in C-G; 200 µm in H, 50 µm in insets.
    Figure Legend Snippet: Tagln- Cre: Tgfbr2 flx/flx embryos develop VBW closure defects. (A,B) Morphological comparison between Tagln- Cre: Tgfbr2 flx/flx and Tagln- Cre: Tgfbr2 flx/wt mouse embryos. (A) E13.5 Tagln- Cre: Tgfbr2 flx/flx embryos show a translucent ventral midline, a more lateral limit to the secondary body wall (arrow) and absence of midline raphe (arrowhead) when compared with Tagln -Cre: Tgfbr2 flx/wt . (B) The ventral midline closure defect in Tagln- Cre: Tgfbr2 flx/flx . A thin membrane covers the VBW cavities, as compared with the nearly closed thoracic midline in the WT (arrow) and the embryos show a large exomphalos compared with the physiological umbilical hernia in the WT (arrowhead). (C) Transverse section in mid-thorax at E14.5 in WT (left) and Tagln -Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. The VBW is composed of a thin sac in the mutant, whereas the two lateral sternebrae are nearly meeting in the midline in the WT. (D) Transverse section at level of the umbilical hernia at E14.5 in WT (left) and Tagln- Cre: Tgfbr2 flx/flx (right), with Alcian Blue staining to delineate ribs and counterstaining with Nuclear Fast Red. In the WT only a small physiological umbilical hernia is present and the small intestine is returning to the abdominal cavity, whereas the mutant shows a large exomphalos defect and very few bowel loops are present in the abdominal cavity. (E-H) Characterisation of cell type in Tagln- Cre: Tgfbr2 flx/flx thoracic (right) and abdominal (left) body wall by immunohistochemistry. (E) E13.5 mutant embryos show normal lateral body wall muscles (MF20 + ) and ribs (SOX9 + ), whereas the ventral midline is made of a thin sac. Condensations of SOX9 + and MF20 + cells (arrow) are seen just lateral to the VBW in the thoracic and abdominal areas, respectively. (F) E14.5 mutant embryo shows very little progression in secondary element migration, and the condensation of chondrocyte and myocyte (arrow) is still seen lateral to the VBW in the thoracic and abdominal compartments, respectively. (G) The VBW of Tagln- Cre: Tgfbr2 flx/flx still expresses TAGLN. (H) The skin covering the premature VBW in Tagln- Cre: Tgfbr2 flx/flx is made of a single layer of squamous epithelial cells (insets P), while in the secondary elements multilayered cuboid epithelium covers the lateral body wall (insets S). Bottom row of insets shows E-cadherin channel. H, heart; L, lungs; LV, liver; IN, intestine; P, primary body wall; S, secondary body wall; TA, transverses abdominis; IO, internal oblique; EO, external oblique; PC, panniculus carnosus; IC, intercostal muscles; R, rib. Scale bars: 1000 µm in A,B; 500 µm in C-G; 200 µm in H, 50 µm in insets.

    Techniques Used: Staining, Mutagenesis, Immunohistochemistry, Migration

    TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P
    Figure Legend Snippet: TGFβ2 and TGFβR2 in the VBW. (A) Transverse section in the abdominal VBW at E14.5 showing expression of TGFβR2 focused in the primary body wall area (labelled by tdTom) in the ventral midline. (A′) Confocal image of the boxed area in A, showing high-level TGFβR2 expression in tdTom + cells beneath the epithelium. (B) Transverse section in the mid thoracic area at E12.5 Tagln -Cre:Rosa26-tdTom mouse embryo stained for TGFβ2 and E-cadherin to label epithelium. TGFβ2 protein is abundant in the midline area of the primary body wall (tdTom channel is removed to expose the TGFβ2 signal). (B′) Confocal image of the primary body wall area (box P) showing strong TGFβ2 expression in the epithelium (arrows) and weaker signalling in the subdermal layer (arrowheads). (B″) Confocal image of the secondary body wall area (box S) showing weak TGFβ2 signal in the subdermal layer (arrows). (C) Midline (ML) and para-midline (PML) ventral wall dissection in an E12.5 WT mouse embryo. (Ca) The embryo was decapitated and the tail excised. (Cb) The dorsal body wall was opened para-sagittal and the thoracic and abdominal organs were exposed. (Cc) The embryo was eviscerated, taking care to preserve the thin primary body wall. (Cd) The thin primary (midline) body wall was carefully dissected from the secondary (para-midline body) wall and sufficient margins were removed from both segments to avoid transitional areas. (D) RT-qPCR comparing Tgfb2 expression in the midline and para-midline of WT mouse embryos between E11.5 and E15.5. There is an anatomical and temporal Tgfb2 gradient in the midline during the closure period. Error bars are s.e.m.; each time point presented is from at least three biological replicates each containing tissue from at least five embryos. (E) Schematic of E14.5 embryo. The VBW delineated by the dashed line was dissected from Tagln -Cre:Rosa26-tdTom embryos and FACS sorted for tdTom signal. (E′) The FACS-sorted cohort. tdTom + cells only accounted for an average of 15% of the total cell population of the VBW (as shown in E). (F) RT-qPCR on the FACS-sorted cells showed higher expression of Tgfbr2 in tdTom + ventral midline cells. Error bars indicate s.e.m.; data presented are from three biological replicates each containing cells from tissue derived from at least seven embryos. ** P

    Techniques Used: Expressing, Staining, Dissection, Quantitative RT-PCR, FACS, Derivative Assay

    20) Product Images from "SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES"

    Article Title: SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2009.11.072

    Relatively passive mechanism for strand displacement synthesis of HIV-1 RT
    Figure Legend Snippet: Relatively passive mechanism for strand displacement synthesis of HIV-1 RT

    Techniques Used:

    Sequence dependent strand displacement synthesis of HIV-1 RT
    Figure Legend Snippet: Sequence dependent strand displacement synthesis of HIV-1 RT

    Techniques Used: Sequencing

    Active and passive mechanisms for strand displacement DNA synthesis by HIV-1 RT near hairpin locations
    Figure Legend Snippet: Active and passive mechanisms for strand displacement DNA synthesis by HIV-1 RT near hairpin locations

    Techniques Used: DNA Synthesis

    DNA replication on flow-stretched ssDNA by HIV-1 RT
    Figure Legend Snippet: DNA replication on flow-stretched ssDNA by HIV-1 RT

    Techniques Used: Flow Cytometry

    21) Product Images from "Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position"

    Article Title: Structural and mechanistic basis for enhanced translational efficiency by 2-thiouridine at the tRNA anticodon wobble position

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2013.05.018

    A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.
    Figure Legend Snippet: A: The total ion chromatogram (TIC) of RNase T1 digested E. coli tRNA Gln and the accompanying extracted ion chromatogram (XIC) of m/z 1004, which corresponds to the expected m/z for the fragment with the sequence U[Um]U[cmnm 5 s 2 U]UGp. B: The MS spectrum of the eluent at 28.2 min. depicting the m/z 1004.25, consistent with the sequence U[Um]U[cmnm 5 s 2 U]UGp. C: The peak in B was selected for MS/MS analysis. The resulting fragmentation is consistent with U[Um]U[cmnm 5 s 2 U]UGp with all identified c- and y-type product ions labeled.

    Techniques Used: Sequencing, Mass Spectrometry, Labeling

    22) Product Images from "Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept"

    Article Title: Co-production of bioethanol and probiotic yeast biomass from agricultural feedstock: application of the rural biorefinery concept

    Journal: AMB Express

    doi: 10.1186/s13568-014-0064-5

    Cell morphology of yeast strains. A = yeast-like growth of turbo ( S. cerevisiae ) showing solitary blastoconidia and normal budding; B = pseudohyphal growth of MYA-796 ( S. boulardii ). Scale bars represent 15 μm.
    Figure Legend Snippet: Cell morphology of yeast strains. A = yeast-like growth of turbo ( S. cerevisiae ) showing solitary blastoconidia and normal budding; B = pseudohyphal growth of MYA-796 ( S. boulardii ). Scale bars represent 15 μm.

    Techniques Used:

    23) Product Images from "Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties"

    Article Title: Structural determinants of human APOBEC3A enzymatic and nucleic acid binding properties

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt945

    Effect of A3A on HIV-1 RT-catalyzed extension of a (−) SSDNA oligonucleotide. The bar graph shows the percent of DNA extension product without (−) or with (+) RT and/or A3A. The positive control reaction with RT only is shown as a white bar. Reactions with RT contain either 5 µM (black bar) or 10 µM (gray bar) A3A (WT and E72Q mutant).
    Figure Legend Snippet: Effect of A3A on HIV-1 RT-catalyzed extension of a (−) SSDNA oligonucleotide. The bar graph shows the percent of DNA extension product without (−) or with (+) RT and/or A3A. The positive control reaction with RT only is shown as a white bar. Reactions with RT contain either 5 µM (black bar) or 10 µM (gray bar) A3A (WT and E72Q mutant).

    Techniques Used: Positive Control, Mutagenesis

    Deamination of dC in ss regions of a 40-bp DNA duplex and the effect of SSB proteins. ( A ) Schematic representation of a series of TBs in a ds nucleic acid. Unpaired bases are located in the center of a 40-bp DNA duplex that contains the TT C A sequence in the ss region of one strand. ( B ) Deaminase assay performed using duplexes (40 bp) containing TBs with different lengths of unpaired bases (1–9 nt). These duplexes were generated by heat annealing the ssDNA substrate (JL913) to oligonucleotides containing 1 nt (JL1088; TB-1), 3 (JL1089; TB-3), 5 (JL1090; TB-5) and 9 (JL1091; TB-9) that are not complementary to the corresponding residues in the other DNA strand. ( C ) Deaminase assay using the ssDNA substrate (JL913; 180 nM) after preincubation with SSB proteins (HIV-1 NC, T4 Gene 32 or E. coli SSB; each protein at 500 nM) for 15 min at 37°C before addition of A3A and incubation for 1 h. Bars: 1, 5 and 9, no proteins (negative control); 2, 6 and 10, A3A only (positive control); 3, 7, 11 and 4, 8, 12, A3A and ssDNA preincubated with 2.5 or 5 µM SSB protein, respectively.
    Figure Legend Snippet: Deamination of dC in ss regions of a 40-bp DNA duplex and the effect of SSB proteins. ( A ) Schematic representation of a series of TBs in a ds nucleic acid. Unpaired bases are located in the center of a 40-bp DNA duplex that contains the TT C A sequence in the ss region of one strand. ( B ) Deaminase assay performed using duplexes (40 bp) containing TBs with different lengths of unpaired bases (1–9 nt). These duplexes were generated by heat annealing the ssDNA substrate (JL913) to oligonucleotides containing 1 nt (JL1088; TB-1), 3 (JL1089; TB-3), 5 (JL1090; TB-5) and 9 (JL1091; TB-9) that are not complementary to the corresponding residues in the other DNA strand. ( C ) Deaminase assay using the ssDNA substrate (JL913; 180 nM) after preincubation with SSB proteins (HIV-1 NC, T4 Gene 32 or E. coli SSB; each protein at 500 nM) for 15 min at 37°C before addition of A3A and incubation for 1 h. Bars: 1, 5 and 9, no proteins (negative control); 2, 6 and 10, A3A only (positive control); 3, 7, 11 and 4, 8, 12, A3A and ssDNA preincubated with 2.5 or 5 µM SSB protein, respectively.

    Techniques Used: Sequencing, Generated, Incubation, Negative Control, Positive Control

    24) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.
    Figure Legend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Techniques Used: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.
    Figure Legend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Techniques Used: Functional Assay, Preserving, Sequencing, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.
    Figure Legend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Techniques Used: Refining, Sequencing

    25) Product Images from "Wnt7a is a novel inducer of β-catenin-independent tumor-suppressive cellular senescence in lung cancer"

    Article Title: Wnt7a is a novel inducer of β-catenin-independent tumor-suppressive cellular senescence in lung cancer

    Journal: Oncogene

    doi: 10.1038/onc.2015.2

    Wnt7a-null mice display reduced cellular senescence. ( a ) Histological sections of the lungs of the indicated genotypes and in the indicated strains were stained for SA-β-galactosidase activity as detailed in the Materials and methods section and are displayed in the figure. The lung sections of wild-type and Wnt7a-null mice showed no differences in the basal staining for SA-β-galactosidase activity. ( b , c ) Lung sections of wild-type ( n =3) and Wnt7a-null mice ( n =3) treated with urethane were stained for SA-β-galactosidase activity as detailed in the Materials and methods section and are displayed in the figure. Lung sections of the Wnt7a-null mice showed reduced staining for SA-β-galactosidase activity in comparison to wild-type littermate controls. ( d ) Lungs of wild-type ( n =3) and Wnt7a-null mice ( n =3) in C57Bl/6J and FVB/NJ strains were harvested, and the cells were stained for Annexin V/propidium iodide double staining followed by flow cytometry. No differences in the percentage of Annexin V-positive cells were observed among wild-type and Wnt7a-null mice. ( e , f ) Representative images of the western blotting analysis for Caspase 3 activity in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( e ) or FVB/NJ ( f ) strains treated with either saline or urethane displayed no detectable cleaved Caspase 3 expression. ( g , h ) Representative images of the western blotting analysis for LC3 expression in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( g ) or FVB/NJ ( h ) strains treated with either saline or urethane displayed no detectable LC3-II band. Scale bar: 100 μm.
    Figure Legend Snippet: Wnt7a-null mice display reduced cellular senescence. ( a ) Histological sections of the lungs of the indicated genotypes and in the indicated strains were stained for SA-β-galactosidase activity as detailed in the Materials and methods section and are displayed in the figure. The lung sections of wild-type and Wnt7a-null mice showed no differences in the basal staining for SA-β-galactosidase activity. ( b , c ) Lung sections of wild-type ( n =3) and Wnt7a-null mice ( n =3) treated with urethane were stained for SA-β-galactosidase activity as detailed in the Materials and methods section and are displayed in the figure. Lung sections of the Wnt7a-null mice showed reduced staining for SA-β-galactosidase activity in comparison to wild-type littermate controls. ( d ) Lungs of wild-type ( n =3) and Wnt7a-null mice ( n =3) in C57Bl/6J and FVB/NJ strains were harvested, and the cells were stained for Annexin V/propidium iodide double staining followed by flow cytometry. No differences in the percentage of Annexin V-positive cells were observed among wild-type and Wnt7a-null mice. ( e , f ) Representative images of the western blotting analysis for Caspase 3 activity in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( e ) or FVB/NJ ( f ) strains treated with either saline or urethane displayed no detectable cleaved Caspase 3 expression. ( g , h ) Representative images of the western blotting analysis for LC3 expression in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( g ) or FVB/NJ ( h ) strains treated with either saline or urethane displayed no detectable LC3-II band. Scale bar: 100 μm.

    Techniques Used: Mouse Assay, Staining, Activity Assay, Double Staining, Flow Cytometry, Cytometry, Western Blot, Expressing

    Wnt7a regulates Rb phosphorylation via inactivation of SKP2. ( a – f ) Representative images of the western blotting analysis for the indicated proteins in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J, or FVB/NJ strains treated with either saline or urethane. ( g ) Human bronchial epithelial cells (Beas2B) were treated with either control siRNA or Wnt7a siRNA for 48 h. The cell lysates were later probed for the indicated proteins, and the representative images are displayed in the figure. ( h – j ) Human lung adenocarcinoma cells (A549 or H358), which are devoid of Wnt7a expression, were stimulated with recombinant Wnt7a for the indicated periods of time. The cells were later analyzed for SA-β-galactosidase activity ( h ) or the expression of indicated senescence markers ( i , j ) as described in the Materials and methods section. Representative blots from three independent highly reproducible experiments are displayed in the figure. For SA-β-galactosidase staining, left panel indicates the number of SA-β-gal-positive cells/field, and representative images are displayed to the right. ** P
    Figure Legend Snippet: Wnt7a regulates Rb phosphorylation via inactivation of SKP2. ( a – f ) Representative images of the western blotting analysis for the indicated proteins in the lung lysates of wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J, or FVB/NJ strains treated with either saline or urethane. ( g ) Human bronchial epithelial cells (Beas2B) were treated with either control siRNA or Wnt7a siRNA for 48 h. The cell lysates were later probed for the indicated proteins, and the representative images are displayed in the figure. ( h – j ) Human lung adenocarcinoma cells (A549 or H358), which are devoid of Wnt7a expression, were stimulated with recombinant Wnt7a for the indicated periods of time. The cells were later analyzed for SA-β-galactosidase activity ( h ) or the expression of indicated senescence markers ( i , j ) as described in the Materials and methods section. Representative blots from three independent highly reproducible experiments are displayed in the figure. For SA-β-galactosidase staining, left panel indicates the number of SA-β-gal-positive cells/field, and representative images are displayed to the right. ** P

    Techniques Used: Western Blot, Mouse Assay, Expressing, Recombinant, Activity Assay, Staining

    Tumor-suppressive effects of Wnt7a are independent of β-catenin signaling. Representative images of the western blotting analysis of β-catenin expression in the lung lysates derived from wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( a ) or FVB/NJ ( b ) strains treated with either saline or urethane. No apparent differences in β-catenin expression were observed among wild-type and Wnt7a-null mice. ( c ) Representative images of the histological sections of mouse lungs derived from Wnt7a +/+ /Axin2 Lacz+/− ( n =3) and Wnt7a −/− /Axin2 Lacz+/− ( n =3) mice stained for the expression of β-galactosidase gene as described in the Materials and methods section. β-Galactosidase expression was similar in the lungs of wild-type and Wnt7a-null mice carrying Axin2 Lacz+/− allele. Human lung adenocarcinoma cells (A549, d ) and human bronchioalveolar carcinoma cell (H358, e ) that are devoid of Wnt7a expression were stimulated with recombinant Wnt7a for the indicated periods of time. The cells were later lysed, and β-catenin expression was determined by probing the blots with anti-β-catenin antibodies. Representative blots from three independent highly reproducible experiments are displayed in the figure. Scale bar: 100 μm.
    Figure Legend Snippet: Tumor-suppressive effects of Wnt7a are independent of β-catenin signaling. Representative images of the western blotting analysis of β-catenin expression in the lung lysates derived from wild-type ( n =5) and Wnt7a-null mice ( n =5) in C57Bl/6J ( a ) or FVB/NJ ( b ) strains treated with either saline or urethane. No apparent differences in β-catenin expression were observed among wild-type and Wnt7a-null mice. ( c ) Representative images of the histological sections of mouse lungs derived from Wnt7a +/+ /Axin2 Lacz+/− ( n =3) and Wnt7a −/− /Axin2 Lacz+/− ( n =3) mice stained for the expression of β-galactosidase gene as described in the Materials and methods section. β-Galactosidase expression was similar in the lungs of wild-type and Wnt7a-null mice carrying Axin2 Lacz+/− allele. Human lung adenocarcinoma cells (A549, d ) and human bronchioalveolar carcinoma cell (H358, e ) that are devoid of Wnt7a expression were stimulated with recombinant Wnt7a for the indicated periods of time. The cells were later lysed, and β-catenin expression was determined by probing the blots with anti-β-catenin antibodies. Representative blots from three independent highly reproducible experiments are displayed in the figure. Scale bar: 100 μm.

    Techniques Used: Western Blot, Expressing, Derivative Assay, Mouse Assay, Staining, Recombinant

    Iloprost stimulates cellular senescence. Human lung adenocarcinoma cells (A549), which are devoid of Wnt7a expression, were stimulated with Ilprost (20 μ M ) for the indicated periods of time either in the presence or absence of recombinant human sFRP1 (0.5 μg/ml). The cells were later analyzed for SA-β-galactosidase activity ( a ) or the expression of the indicated senescence markers ( b ) as described in the Materials and methods section. Representative blots from three independent highly reproducible experiments are displayed in the figure. For SA-β-galactosidase staining, left panel indicates the number of SA-β-gal-positive cells/field and the representative images are displayed in the right panel. ** P
    Figure Legend Snippet: Iloprost stimulates cellular senescence. Human lung adenocarcinoma cells (A549), which are devoid of Wnt7a expression, were stimulated with Ilprost (20 μ M ) for the indicated periods of time either in the presence or absence of recombinant human sFRP1 (0.5 μg/ml). The cells were later analyzed for SA-β-galactosidase activity ( a ) or the expression of the indicated senescence markers ( b ) as described in the Materials and methods section. Representative blots from three independent highly reproducible experiments are displayed in the figure. For SA-β-galactosidase staining, left panel indicates the number of SA-β-gal-positive cells/field and the representative images are displayed in the right panel. ** P

    Techniques Used: Expressing, Recombinant, Activity Assay, Staining

    Lung-specific gene expression in wild-type and Wnt7a-null mice. ( a ) Total RNA isolated from urethane-treated wild-type ( n =3) and Wnt7a-null mice ( n =3) in C57Bl/6J strain were subjected to transcriptome analysis using RNA-seq. Genes that showed a false discovery rate of
    Figure Legend Snippet: Lung-specific gene expression in wild-type and Wnt7a-null mice. ( a ) Total RNA isolated from urethane-treated wild-type ( n =3) and Wnt7a-null mice ( n =3) in C57Bl/6J strain were subjected to transcriptome analysis using RNA-seq. Genes that showed a false discovery rate of

    Techniques Used: Expressing, Mouse Assay, Isolation, RNA Sequencing Assay

    Loss of Wnt7a leads to decrease in lung epithelial cell markers and increased lung tumorigenesis. Representative images of the western blotting analysis of Wnt7a expression in lung lysates derived from wild-type and Wnt7a-null mice either in C57Bl/6J ( a ), or FVB/NJ ( c ) strains. ( b , d , e ) Representative images of the histological sections of the lungs of the indicated genotypes that were stained with hematoxylin and eosin (H E) stain displaying no gross differences in the lung architectures among Wnt7a-null mice and wild-type littermate controls. Scale bar: 100 μm. ( f ) Representative western blotting images of the lung lysates of wild-type ( n =3) and Wnt7a-null mice ( n =3) displaying reduced expression of epithelial cell marker (E-cadherin) and increased expression of mesenchymal cell marker (N-cadherin) in the Wnt7a-null mice. ( g ) Histological sections of the lungs were fixed and stained with either E-cadherin or N-cadherin antibodies, and the expression of E-cadherin and N-cadherin were visualized by indirect immunofluorescence and confocal microscopy. Representative images of Wnt7a-null mice and wild-type littermate controls are displayed in the figure. Scale bar: 10 μm. ( h , i ) Wild-type and Wnt7a-null mice in C57Bl/6J ( h ) or FVB/NJ ( i ) strains were given urethane as described in the Materials and methods section. The mice were later euthanized and dissected to assess lung tumorigenesis. Box plots display the number of lung tumors developed in the wild-type and Wnt7a-null mice in response to urethane. Statistical significance was determined using Mann–Whitney non-parametric test. ( j ) Representative images of the lung tumors from wild-type and Wnt7a-null mice. ( k , l ) Representative images of the histological sections of the lung tissues with tumors stained with H E as detailed in the Materials and methods section. The edges of the tumors were identified with a broken line. Although wild-type mice developed adenomas with soft borders, majority of Wnt7a-null mice developed adenocarcinomas, as evidenced by spiculated borders. Scale bar: 100 μm. ( m ) Wnt7a expression was lost in the majority of NSCLCs as determined from the Oncomine database search.
    Figure Legend Snippet: Loss of Wnt7a leads to decrease in lung epithelial cell markers and increased lung tumorigenesis. Representative images of the western blotting analysis of Wnt7a expression in lung lysates derived from wild-type and Wnt7a-null mice either in C57Bl/6J ( a ), or FVB/NJ ( c ) strains. ( b , d , e ) Representative images of the histological sections of the lungs of the indicated genotypes that were stained with hematoxylin and eosin (H E) stain displaying no gross differences in the lung architectures among Wnt7a-null mice and wild-type littermate controls. Scale bar: 100 μm. ( f ) Representative western blotting images of the lung lysates of wild-type ( n =3) and Wnt7a-null mice ( n =3) displaying reduced expression of epithelial cell marker (E-cadherin) and increased expression of mesenchymal cell marker (N-cadherin) in the Wnt7a-null mice. ( g ) Histological sections of the lungs were fixed and stained with either E-cadherin or N-cadherin antibodies, and the expression of E-cadherin and N-cadherin were visualized by indirect immunofluorescence and confocal microscopy. Representative images of Wnt7a-null mice and wild-type littermate controls are displayed in the figure. Scale bar: 10 μm. ( h , i ) Wild-type and Wnt7a-null mice in C57Bl/6J ( h ) or FVB/NJ ( i ) strains were given urethane as described in the Materials and methods section. The mice were later euthanized and dissected to assess lung tumorigenesis. Box plots display the number of lung tumors developed in the wild-type and Wnt7a-null mice in response to urethane. Statistical significance was determined using Mann–Whitney non-parametric test. ( j ) Representative images of the lung tumors from wild-type and Wnt7a-null mice. ( k , l ) Representative images of the histological sections of the lung tissues with tumors stained with H E as detailed in the Materials and methods section. The edges of the tumors were identified with a broken line. Although wild-type mice developed adenomas with soft borders, majority of Wnt7a-null mice developed adenocarcinomas, as evidenced by spiculated borders. Scale bar: 100 μm. ( m ) Wnt7a expression was lost in the majority of NSCLCs as determined from the Oncomine database search.

    Techniques Used: Western Blot, Expressing, Derivative Assay, Mouse Assay, Staining, H&E Stain, Marker, Immunofluorescence, Confocal Microscopy, MANN-WHITNEY

    Wnt7a regulates SASP. Equal amounts of protein lysates of wild-type and Wnt7a-null mice in FVB/NJ strain were probed against a mouse cytokine array ( a ) (ARY006, R D) to profile the relative levels of select cytokines and chemokines in wild-type and Wnt7a-null mice as per the manufacturer's recommendations. A representative image of the cytokine array identifying differentially expressed cytokines was displayed in the image. ( b , c ). Total RNA isolated from wild-type ( n =3) and Wnt7a-null mice ( n =3) were used to generate cDNAs. The cDNAs were later used to determine the expression of IL1α ( b ) and IL6 ( c ) via quantitative PCR. The expression of IL1α and IL6 were normalized to the expression of 18S ribosomal RNA and are displayed in the figure. ## P
    Figure Legend Snippet: Wnt7a regulates SASP. Equal amounts of protein lysates of wild-type and Wnt7a-null mice in FVB/NJ strain were probed against a mouse cytokine array ( a ) (ARY006, R D) to profile the relative levels of select cytokines and chemokines in wild-type and Wnt7a-null mice as per the manufacturer's recommendations. A representative image of the cytokine array identifying differentially expressed cytokines was displayed in the image. ( b , c ). Total RNA isolated from wild-type ( n =3) and Wnt7a-null mice ( n =3) were used to generate cDNAs. The cDNAs were later used to determine the expression of IL1α ( b ) and IL6 ( c ) via quantitative PCR. The expression of IL1α and IL6 were normalized to the expression of 18S ribosomal RNA and are displayed in the figure. ## P

    Techniques Used: Mouse Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction

    26) Product Images from "CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen"

    Article Title: CD33 recruitment inhibits IgE-mediated anaphylaxis and desensitizes mast cells to allergen

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI125456

    Antigenic liposomes with CD33L desensitize CD33-Tg mice to antigen challenge. ( A ) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). ( B ) Changes in rectal temperature induced by treatment or the challenges indicated in A . ( C ) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). ( D ) Rectal temperature induced by the treatment or challenge illustrated in C . ( B and D ) Values are plotted as the mean ± SEM. ( E ) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. ( F ) Frequencies of mast cells from peritoneal fluid from mice treated in E . Mast cell frequencies were determined by c-Kit + CD45 + PI – cells. ( G ) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C . ( H ) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G . The background was determined using untreated cells from a naive mouse. ( I ) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 ( F – H ) or 3 ( I ) independent experiments. ** P
    Figure Legend Snippet: Antigenic liposomes with CD33L desensitize CD33-Tg mice to antigen challenge. ( A ) Injection scheme for desensitization to TNP. CD33-Tg mice were used in the TNP-LP-CD33L–treated group (red). Both CD33-Tg and control-Tg mice were used in the 2 untreated groups (black, gray). ( B ) Changes in rectal temperature induced by treatment or the challenges indicated in A . ( C ) Injection scheme to determine antigen specificity of desensitization. CD33-Tg mice were used in the OVA-LP-CD33L–treated group (red circles and squares). Both CD33-Tg and control-Tg mice were used in the untreated group (gray circles and squares). ( D ) Rectal temperature induced by the treatment or challenge illustrated in C . ( B and D ) Values are plotted as the mean ± SEM. ( E ) Injection scheme used to evaluate the impact of TNP-LP-CD33L on mast cell frequency and anti–TNP-IgE on mast cells. Control mice received 200 μl PBS. ( F ) Frequencies of mast cells from peritoneal fluid from mice treated in E . Mast cell frequencies were determined by c-Kit + CD45 + PI – cells. ( G ) In vitro binding of fluorescent TNP-LP (20 μM) to peritoneal mast cells harvested from mice treated as illustrated in C . ( H ) MFI of fluorescent TNP-LP binding to peritoneal mast cells quantified in G . The background was determined using untreated cells from a naive mouse. ( I ) Serum anti–TNP-IgE quantified prior to and 6 hours and 24 hours after treatment with TNP-LP-CD33L (450 μg) using CD33-Tg mice sensitized with 10 μg anti–TNP-IgE. Control mice received 200 μl PBS. Data in B were compiled from 2 experiments. Data are representative of 2 ( F – H ) or 3 ( I ) independent experiments. ** P

    Techniques Used: Mouse Assay, Injection, In Vitro, Binding Assay

    Display of CD33L on antigenic liposomes suppresses IgE-dependent degranulation of LAD2 cells. ( A ) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). ( B ) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. ( C ) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). ( D ) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. ( E ) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release ( n = 3 per condition; values are plotted as the mean ± SD). ( F ) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). ( G ) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. ( H ) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). ( I ) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). ( J ) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E – J are representative of 3 independent experiments. *** P
    Figure Legend Snippet: Display of CD33L on antigenic liposomes suppresses IgE-dependent degranulation of LAD2 cells. ( A ) Schematic representation of an antigenic liposome (TNP-LP, left) or an antigenic liposome displaying human CD33 ligands (TNP-LP-CD33L, right). ( B ) Antibody staining of various Siglecs (Sig-) on LAD2 cells analyzed by flow cytometry. ( C ) Flow cytometric analysis of binding of fluorescent liposomes with or without CD33L (20 μM) to LAD2 cells pretreated with isotype control or anti-CD33 (clone WM53). ( D ) Calcium flux of LAD2 cells induced by addition (arrow) of TNP-LP or TNP-LP-CD33 (2.5 μM) or PBS (1 μl). Graph shows quantification of the AUC of calcium flux induced by 2.5 μM TNP-LP or TNP-LP-CD33L. Results were combined from 2 independent experiments. ( E ) Degranulation induced by TNP-LP or TNP-LP-CD33L as measured by the percentage of β-hex release ( n = 3 per condition; values are plotted as the mean ± SD). ( F ) Degranulation induced by TNP-LP (30 μM), TNP-LP-CD33L (30 μM), or a mixture of TNP-LP and LP-CD33L (30 μM each). ( G ) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of LP-CD33L (10 μM). Control cells received buffer only. ( H ) Degranulation induced by TNP-LP or TNP-LP-CD33L (30 μM) in the presence of isotype or anti-CD33 (clone WM53, 1 μg/ml). ( I ) Degranulation induced by Ah2-LP or Ah2-LP-CD33L (30 μM), with final Ah2 at 750 ng/ml using LAD2 cells sensitized with atopic plasma reactive to peanut (PlasmaLab). ( J ) Degranulation induced by OVA-LP or OVA-LP-CD33L (30 μM), with the final OVA dose at 1.5 μg/ml using LAD2 cells sensitized with human anti–OVA-IgE. Results in E – J are representative of 3 independent experiments. *** P

    Techniques Used: Staining, Flow Cytometry, Cytometry, Binding Assay

    27) Product Images from "Diagnostic value of antigenemia assay for cytomegalovirus gastrointestinal disease in immunocompromised patients"

    Article Title: Diagnostic value of antigenemia assay for cytomegalovirus gastrointestinal disease in immunocompromised patients

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v17.i9.1185

    Study design. CMV: Cytomegalovirus; PCR: Polymerase chain reaction.
    Figure Legend Snippet: Study design. CMV: Cytomegalovirus; PCR: Polymerase chain reaction.

    Techniques Used: Polymerase Chain Reaction

    Pathological features in cytomegalovirus gastrointestinal disease. A: Large cells with intranuclear inclusions or associated with granular cytoplasmic inclusions (hematoxylin and eosin stain); B: Cytomegalovirus (CMV)-infected cells (arrows) show brown
    Figure Legend Snippet: Pathological features in cytomegalovirus gastrointestinal disease. A: Large cells with intranuclear inclusions or associated with granular cytoplasmic inclusions (hematoxylin and eosin stain); B: Cytomegalovirus (CMV)-infected cells (arrows) show brown

    Techniques Used: H&E Stain, Infection

    Endoscopic features in cytomegalovirus gastrointestinal disease. A: Deep, punched-out ulcer in the esophagus; B: Multiple, shallow ulcers in the gastric antrum; C: Large, deep ulcer in the duodenum; D: Multiple erosions and edematous mucosa with ulcer
    Figure Legend Snippet: Endoscopic features in cytomegalovirus gastrointestinal disease. A: Deep, punched-out ulcer in the esophagus; B: Multiple, shallow ulcers in the gastric antrum; C: Large, deep ulcer in the duodenum; D: Multiple erosions and edematous mucosa with ulcer

    Techniques Used:

    28) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.
    Figure Legend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Techniques Used: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.
    Figure Legend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Techniques Used: Functional Assay, Preserving, Sequencing, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.
    Figure Legend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Techniques Used: Refining, Sequencing

    29) Product Images from "Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration"

    Article Title: Continuous Signal Enhancement for Sensitive Aptamer Affinity Probe Electrophoresis Assay Using Electrokinetic Concentration

    Journal: Analytical chemistry

    doi: 10.1021/ac201307d

    a,b) Electropherogram demonstrating detection of 6.5 pM HIV-1 RT in buffer, c) Dose response curve of anti-HIV-1RT aptamer with HIV-RT spiked in buffer, error bars represent standard error from duplicate experiments, d) Linear relationship in the log-log
    Figure Legend Snippet: a,b) Electropherogram demonstrating detection of 6.5 pM HIV-1 RT in buffer, c) Dose response curve of anti-HIV-1RT aptamer with HIV-RT spiked in buffer, error bars represent standard error from duplicate experiments, d) Linear relationship in the log-log

    Techniques Used:

    30) Product Images from "An Upstream Activator Sequence Regulates the Murine Pgk-1 Promoter and Binds Multiple Nuclear Proteins"

    Article Title: An Upstream Activator Sequence Regulates the Murine Pgk-1 Promoter and Binds Multiple Nuclear Proteins

    Journal: Gene Expression

    doi:

    Diagram of the Pgk-1 promoter. The solid line represents the DNA sequence upstream of the Pgk-1 ). The cross-hatched bars labeled ES, EA, and AS are the probes used for EMSA and DNasel footprinting experiments. The slashed, black, and gray boxes labeled R1, R2, and R3 refer to the regions within the UAS that bind nuclear proteins. The black box labeled R2 was defined by both DNase1 footprinting and EMSA whereas the slashed and gray boxes labeled R1 and R3 were shown to bind protein by EMSA only; no footprints were obtained over these regions. The locations of R1 and R3 were established by EMSA with oligonucleotide probes or with DNA fragments from the promoter region. The distribution of nuclear DNA binding factors is indicated below the promoter diagram where the intensity of the signal is roughly proportional to the size of the bars.
    Figure Legend Snippet: Diagram of the Pgk-1 promoter. The solid line represents the DNA sequence upstream of the Pgk-1 ). The cross-hatched bars labeled ES, EA, and AS are the probes used for EMSA and DNasel footprinting experiments. The slashed, black, and gray boxes labeled R1, R2, and R3 refer to the regions within the UAS that bind nuclear proteins. The black box labeled R2 was defined by both DNase1 footprinting and EMSA whereas the slashed and gray boxes labeled R1 and R3 were shown to bind protein by EMSA only; no footprints were obtained over these regions. The locations of R1 and R3 were established by EMSA with oligonucleotide probes or with DNA fragments from the promoter region. The distribution of nuclear DNA binding factors is indicated below the promoter diagram where the intensity of the signal is roughly proportional to the size of the bars.

    Techniques Used: Sequencing, Labeling, Footprinting, Binding Assay

    31) Product Images from "In vivo gene delivery and expression by bacteriophage lambda vectors"

    Article Title: In vivo gene delivery and expression by bacteriophage lambda vectors

    Journal: Journal of Applied Microbiology

    doi: 10.1111/j.1365-2672.2006.03182.x

    Luciferase (luc)-encoding phage is resistant to DNase I treatment. Mice (eight per group) were injected ID at the tail base with 1 × 10 11 PFU of gpD (luc) phage that were either treated with 10 U of DNase I for 30 min at 37°C prior to injection, or not exposed to DNase I. As a control, mice (four per group) were injected with 1 × 10 11 PFU of matching phage that lacked the luc expression cassette (no luc), which were physically mixed with 5 μ g of lambda luc DNA and then either treated with 10 U of DNase I for 30 min at 37°C prior to injection, or not exposed to DNase I. Twenty-four hours later, luc expression was measured at the tail base site of injection. DNase I treatment had no detectable effect on luc expression levels in mice that were injected with gpD (luc) phage ( P > 0·05, Student's two-tailed t -test). However, the amount of DNase I added was sufficient to degrade 5 μ g of exogenous DNA (compare luc expression levels in the two no luc groups).
    Figure Legend Snippet: Luciferase (luc)-encoding phage is resistant to DNase I treatment. Mice (eight per group) were injected ID at the tail base with 1 × 10 11 PFU of gpD (luc) phage that were either treated with 10 U of DNase I for 30 min at 37°C prior to injection, or not exposed to DNase I. As a control, mice (four per group) were injected with 1 × 10 11 PFU of matching phage that lacked the luc expression cassette (no luc), which were physically mixed with 5 μ g of lambda luc DNA and then either treated with 10 U of DNase I for 30 min at 37°C prior to injection, or not exposed to DNase I. Twenty-four hours later, luc expression was measured at the tail base site of injection. DNase I treatment had no detectable effect on luc expression levels in mice that were injected with gpD (luc) phage ( P > 0·05, Student's two-tailed t -test). However, the amount of DNase I added was sufficient to degrade 5 μ g of exogenous DNA (compare luc expression levels in the two no luc groups).

    Techniques Used: Luciferase, Mouse Assay, Injection, Expressing, Two Tailed Test

    32) Product Images from "G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases"

    Article Title: G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases

    Journal: eLife

    doi: 10.7554/eLife.04871

    A ternary complex of DNase I, G-actin, PP1G and PPP1R15A retains its eIF2a P -directed phosphatase activity. ( A ) UV protein absorbance trace of a PPP1R15A (539–614)-PP1G(7–323)-G-actin and DNase I complex assembled from the bacterially-expressed binary complex, rabbit muscle G-actin and bovine pancreatic DNase I, resolved by size-exclusion chromatography. The indicated fractions from the chromatogram are presented in the Coomassie-stained SDS-PAGE below. The positions of G-actin, PP1, DNase I, and the PPP1R15A peptide are indicated. ( B ) Cartoon representation of a model of the PPP1R15B, PP1G, and G-actin ternary complex with DNase I placed by superimposing the actin and DNase I complex (PDB: 2A41) (  Chereau et al., 2005 ) onto the PPP1R15B, PP1G and G-actin ternary complex (PDB: 4V0U). Note that DNase I is bound to the backside of the ternary complex, facing away from the PP1 active site (arrow). ( C ) Images of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated and dephosphorylated eIF2a (eIF2a P  and eIF2a 0 ) have been resolved. Escalating amounts of G-actin or a complex of G-actin and DNase I (final concentration, 10 nM–1 µM) were added to a reaction containing 25 nM PPP1R15B-MBP and PP1G complex (as in   Figure 3A ) and 2 µM eIF2a P  substrate for 20 min. DOI: http://dx.doi.org/10.7554/eLife.04871.022
    Figure Legend Snippet: A ternary complex of DNase I, G-actin, PP1G and PPP1R15A retains its eIF2a P -directed phosphatase activity. ( A ) UV protein absorbance trace of a PPP1R15A (539–614)-PP1G(7–323)-G-actin and DNase I complex assembled from the bacterially-expressed binary complex, rabbit muscle G-actin and bovine pancreatic DNase I, resolved by size-exclusion chromatography. The indicated fractions from the chromatogram are presented in the Coomassie-stained SDS-PAGE below. The positions of G-actin, PP1, DNase I, and the PPP1R15A peptide are indicated. ( B ) Cartoon representation of a model of the PPP1R15B, PP1G, and G-actin ternary complex with DNase I placed by superimposing the actin and DNase I complex (PDB: 2A41) ( Chereau et al., 2005 ) onto the PPP1R15B, PP1G and G-actin ternary complex (PDB: 4V0U). Note that DNase I is bound to the backside of the ternary complex, facing away from the PP1 active site (arrow). ( C ) Images of Coomassie-stained Phos-Tag SDS-PAGE in which phosphorylated and dephosphorylated eIF2a (eIF2a P and eIF2a 0 ) have been resolved. Escalating amounts of G-actin or a complex of G-actin and DNase I (final concentration, 10 nM–1 µM) were added to a reaction containing 25 nM PPP1R15B-MBP and PP1G complex (as in Figure 3A ) and 2 µM eIF2a P substrate for 20 min. DOI: http://dx.doi.org/10.7554/eLife.04871.022

    Techniques Used: Activity Assay, Size-exclusion Chromatography, Staining, SDS Page, Concentration Assay

    33) Product Images from "Transcriptional regulatory logic of the diurnal cycle in the mouse liver"

    Article Title: Transcriptional regulatory logic of the diurnal cycle in the mouse liver

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001069

    DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.
    Figure Legend Snippet: DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.

    Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Activation Assay

    Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].
    Figure Legend Snippet: Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].

    Techniques Used: Binding Assay

    Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p
    Figure Legend Snippet: Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, DNA Sequencing, Binding Assay, Expressing

    BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.
    Figure Legend Snippet: BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, DNA Sequencing, Mouse Assay

    Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p
    Figure Legend Snippet: Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p

    Techniques Used: Mouse Assay, Western Blot

    Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p
    Figure Legend Snippet: Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p

    Techniques Used: Genome Wide

    34) Product Images from "A rigorous method to enrich for exosomes from brain tissue"

    Article Title: A rigorous method to enrich for exosomes from brain tissue

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2017.1348885

    Schematic of the exosome isolation protocol from solid brain tissue. Fresh frozen (−80°C) human frontal cortex was sliced with a razor blade on ice while frozen to generate 1–2 cm long, 2–3 mm wide sections. The cut sections are dissociated while partially frozen in 75 U/ml of collagenase type 3 in Hibernate-E at 37°C for a total of 20 min. The tissue is returned to ice immediately after incubation and protease and phosphatase inhibitors are added. The tissue is spun at 300 × g for 5 min at 4°C (pellet is used as the brain homogenate + collagenase control), the supernatant transferred to a fresh tube, spun at 2000 × g for 10 min at 4°C, then at 10,000 × g for 30 min at 4°C. The EV-containing supernatant is overlaid on a triple sucrose cushion (0.6 M, 1.3 M, 2.5 M) and ultracentrifuged for 3 h at 180,000 × g to separate vesicles based on density. The top of the gradient is discarded and fractions designated 1, 2 and 3 are collected and the refractive index is measured. Each fraction is further ultracentrifuged at 100,000 × g to pellet the vesicles contained in each fraction. Each preparation is validated by a combination of techniques including electron microscopy and RNA and protein analysis. Note – some tissue samples will not be amenable to this method. Post-mortem delay, storage time and the number of freeze-thaw cycles will negatively impact on tissue quality and result in contamination of the fractions with cellular debris and non-exosome vesicles.
    Figure Legend Snippet: Schematic of the exosome isolation protocol from solid brain tissue. Fresh frozen (−80°C) human frontal cortex was sliced with a razor blade on ice while frozen to generate 1–2 cm long, 2–3 mm wide sections. The cut sections are dissociated while partially frozen in 75 U/ml of collagenase type 3 in Hibernate-E at 37°C for a total of 20 min. The tissue is returned to ice immediately after incubation and protease and phosphatase inhibitors are added. The tissue is spun at 300 × g for 5 min at 4°C (pellet is used as the brain homogenate + collagenase control), the supernatant transferred to a fresh tube, spun at 2000 × g for 10 min at 4°C, then at 10,000 × g for 30 min at 4°C. The EV-containing supernatant is overlaid on a triple sucrose cushion (0.6 M, 1.3 M, 2.5 M) and ultracentrifuged for 3 h at 180,000 × g to separate vesicles based on density. The top of the gradient is discarded and fractions designated 1, 2 and 3 are collected and the refractive index is measured. Each fraction is further ultracentrifuged at 100,000 × g to pellet the vesicles contained in each fraction. Each preparation is validated by a combination of techniques including electron microscopy and RNA and protein analysis. Note – some tissue samples will not be amenable to this method. Post-mortem delay, storage time and the number of freeze-thaw cycles will negatively impact on tissue quality and result in contamination of the fractions with cellular debris and non-exosome vesicles.

    Techniques Used: Isolation, Incubation, Electron Microscopy

    35) Product Images from "Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing"

    Article Title: Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1462654

    Reverse transcription through CMC-modified Ψ. (A) Chemical structures of Ψ and CMC-modified Ψ (CMC-Ψ). (B) Reverse transcription of a synthetic RNA oligo (Oligo Ψa) containing Ψ or CMC-Ψ with AMV RT or HIV RT. RNA oligo sequence: 5′- UACACUCAGXUCGGACUAAAGCUGCUC (X = Ψ or CMC-Ψ). (C) Quantification of Ψ or CMC-Ψ read-through by different reverse transcriptase enzymes under varying divalent cation conditions.
    Figure Legend Snippet: Reverse transcription through CMC-modified Ψ. (A) Chemical structures of Ψ and CMC-modified Ψ (CMC-Ψ). (B) Reverse transcription of a synthetic RNA oligo (Oligo Ψa) containing Ψ or CMC-Ψ with AMV RT or HIV RT. RNA oligo sequence: 5′- UACACUCAGXUCGGACUAAAGCUGCUC (X = Ψ or CMC-Ψ). (C) Quantification of Ψ or CMC-Ψ read-through by different reverse transcriptase enzymes under varying divalent cation conditions.

    Techniques Used: Modification, Sequencing

    36) Product Images from "Therapeutic suppression of pulmonary neutrophilia and allergic airway hyperresponsiveness by an ROR γt inverse agonist"

    Article Title: Therapeutic suppression of pulmonary neutrophilia and allergic airway hyperresponsiveness by an ROR γt inverse agonist

    Journal: JCI Insight

    doi: 10.1172/jci.insight.125528

    Effect of VTP on developing Th17 responses in regional mLNs. ( A ) Timeline for adoptive cell transfer, VTP administration, allergic sensitization, and analysis of cytokine production in cultures of cells from mLNs. ( B ) Cytokine concentrations in OVA-stimulated cultures of LNs from mice sensitized using OVA/LPS (O/L) or PBS (P) and given VTP or vehicle alone (veh). n = 6/group. ( C and D ) Analysis of IL-17A fate-mapping cells in mLNs. ( C ) Gating strategy for Th17 (Tomato + ) cells within a CD4 + gate. ( D ) Total numbers (left) and percentages (right) of Tomato + CD4 + cells in mLNs. n = 6/group. Data shown represent mean ± SEM from single experiments, each representative of 2. * P
    Figure Legend Snippet: Effect of VTP on developing Th17 responses in regional mLNs. ( A ) Timeline for adoptive cell transfer, VTP administration, allergic sensitization, and analysis of cytokine production in cultures of cells from mLNs. ( B ) Cytokine concentrations in OVA-stimulated cultures of LNs from mice sensitized using OVA/LPS (O/L) or PBS (P) and given VTP or vehicle alone (veh). n = 6/group. ( C and D ) Analysis of IL-17A fate-mapping cells in mLNs. ( C ) Gating strategy for Th17 (Tomato + ) cells within a CD4 + gate. ( D ) Total numbers (left) and percentages (right) of Tomato + CD4 + cells in mLNs. n = 6/group. Data shown represent mean ± SEM from single experiments, each representative of 2. * P

    Techniques Used: Mouse Assay

    Effect of VTP given before allergen challenge of previously sensitized mice. ( A ) Timeline for OVA/LPS–mediated sensitization of mice given VTP or the vehicle (veh) alone before OVA challenge. PCLS, precision-cut lung slice. ( B ) Mean concentrations ± SEM of cytokines in BALF at 4 hours after allergen challenge. n = 10–12 mice/group; data are combined from 2 experiments. ( C ) CyTOF analysis of lung cells showing the indicated staining for the following populations: 1, Th17 cells; 2, other CD4 + T cells; 3, CD8 + T cells; 4, TCRγδ + T cells; 5, neutrophils; 6, NK T cells; 7, CD103 + DCs; 8, CD11b + DCs; 9, interstitial macrophages; 10, alveolar macrophages; and 11, B cells. ( D ) Percentages of IL-17 + cells within a Tomato + Th17 gate at 24 hours after challenge. ( E ) Confocal image of a PCLS showing Th17 cells (red), IL-17 (green), IL-17–expressing Th17 cells (yellow), CD11c + antigen-presenting cells (white), and epithelial cells (blue). ( F ) Numbers of cells corresponding to the indicated leukocyte subsets in lung lavages at 48 hours after challenge. Data shown are from a single experiment, representative of 2. n = 6/group. * P
    Figure Legend Snippet: Effect of VTP given before allergen challenge of previously sensitized mice. ( A ) Timeline for OVA/LPS–mediated sensitization of mice given VTP or the vehicle (veh) alone before OVA challenge. PCLS, precision-cut lung slice. ( B ) Mean concentrations ± SEM of cytokines in BALF at 4 hours after allergen challenge. n = 10–12 mice/group; data are combined from 2 experiments. ( C ) CyTOF analysis of lung cells showing the indicated staining for the following populations: 1, Th17 cells; 2, other CD4 + T cells; 3, CD8 + T cells; 4, TCRγδ + T cells; 5, neutrophils; 6, NK T cells; 7, CD103 + DCs; 8, CD11b + DCs; 9, interstitial macrophages; 10, alveolar macrophages; and 11, B cells. ( D ) Percentages of IL-17 + cells within a Tomato + Th17 gate at 24 hours after challenge. ( E ) Confocal image of a PCLS showing Th17 cells (red), IL-17 (green), IL-17–expressing Th17 cells (yellow), CD11c + antigen-presenting cells (white), and epithelial cells (blue). ( F ) Numbers of cells corresponding to the indicated leukocyte subsets in lung lavages at 48 hours after challenge. Data shown are from a single experiment, representative of 2. n = 6/group. * P

    Techniques Used: Mouse Assay, Staining, Expressing

    37) Product Images from "Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment"

    Article Title: Gene signatures of quiescent glioblastoma cells reveal mesenchymal shift and interactions with niche microenvironment

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.03.064

    Tracking cell division with iH2B-GFP reporter identifies quiescent population in GBM organoids. a) Targeting strategy for iH2B-GFP reporter knock-in by CRISPR-assisted homologous recombination into AAVS1 locus (gene symbol PPP1R12C ). SA: splice acceptor; Neo: Neomycin resistance gene; pA: poly-adenylation signal; CAG: CAG promoter; rtTA: reverse tetracycline-controlled transactivator; H2B-GFP: histone2B-green fluorescent protein; tetO: tet operator. b) Principle of doxycyline (Dox)-inducible expression of H2B-GFP. c) Schematic depiction of divisional dilution of H2B-GFP label during -Dox chase period. Quiescent cells retain H2B-GFP label (GFP high ), while proliferative cells dilute the label (GFP low ). d) GBM cell line SD3-iH2B-GFP grown as proliferative culture on 2D laminin-coated dishes. In the presence of doxycycline (+Dox), nuclei are uniformly labeled with H2B-GFP. Cells dilute H2B-GFP label during -Dox chase periods (5, 10, and 20 days shown) by cell division. DAPI is used for nuclear counter staining. e) Flow cytometry analysis of SD3-iH2B-GFP cells grown on 2D laminin for the indicated -Dox chase periods. A small fraction of SD3-iH2B-GFP cells remained GFP-negative even in +Dox conditions (denoted as “[s]”), possibly due to sporadic silencing of transgene. Histograms are normalized on y-axis to modal scale (FlowJo). f) Experimental design for isolation of quiescent GBM cells from 3D GBM organoids. GBM organoids are generated by seeding cells in Matrigel droplets and expanding them as floating cultures. After growth for 2 weeks with +Dox pulse, organoids are chased for 2 or 4 weeks in -Dox conditions. Dissociated cells are separated into GFP high and GFP low populations by FACS. g) Images of GBM organoids in culture dishes, after 2 or 4 week -Dox chase periods. h) Fluorescence images of sections of 3D GBM organoids show a declining number of label-retaining GFP high cells during organoid expansion. i) Immunofluorescence images show absence of proliferation markers Ki67 and phospho-Vimentin (pVim) in GFP high cells (arrows), confirming slow dividing nature of GFP high cells. Notice debris from dead cells accumulates during organoid culture, which is more prominent after 4 week chase. j) Representative FACS results of GBM organoids analyzed after 2 or 4 week -Dox chase. After 2 week -Dox chase, 3·1% of cells remained GFP high ; after 4 week chase, only 0·4% of cells remained GFP high . Three independent experiments (10–12 pooled organoids per experiment) yielded similar results. X-axis in left histograms shows red auto-fluorescence of cells. Histograms are normalized on y-axis to modal scale (FlowJo). Scale bars: 50 μm (d), 10 mm (g), 200 μm (h), 20 μm (i).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
    Figure Legend Snippet: Tracking cell division with iH2B-GFP reporter identifies quiescent population in GBM organoids. a) Targeting strategy for iH2B-GFP reporter knock-in by CRISPR-assisted homologous recombination into AAVS1 locus (gene symbol PPP1R12C ). SA: splice acceptor; Neo: Neomycin resistance gene; pA: poly-adenylation signal; CAG: CAG promoter; rtTA: reverse tetracycline-controlled transactivator; H2B-GFP: histone2B-green fluorescent protein; tetO: tet operator. b) Principle of doxycyline (Dox)-inducible expression of H2B-GFP. c) Schematic depiction of divisional dilution of H2B-GFP label during -Dox chase period. Quiescent cells retain H2B-GFP label (GFP high ), while proliferative cells dilute the label (GFP low ). d) GBM cell line SD3-iH2B-GFP grown as proliferative culture on 2D laminin-coated dishes. In the presence of doxycycline (+Dox), nuclei are uniformly labeled with H2B-GFP. Cells dilute H2B-GFP label during -Dox chase periods (5, 10, and 20 days shown) by cell division. DAPI is used for nuclear counter staining. e) Flow cytometry analysis of SD3-iH2B-GFP cells grown on 2D laminin for the indicated -Dox chase periods. A small fraction of SD3-iH2B-GFP cells remained GFP-negative even in +Dox conditions (denoted as “[s]”), possibly due to sporadic silencing of transgene. Histograms are normalized on y-axis to modal scale (FlowJo). f) Experimental design for isolation of quiescent GBM cells from 3D GBM organoids. GBM organoids are generated by seeding cells in Matrigel droplets and expanding them as floating cultures. After growth for 2 weeks with +Dox pulse, organoids are chased for 2 or 4 weeks in -Dox conditions. Dissociated cells are separated into GFP high and GFP low populations by FACS. g) Images of GBM organoids in culture dishes, after 2 or 4 week -Dox chase periods. h) Fluorescence images of sections of 3D GBM organoids show a declining number of label-retaining GFP high cells during organoid expansion. i) Immunofluorescence images show absence of proliferation markers Ki67 and phospho-Vimentin (pVim) in GFP high cells (arrows), confirming slow dividing nature of GFP high cells. Notice debris from dead cells accumulates during organoid culture, which is more prominent after 4 week chase. j) Representative FACS results of GBM organoids analyzed after 2 or 4 week -Dox chase. After 2 week -Dox chase, 3·1% of cells remained GFP high ; after 4 week chase, only 0·4% of cells remained GFP high . Three independent experiments (10–12 pooled organoids per experiment) yielded similar results. X-axis in left histograms shows red auto-fluorescence of cells. Histograms are normalized on y-axis to modal scale (FlowJo). Scale bars: 50 μm (d), 10 mm (g), 200 μm (h), 20 μm (i).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

    Techniques Used: Knock-In, CRISPR, Homologous Recombination, Expressing, Labeling, Staining, Flow Cytometry, Cytometry, Isolation, Generated, FACS, Fluorescence, Immunofluorescence

    38) Product Images from "Functional amyloid formation by Streptococcus mutans"

    Article Title: Functional amyloid formation by Streptococcus mutans

    Journal: Microbiology

    doi: 10.1099/mic.0.060855-0

    (a) Green birefringence of  S. mutans  extracellular proteins derived from wild-type strain NG8 and P1- and SrtA-deficient mutants. (b) Amyloid material derived from  S. mutans  NG8 culture supernatant treated with formic acid or proteinase K. (c) Green birefringence
    Figure Legend Snippet: (a) Green birefringence of S. mutans extracellular proteins derived from wild-type strain NG8 and P1- and SrtA-deficient mutants. (b) Amyloid material derived from S. mutans NG8 culture supernatant treated with formic acid or proteinase K. (c) Green birefringence

    Techniques Used: Derivative Assay

    39) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    40) Product Images from "Splashing transients of 2D plasmons launched by swift electrons"

    Article Title: Splashing transients of 2D plasmons launched by swift electrons

    Journal: Science Advances

    doi: 10.1126/sciadv.1601192

    Time evolution of the deviation of the electron density from its average value on graphene plane δ n ( r ¯ , t ) when a swift electron penetrates through a graphene monolayer. The electron is located ( A and B ) above graphene, ( C ) at graphene, and ( D to H ) below graphene.
    Figure Legend Snippet: Time evolution of the deviation of the electron density from its average value on graphene plane δ n ( r ¯ , t ) when a swift electron penetrates through a graphene monolayer. The electron is located ( A and B ) above graphene, ( C ) at graphene, and ( D to H ) below graphene.

    Techniques Used:

    Time evolution of magnetic field H φ ( r ¯ , t ) when a swift electron perpendicularly penetrates through a graphene monolayer. The green dashed line represents graphene. The electron is located ( A ) above graphene, ( B ) at graphene, and ( C ) below graphene.
    Figure Legend Snippet: Time evolution of magnetic field H φ ( r ¯ , t ) when a swift electron perpendicularly penetrates through a graphene monolayer. The green dashed line represents graphene. The electron is located ( A ) above graphene, ( B ) at graphene, and ( C ) below graphene.

    Techniques Used:

    Schematic of 2D plasmons launching with a swift electron penetrating through a graphene monolayer. L f 1 and L f 2 are the lengths of the formation zone in the region above and below the graphene layer, respectively.
    Figure Legend Snippet: Schematic of 2D plasmons launching with a swift electron penetrating through a graphene monolayer. L f 1 and L f 2 are the lengths of the formation zone in the region above and below the graphene layer, respectively.

    Techniques Used:

    Energy dissipation during the plasmonic formation time. ( A ) Time evolution of emitted photon energy and the induced field energy [related to the induced field strength ( E κ ¯ ⊥ , ω 1 , 2 ) 2 ]. ( B ) Energy spectra of graphene plasmons by taking t = ∞ in the lossless case and by taking t = L f 2 / v in the lossy case.
    Figure Legend Snippet: Energy dissipation during the plasmonic formation time. ( A ) Time evolution of emitted photon energy and the induced field energy [related to the induced field strength ( E κ ¯ ⊥ , ω 1 , 2 ) 2 ]. ( B ) Energy spectra of graphene plasmons by taking t = ∞ in the lossless case and by taking t = L f 2 / v in the lossy case.

    Techniques Used:

    Related Articles

    In Vitro:

    Article Title: Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis
    Article Snippet: .. Treatment with DNase I eliminated the presence of released DNA, but did not block killing of the larvae by mouse neutrophils and macrophages in vitro. .. This observation suggests that in contrast to human neutrophils and macrophages, mouse cells do not require ET formation in vitro to kill the worms.

    Blocking Assay:

    Article Title: Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis
    Article Snippet: .. Treatment with DNase I eliminated the presence of released DNA, but did not block killing of the larvae by mouse neutrophils and macrophages in vitro. .. This observation suggests that in contrast to human neutrophils and macrophages, mouse cells do not require ET formation in vitro to kill the worms.

    Produced:

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay
    Article Snippet: .. The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0. .. For evaluation of the quality of the assay, Z’ values were calculated using Equation 2: Z\u2019 =\u00a01\u00a0\u2212\u00a0(3SDC +\u00a03SDB )/(MC \u00a0\u2212\u00a0MB ) (2) where M = mean value; SD = standard deviation; C = control; and B = background.

    Concentration Assay:

    Article Title: Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA
    Article Snippet: .. DNase I (Worthington) diluted in DNase I dilution buffer (2.5 mM MgCl2 , 0.5 mM CaCl2 , 10 mM Tris-HCl, pH 7.6, 0.1 mg/ml BSA) to a final concentration of 0.0625 μg/ml was incubated with reaction mixtures for 1 min. ..

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay
    Article Snippet: .. The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0. .. For evaluation of the quality of the assay, Z’ values were calculated using Equation 2: Z\u2019 =\u00a01\u00a0\u2212\u00a0(3SDC +\u00a03SDB )/(MC \u00a0\u2212\u00a0MB ) (2) where M = mean value; SD = standard deviation; C = control; and B = background.

    Incubation:

    Article Title: Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle
    Article Snippet: .. Nuclei were then resuspended in RSB buffer supplemented with 1 mM CaCl2 and incubated with increasing concentrations of DNase I (DPRF; Worthington Biochemical Corporation, Lakewood, NJ) for 10 min at room temperature with gentle agitation. ..

    Article Title: Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA
    Article Snippet: .. DNase I (Worthington) diluted in DNase I dilution buffer (2.5 mM MgCl2 , 0.5 mM CaCl2 , 10 mM Tris-HCl, pH 7.6, 0.1 mg/ml BSA) to a final concentration of 0.0625 μg/ml was incubated with reaction mixtures for 1 min. ..

    other:

    Article Title: Interactions of NBU1 IntN1 and Orf2x Proteins with Attachment Site DNA
    Article Snippet: The region is dA+dT rich, and protection seen from positions +126 to +129 is subtle because DNase I does not cleave DNA efficiently in this region.

    Article Title: Identification of Viral Peptide Fragments for Vaccine Development
    Article Snippet: In the random digestion step, the use of DNase I in the presence of MnCl2 is critical as this protocol will generate DNA fragments of relatively uniform sizes, which facilitates the reassembly step ( 17 , also see Note ).

    Activity Assay:

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay
    Article Snippet: .. The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0. .. For evaluation of the quality of the assay, Z’ values were calculated using Equation 2: Z\u2019 =\u00a01\u00a0\u2212\u00a0(3SDC +\u00a03SDB )/(MC \u00a0\u2212\u00a0MB ) (2) where M = mean value; SD = standard deviation; C = control; and B = background.

    Expressing:

    Article Title: Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle
    Article Snippet: .. Analysis of nuclease sensitivity ( ) of the genomic histone HIST2H4 locus to DNase I reveals changes in chromatin structure that accompany increased histone gene expression in hES cells. ..

    Recombinant:

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay
    Article Snippet: .. The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0. .. For evaluation of the quality of the assay, Z’ values were calculated using Equation 2: Z\u2019 =\u00a01\u00a0\u2212\u00a0(3SDC +\u00a03SDB )/(MC \u00a0\u2212\u00a0MB ) (2) where M = mean value; SD = standard deviation; C = control; and B = background.

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    Worthington Biochemical e coli dna polymerase
    MALDI-TOF mass spectra of <t>SVPDE</t> digests of modified <t>DNA</t> 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.
    E Coli Dna Polymerase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical bfu e
    Analysis of <t>BFU-E</t> in the bone marrow and liver during the recovery from PHZ induced anemia
    Bfu E, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Worthington Biochemical e coli total trna
    CID-based sequencing of the double carbodiimide-tagged <t>RNase</t> T1 digestion product from the anticodon of E. coli <t>tRNA</t> Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with two carbodiimide units (+502 Da, m / z 1506.6). The appearance
    E Coli Total Trna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Journal: Nucleic Acids Research

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

    doi:

    Figure Lengend Snippet: MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Article Snippet: SVPDE and E.coli DNA polymerase I were purchased from Worthington Biochemicals (Lakewood, NJ).

    Techniques: Modification, Sequencing

    Analysis of BFU-E in the bone marrow and liver during the recovery from PHZ induced anemia

    Journal: Experimental hematology

    Article Title: Extramedullary erythropoiesis in the adult liver requires BMP4/Smad5 dependent signaling

    doi: 10.1016/j.exphem.2009.01.004

    Figure Lengend Snippet: Analysis of BFU-E in the bone marrow and liver during the recovery from PHZ induced anemia

    Article Snippet: For the analysis of BFU-E in liver, single cell suspensions of liver cells were generated by treating liver tissue with Type I collagenase (440 ug/ml) (Worthington, Lakewood, NJ).

    Techniques:

    CID-based sequencing of the double carbodiimide-tagged RNase T1 digestion product from the anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with two carbodiimide units (+502 Da, m / z 1506.6). The appearance

    Journal: The Journal of Biological Chemistry

    Article Title: Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF *

    doi: 10.1074/jbc.M116.747865

    Figure Lengend Snippet: CID-based sequencing of the double carbodiimide-tagged RNase T1 digestion product from the anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with two carbodiimide units (+502 Da, m / z 1506.6). The appearance

    Article Snippet: Two micrograms of E. coli tRNATyr I or II (R0383 or R0258, Sigma-Aldrich) or 4 μg of E. coli total tRNA were digested with purified RNase T1 (25 units/μg of RNA) and BAP (0.01 unit/μg of RNA) (Worthington Biochemical Corp.) in 120 m m ammonium acetate, pH 6.5, at 37 °C for 2 h ( , ).

    Techniques: Sequencing, Mass Spectrometry

    LC-MS analysis of the carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . A , total ion chromatogram representing the elution pattern of all the oligonucleotide digestion products detected as anions. B , XIC for m

    Journal: The Journal of Biological Chemistry

    Article Title: Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF *

    doi: 10.1074/jbc.M116.747865

    Figure Lengend Snippet: LC-MS analysis of the carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . A , total ion chromatogram representing the elution pattern of all the oligonucleotide digestion products detected as anions. B , XIC for m

    Article Snippet: Two micrograms of E. coli tRNATyr I or II (R0383 or R0258, Sigma-Aldrich) or 4 μg of E. coli total tRNA were digested with purified RNase T1 (25 units/μg of RNA) and BAP (0.01 unit/μg of RNA) (Worthington Biochemical Corp.) in 120 m m ammonium acetate, pH 6.5, at 37 °C for 2 h ( , ).

    Techniques: Liquid Chromatography with Mass Spectroscopy

    CID-based sequencing of the single carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with one carbodiimide ( m / z 1422.8) tagged at either one of two

    Journal: The Journal of Biological Chemistry

    Article Title: Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF *

    doi: 10.1074/jbc.M116.747865

    Figure Lengend Snippet: CID-based sequencing of the single carbodiimide-tagged RNase T1 digestion product the from anticodon of E. coli tRNA Tyr(QUA) . Shown is the CID MS/MS spectrum of ACU[Q]UA[ms 2 i 6 A]AΨCUG with one carbodiimide ( m / z 1422.8) tagged at either one of two

    Article Snippet: Two micrograms of E. coli tRNATyr I or II (R0383 or R0258, Sigma-Aldrich) or 4 μg of E. coli total tRNA were digested with purified RNase T1 (25 units/μg of RNA) and BAP (0.01 unit/μg of RNA) (Worthington Biochemical Corp.) in 120 m m ammonium acetate, pH 6.5, at 37 °C for 2 h ( , ).

    Techniques: Sequencing, Mass Spectrometry