total rna  (Qiagen)


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    RNeasy Micro Kit
    Description:
    For purification of up to 45 µg total RNA from cell and tissue samples Kit contents Qiagen RNeasy Micro Kit 50 preps 10 to 14L Elution Volume 5mg Sample Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes 50 RNeasy MinElute Spin Columns Collection Tubes 1 5mL and 2mL RNase free DNase I Carrier RNA RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from very small amounts of starting material No phenol chloroform extraction No CsCl gradients no LiCl or ethanol precipitation
    Catalog Number:
    74004
    Price:
    492
    Category:
    RNeasy Micro Kit
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    Structured Review

    Qiagen total rna
    RNeasy Micro Kit
    For purification of up to 45 µg total RNA from cell and tissue samples Kit contents Qiagen RNeasy Micro Kit 50 preps 10 to 14L Elution Volume 5mg Sample Tissue Cells Sample Total RNA Purification Spin Column Format Silica Technology Ideal for Northern Dot and Slot Blotting End point RT PCR Quantitative Real time RT PCR Includes 50 RNeasy MinElute Spin Columns Collection Tubes 1 5mL and 2mL RNase free DNase I Carrier RNA RNase free Reagents and Buffers Benefits Fast procedure delivering high quality total RNA in minutes Ready to use RNA for high performance in any downstream application Consistent RNA yields from very small amounts of starting material No phenol chloroform extraction No CsCl gradients no LiCl or ethanol precipitation
    https://www.bioz.com/result/total rna/product/Qiagen
    Average 99 stars, based on 29687 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-04
    99/100 stars

    Images

    1) Product Images from "T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1"

    Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

    Journal: eLife

    doi: 10.7554/eLife.38314

    Cells in the HAVCR2 high subgroup are in a quiescent cell cycle state. ( A ) Left panel: GSEA analysis shows signaling pathways enriched in the HAVCR2 high and blast subpopulations. Right panel: Percentage of cells in each phase of the cell cycle based on single-cell RNA-seq; ( B ) Intracellular FACS analyses of MYC levels in the HAVCR2 high ,HAVCR2 mid , HAVCR2 low and blast subgroups. Gray line: isotype control.
    Figure Legend Snippet: Cells in the HAVCR2 high subgroup are in a quiescent cell cycle state. ( A ) Left panel: GSEA analysis shows signaling pathways enriched in the HAVCR2 high and blast subpopulations. Right panel: Percentage of cells in each phase of the cell cycle based on single-cell RNA-seq; ( B ) Intracellular FACS analyses of MYC levels in the HAVCR2 high ,HAVCR2 mid , HAVCR2 low and blast subgroups. Gray line: isotype control.

    Techniques Used: RNA Sequencing Assay, FACS

    2) Product Images from "IL-33 promotes the egress of group 2 innate lymphoid cells from the bone marrow"

    Article Title: IL-33 promotes the egress of group 2 innate lymphoid cells from the bone marrow

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20170449

    Allergic airway inflammation induced by the fungal aeroallergen A. alternata promotes increased serum IL-33 and ILC2P egress from the bone marrow. (A) Adult WT mice were treated for four consecutive days with an intranasal challenge of A. alternata extract or PBS vehicle and killed 24 h after the final treatment. (B) Representative gating for ILC2Ps in the bone marrow. (C) The total number of viable bone marrow cells. (D) The total number of FSC-A lo SSC-A lo cells in the bone marrow. (E) ILC2P frequency among live bone marrow cells. (F) The total number of ILC2Ps in the bone marrow. (G) The concentration of IL-33 in the serum as measured by ELISA. (H) The total number of FSC-A lo SSC-A lo cells. (I) ILC2Ps in WT and St2 −/− mice treated for four consecutive days with Alternaria extract. For H and I, PBS-treated WT and St2 −/− mice were normalized to 100%, and Alternaria extract–treated mice are displayed as a percentage of PBS-treated within each genotype (WT and St2 −/− ). Data are combined from two (H and I, n = 7) or three independent experiments (C–G, n = 13–15) or representative of three independent experiments (B) and displayed as the mean ± SEM. *, P
    Figure Legend Snippet: Allergic airway inflammation induced by the fungal aeroallergen A. alternata promotes increased serum IL-33 and ILC2P egress from the bone marrow. (A) Adult WT mice were treated for four consecutive days with an intranasal challenge of A. alternata extract or PBS vehicle and killed 24 h after the final treatment. (B) Representative gating for ILC2Ps in the bone marrow. (C) The total number of viable bone marrow cells. (D) The total number of FSC-A lo SSC-A lo cells in the bone marrow. (E) ILC2P frequency among live bone marrow cells. (F) The total number of ILC2Ps in the bone marrow. (G) The concentration of IL-33 in the serum as measured by ELISA. (H) The total number of FSC-A lo SSC-A lo cells. (I) ILC2Ps in WT and St2 −/− mice treated for four consecutive days with Alternaria extract. For H and I, PBS-treated WT and St2 −/− mice were normalized to 100%, and Alternaria extract–treated mice are displayed as a percentage of PBS-treated within each genotype (WT and St2 −/− ). Data are combined from two (H and I, n = 7) or three independent experiments (C–G, n = 13–15) or representative of three independent experiments (B) and displayed as the mean ± SEM. *, P

    Techniques Used: Mouse Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

    IL-33 negatively regulates CXCR4 to promote efficient egress of ILC2Ps. (A) ILC2Ps were magnetically enriched and FACS-purified from the bone marrow of WT and St2 −/− mice for quantitative RT-PCR of chemokine receptors and signals. Data were normalized to a pooled set of three housekeeping genes, and differential expression was assessed by the ΔΔCt method. Data are shown as the expression level in St2 −/− ILC2Ps compared with WT ILC2Ps and are displayed as a log 2 fold change. (B) Cxcr4 expression by quantitative RT-PCR in purified ILC2Ps from WT and Il33 −/− mice. (C) Purified ILC2Ps from WT mice were treated for 24 h in vitro with IL-2 in combination with varying doses of IL-33 and assessed for CXCR4 expression. (D) Quantification of C. (E) CXCR4 expression in purified ILC2Ps from WT, Il33 −/− , and St2 −/− mice that were treated for 24 h in vitro with 1 ng/ml IL-33. For D and E, IL-2–treated WT, Il33 −/− , and St2 −/− samples are normalized to 100%, and IL-33–treated samples are displayed as a percentage of IL-2–treated samples within each genotype (WT, Il33 −/− , and St2 −/− ). (F) Mean fluorescence intensity (MFI) of CXCR4 on ILC2Ps and the percentage of ILC2Ps expressing CXCR4 in adult female mice that were treated intravenously with 4 µg rIL-33 or vehicle and killed 24 h later. (G) WT and St2 −/− mice were treated intraperitoneally with three doses of AMD3100 (10 mg/kg) or PBS vehicle given every 6 h, and bone marrow was collected for flow cytometric analysis 4 h after the final AMD3100 dose. (H) Total number of ILC2Ps in the bone marrow from adult female WT and St2 −/− mice treated as in G. (I) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P2 WT and St2 −/− mice. (J) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P15 WT and St2 −/− mice. (K) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P15 WT and Il33 −/− mice. (L) Total number of ILC2Ps in the bone marrow of P15 WT and St2 −/− mice treated as in G. Data are combined from two (B, n = 6–8; L, n = 7–11) or three (A, n = 12; H, n = 10–11) independent experiments or are representative of two (E, n = 3; F, n = 4; I, n = 5; K, n = 5–6) or three (C and D, n = 4; J, n = 4–5) independent experiments and displayed as the mean ± SEM. *, P
    Figure Legend Snippet: IL-33 negatively regulates CXCR4 to promote efficient egress of ILC2Ps. (A) ILC2Ps were magnetically enriched and FACS-purified from the bone marrow of WT and St2 −/− mice for quantitative RT-PCR of chemokine receptors and signals. Data were normalized to a pooled set of three housekeeping genes, and differential expression was assessed by the ΔΔCt method. Data are shown as the expression level in St2 −/− ILC2Ps compared with WT ILC2Ps and are displayed as a log 2 fold change. (B) Cxcr4 expression by quantitative RT-PCR in purified ILC2Ps from WT and Il33 −/− mice. (C) Purified ILC2Ps from WT mice were treated for 24 h in vitro with IL-2 in combination with varying doses of IL-33 and assessed for CXCR4 expression. (D) Quantification of C. (E) CXCR4 expression in purified ILC2Ps from WT, Il33 −/− , and St2 −/− mice that were treated for 24 h in vitro with 1 ng/ml IL-33. For D and E, IL-2–treated WT, Il33 −/− , and St2 −/− samples are normalized to 100%, and IL-33–treated samples are displayed as a percentage of IL-2–treated samples within each genotype (WT, Il33 −/− , and St2 −/− ). (F) Mean fluorescence intensity (MFI) of CXCR4 on ILC2Ps and the percentage of ILC2Ps expressing CXCR4 in adult female mice that were treated intravenously with 4 µg rIL-33 or vehicle and killed 24 h later. (G) WT and St2 −/− mice were treated intraperitoneally with three doses of AMD3100 (10 mg/kg) or PBS vehicle given every 6 h, and bone marrow was collected for flow cytometric analysis 4 h after the final AMD3100 dose. (H) Total number of ILC2Ps in the bone marrow from adult female WT and St2 −/− mice treated as in G. (I) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P2 WT and St2 −/− mice. (J) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P15 WT and St2 −/− mice. (K) MFI of CXCR4 on ILC2Ps, and the percentage of ILC2Ps expressing CXCR4 in P15 WT and Il33 −/− mice. (L) Total number of ILC2Ps in the bone marrow of P15 WT and St2 −/− mice treated as in G. Data are combined from two (B, n = 6–8; L, n = 7–11) or three (A, n = 12; H, n = 10–11) independent experiments or are representative of two (E, n = 3; F, n = 4; I, n = 5; K, n = 5–6) or three (C and D, n = 4; J, n = 4–5) independent experiments and displayed as the mean ± SEM. *, P

    Techniques Used: FACS, Purification, Mouse Assay, Quantitative RT-PCR, Expressing, In Vitro, Fluorescence, Flow Cytometry

    Direct intravenous administration of IL-33 decreases ILC2P frequency in the bone marrow. Adult naive WT mice were treated intravenously with rIL-33 or vehicle (0.1% BSA in PBS), and bone marrow was harvested 24 h later for flow cytometric analysis. (A) Representative gating for bone marrow ILC2Ps from mice treated with vehicle or 1 µg rIL-33. (B) The total number of viable bone marrow cells. (C) ILC2P frequency among live bone marrow cells. (D) The total number of ILC2Ps in the bone marrow. (E) The total number of ILC2Ps in the bone marrow of mice treated with vehicle or 0.25, 1, or 4 µg rIL-33 for 24 h. Data are combined from two independent experiments (B–D, n = 8–10; E, n = 7) or representative of two independent experiments (A) and are displayed as the mean ± SEM. *, P
    Figure Legend Snippet: Direct intravenous administration of IL-33 decreases ILC2P frequency in the bone marrow. Adult naive WT mice were treated intravenously with rIL-33 or vehicle (0.1% BSA in PBS), and bone marrow was harvested 24 h later for flow cytometric analysis. (A) Representative gating for bone marrow ILC2Ps from mice treated with vehicle or 1 µg rIL-33. (B) The total number of viable bone marrow cells. (C) ILC2P frequency among live bone marrow cells. (D) The total number of ILC2Ps in the bone marrow. (E) The total number of ILC2Ps in the bone marrow of mice treated with vehicle or 0.25, 1, or 4 µg rIL-33 for 24 h. Data are combined from two independent experiments (B–D, n = 8–10; E, n = 7) or representative of two independent experiments (A) and are displayed as the mean ± SEM. *, P

    Techniques Used: Mouse Assay, Flow Cytometry

    WT and IL-33–deficient ILC2Ps are comparably functional and develop at a similar rate. ILC2Ps from the bone marrow of WT and Il33 −/− mice were enriched by magnetic separation and purified by FACS. Sorted ILC2Ps were stained with the dilution-based proliferation dye CellTrace Violet and cultured in supplemented RPMI medium in the presence of IL-2 (10 ng/ml) ± IL-33 (10 ng/ml) for 5 d. (A) Representative CellTrace Violet dilution peaks. (B) Proliferation index is a measure derived from CellTrace Violet staining for quantifying the mean number of proliferation events undergone by each ILC2P that was initially cultured. (C) Cell counts of ILC2Ps poststimulation. (D and E) IL-5 (D) and IL-13 (E) concentrations in the supernatants as measured by ELISA. Adult naive WT and St2 −/− mice were killed, and bone marrow from one tibia and femur was prepared for flow cytometric analysis. (F–I) The total number of progenitors in the ILC2 lineage was quantified: LMPPs (F), CLPs (G), CHILPs (H), and ILCP (I). (J) WT and St2 −/− mice were treated daily with 1 mg BrdU intraperitoneally for 5 d and harvested 24 h after the final dose. (K) The total number of BrdU + ILC2Ps in the bone marrow from J. Data are combined from two independent experiments (B, n = 9; F–I, n = 8; K, n = 9–10) or representative of two similar experiments (A and C–E, n = 4). Data are displayed as the mean ± SEM. *, P
    Figure Legend Snippet: WT and IL-33–deficient ILC2Ps are comparably functional and develop at a similar rate. ILC2Ps from the bone marrow of WT and Il33 −/− mice were enriched by magnetic separation and purified by FACS. Sorted ILC2Ps were stained with the dilution-based proliferation dye CellTrace Violet and cultured in supplemented RPMI medium in the presence of IL-2 (10 ng/ml) ± IL-33 (10 ng/ml) for 5 d. (A) Representative CellTrace Violet dilution peaks. (B) Proliferation index is a measure derived from CellTrace Violet staining for quantifying the mean number of proliferation events undergone by each ILC2P that was initially cultured. (C) Cell counts of ILC2Ps poststimulation. (D and E) IL-5 (D) and IL-13 (E) concentrations in the supernatants as measured by ELISA. Adult naive WT and St2 −/− mice were killed, and bone marrow from one tibia and femur was prepared for flow cytometric analysis. (F–I) The total number of progenitors in the ILC2 lineage was quantified: LMPPs (F), CLPs (G), CHILPs (H), and ILCP (I). (J) WT and St2 −/− mice were treated daily with 1 mg BrdU intraperitoneally for 5 d and harvested 24 h after the final dose. (K) The total number of BrdU + ILC2Ps in the bone marrow from J. Data are combined from two independent experiments (B, n = 9; F–I, n = 8; K, n = 9–10) or representative of two similar experiments (A and C–E, n = 4). Data are displayed as the mean ± SEM. *, P

    Techniques Used: Functional Assay, Mouse Assay, Purification, FACS, Staining, Cell Culture, Derivative Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    IL-33 regulates ILC2/ILC2P frequencies in the bone marrow and lung of early postnatal mice. Age-matched pups were obtained from timed breeding pairs of WT and St2 −/− mice. (A–D) Representative gating for ILC2s and ILC2Ps in P2 bone marrow (A), P2 lungs (B), P15 bone marrow (C), and P15 lungs (D). (E) The total number of ILC2Ps in the bone marrow and ILC2s in the lungs of P2 mice. (F) The total number of ILC2Ps in the bone marrow and ILC2s in the lungs of P15 mice. Data are representative of two (C and D) or three (A and B) independent experiments or combined from two (F, n = 6–10) or three (E, n = 12–15) independent experiments and displayed as the mean ± SEM. *, P
    Figure Legend Snippet: IL-33 regulates ILC2/ILC2P frequencies in the bone marrow and lung of early postnatal mice. Age-matched pups were obtained from timed breeding pairs of WT and St2 −/− mice. (A–D) Representative gating for ILC2s and ILC2Ps in P2 bone marrow (A), P2 lungs (B), P15 bone marrow (C), and P15 lungs (D). (E) The total number of ILC2Ps in the bone marrow and ILC2s in the lungs of P2 mice. (F) The total number of ILC2Ps in the bone marrow and ILC2s in the lungs of P15 mice. Data are representative of two (C and D) or three (A and B) independent experiments or combined from two (F, n = 6–10) or three (E, n = 12–15) independent experiments and displayed as the mean ± SEM. *, P

    Techniques Used: Mouse Assay

    Deficiency in IL-33 signaling leads to an accumulation of ILC2Ps in the bone marrow. Adult naive WT BALB/c, Il33 −/− , and St2 −/− mice were killed, and bone marrow from one tibia and femur was prepared for flow cytometric analysis. (A) Gating strategy and representative gating of WT, Il33 −/− , and St2 −/− ILC2Ps. ILC2Ps were defined as viable CD45 + FSC-A lo SSC-A lo Lin − IL-25R + CD25 + CD127 + cells. (B) The total number of viable bone marrow cells. (C) ILC2P frequency among live bone marrow cells. (D) The total number of ILC2Ps in the bone marrow. (E) The total number of ILC2Ps in the bone marrow of WT C57BL/6 and Il33 −/− mice on a C57BL/6 background. Data are representative of three independent experiments (A) or combined from two (E, n = 8–9) or three (B-D, n = 10–13) independent experiments and displayed as the mean ± SEM. **, P
    Figure Legend Snippet: Deficiency in IL-33 signaling leads to an accumulation of ILC2Ps in the bone marrow. Adult naive WT BALB/c, Il33 −/− , and St2 −/− mice were killed, and bone marrow from one tibia and femur was prepared for flow cytometric analysis. (A) Gating strategy and representative gating of WT, Il33 −/− , and St2 −/− ILC2Ps. ILC2Ps were defined as viable CD45 + FSC-A lo SSC-A lo Lin − IL-25R + CD25 + CD127 + cells. (B) The total number of viable bone marrow cells. (C) ILC2P frequency among live bone marrow cells. (D) The total number of ILC2Ps in the bone marrow. (E) The total number of ILC2Ps in the bone marrow of WT C57BL/6 and Il33 −/− mice on a C57BL/6 background. Data are representative of three independent experiments (A) or combined from two (E, n = 8–9) or three (B-D, n = 10–13) independent experiments and displayed as the mean ± SEM. **, P

    Techniques Used: Mouse Assay, Flow Cytometry

    Tissue frequencies of ILC2s/ILC2Ps are established by a cell-intrinsic, ST2-dependent mechanism. 6-wk-old heterozygous CD45.1 + CD45.2 + WT mice were lethally irradiated and reconstituted with 10 million cells of a 1:1 mixture of CD45.1 + WT and CD45.2 + St2 −/− total bone marrow cells. (A) Experimental design. (B) ILC2P representative gating and pooled analyses of WT and St2 −/− -derived ILC2Ps displayed as frequencies of donor-derived ILC2Ps. (C–E) ILC2 representative gating and pooled analyses of WT and St2 −/− -derived ILC2s displayed as frequencies of donor-derived ILC2s in the lungs (C), skin (D), and mLNs (E). (F) Bone marrow chimeric mice were treated intravenously with 4 µg rIL-33 or vehicle (0.1% BSA in PBS), and cells were harvested 24 h later. ILC2P total cell numbers in the vehicle-treated mice are normalized to 100% for each genotype (WT or St2 −/− ). ILC2Ps in the rIL-33–treated mice are displayed as the percentage of vehicle-treated mice within their respective genotype. Data are combined from two (D, n = 7; E, n = 5; F, n = 6) or three (B and C, n = 13) independent experiments and displayed as the mean ± SEM. *, P
    Figure Legend Snippet: Tissue frequencies of ILC2s/ILC2Ps are established by a cell-intrinsic, ST2-dependent mechanism. 6-wk-old heterozygous CD45.1 + CD45.2 + WT mice were lethally irradiated and reconstituted with 10 million cells of a 1:1 mixture of CD45.1 + WT and CD45.2 + St2 −/− total bone marrow cells. (A) Experimental design. (B) ILC2P representative gating and pooled analyses of WT and St2 −/− -derived ILC2Ps displayed as frequencies of donor-derived ILC2Ps. (C–E) ILC2 representative gating and pooled analyses of WT and St2 −/− -derived ILC2s displayed as frequencies of donor-derived ILC2s in the lungs (C), skin (D), and mLNs (E). (F) Bone marrow chimeric mice were treated intravenously with 4 µg rIL-33 or vehicle (0.1% BSA in PBS), and cells were harvested 24 h later. ILC2P total cell numbers in the vehicle-treated mice are normalized to 100% for each genotype (WT or St2 −/− ). ILC2Ps in the rIL-33–treated mice are displayed as the percentage of vehicle-treated mice within their respective genotype. Data are combined from two (D, n = 7; E, n = 5; F, n = 6) or three (B and C, n = 13) independent experiments and displayed as the mean ± SEM. *, P

    Techniques Used: Mouse Assay, Irradiation, Derivative Assay

    3) Product Images from "Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling"

    Article Title: Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201511061

    Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2 Y324C . (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-catenin GOF/+ ; Pou5f1 +/+ ) and βcat GOF ( β-catenin GOF/+ ; Pou5f1 Cre-ER/+ ) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcat GOF embryos at different ages. n = 13 Cnt embryos and 15 βcat GOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcat GOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP + PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcat GOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P
    Figure Legend Snippet: Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2 Y324C . (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-catenin GOF/+ ; Pou5f1 +/+ ) and βcat GOF ( β-catenin GOF/+ ; Pou5f1 Cre-ER/+ ) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcat GOF embryos at different ages. n = 13 Cnt embryos and 15 βcat GOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcat GOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP + PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcat GOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P

    Techniques Used: In Vivo, Ex Vivo, In Vitro, Cell Culture

    Ror2 Y324C downstream targets overlap with cell cycle–associated genes. (A) Schematic of experimental design. Single embryos were collected at E9.5, and Oct4-ΔPE-GFP + PGCs were isolated by FACS. RNA was extracted from WT ( Ror2 +/+ ) and Ror2 Y324C PGCs, converted to cDNA, amplified, and hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Age and number of PGCs collected per embryo are shown in the table. (B) Microarray data from Ror2 Y324C PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (dChip). Each dot represents a single probe. Purple dots show genes with a P
    Figure Legend Snippet: Ror2 Y324C downstream targets overlap with cell cycle–associated genes. (A) Schematic of experimental design. Single embryos were collected at E9.5, and Oct4-ΔPE-GFP + PGCs were isolated by FACS. RNA was extracted from WT ( Ror2 +/+ ) and Ror2 Y324C PGCs, converted to cDNA, amplified, and hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Age and number of PGCs collected per embryo are shown in the table. (B) Microarray data from Ror2 Y324C PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (dChip). Each dot represents a single probe. Purple dots show genes with a P

    Techniques Used: Isolation, FACS, Amplification, Microarray

    Increased nuclear β-catenin levels in PGCs during their migratory progression and perturbation in Ror2 Y324C PGCs. (A) Nuclear β-catenin (n-βcat, red) immunofluorescence in transverse histological sections of an E9.75 WT embryo treated with Ficin enzyme to disrupt E-cadherin/β-catenin membrane staining. The hindgut (hg), neural tube (nt), and mesonephric duct (mes) are indicated. Bar, 30 µm. (i) PGCs are identified by expression of Oct4-ΔPE-GFP (green; white arrows in A). Bar, 30 µm. (ii) Pseudocoloring (rainbow) indicates individually selected Oct4-ΔPE-GFP + PGCs for quantitative measurement of n-βcat. (iii) Pseudocoloring (rainbow) indicates DAPI-selected nuclei of all cells in the field ( > 600 counted) used to obtain the mean intensity of n-βcat. (iv) Inset from box in A and i to exemplify differences in n-βcat levels in PGCs (dashed white lines) relative to the mean n-βcat levels in all nuclei in the field. Fold differences in staining intensity are indicated. Bar, 10 µm. (B) Quantification of n-βcat in all E9.5 Ror2 Y324C PGCs relative to WT/het littermates shows an increase in accumulation of n-βcat. Each dot represents a single PGC; bars indicate the mean and boxes denote the middle 50% of data points. n = 100–134 cells from two embryos per group; **, P
    Figure Legend Snippet: Increased nuclear β-catenin levels in PGCs during their migratory progression and perturbation in Ror2 Y324C PGCs. (A) Nuclear β-catenin (n-βcat, red) immunofluorescence in transverse histological sections of an E9.75 WT embryo treated with Ficin enzyme to disrupt E-cadherin/β-catenin membrane staining. The hindgut (hg), neural tube (nt), and mesonephric duct (mes) are indicated. Bar, 30 µm. (i) PGCs are identified by expression of Oct4-ΔPE-GFP (green; white arrows in A). Bar, 30 µm. (ii) Pseudocoloring (rainbow) indicates individually selected Oct4-ΔPE-GFP + PGCs for quantitative measurement of n-βcat. (iii) Pseudocoloring (rainbow) indicates DAPI-selected nuclei of all cells in the field ( > 600 counted) used to obtain the mean intensity of n-βcat. (iv) Inset from box in A and i to exemplify differences in n-βcat levels in PGCs (dashed white lines) relative to the mean n-βcat levels in all nuclei in the field. Fold differences in staining intensity are indicated. Bar, 10 µm. (B) Quantification of n-βcat in all E9.5 Ror2 Y324C PGCs relative to WT/het littermates shows an increase in accumulation of n-βcat. Each dot represents a single PGC; bars indicate the mean and boxes denote the middle 50% of data points. n = 100–134 cells from two embryos per group; **, P

    Techniques Used: Immunofluorescence, Staining, Expressing, Pyrolysis Gas Chromatography

    4) Product Images from "Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway"

    Article Title: Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060518774687

    Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.
    Figure Legend Snippet: Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Mutagenesis, Staining, In Situ Hybridization

    (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P
    Figure Legend Snippet: (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P

    Techniques Used: Staining, Mouse Assay

    (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.
    Figure Legend Snippet: (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

    (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.
    Figure Legend Snippet: (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.

    Techniques Used: Mouse Assay

    5) Product Images from "Differentiation of human‐induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering, et al. Differentiation of human‐induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering"

    Article Title: Differentiation of human‐induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering, et al. Differentiation of human‐induced pluripotent stem cell under flow conditions to mature hepatocytes for liver tissue engineering

    Journal: Journal of Tissue Engineering and Regenerative Medicine

    doi: 10.1002/term.2659

    Expression of different hepatic genes by definitive endoderm (DE) cells cultured and differentiated under different conditions. Data are given for each individual sample to appreciate the variation within each condition and the overlap between the different conditions. Data are presented as Ct values of the respective genes normalized to Ct values of the housekeeping gene CREBBP. Results are from four independent differentiation experiments and seven donors. Due to poor RNA yield, some genes where only analysed in two (P‐gp) or three (CK7, BSEP, and BGP) of the cultures. PCLS = precision‐cut liver slices; PDMS = polydimethylsiloxane; PS = polystyrene [Colour figure can be viewed at http://wileyonlinelibrary.com ]
    Figure Legend Snippet: Expression of different hepatic genes by definitive endoderm (DE) cells cultured and differentiated under different conditions. Data are given for each individual sample to appreciate the variation within each condition and the overlap between the different conditions. Data are presented as Ct values of the respective genes normalized to Ct values of the housekeeping gene CREBBP. Results are from four independent differentiation experiments and seven donors. Due to poor RNA yield, some genes where only analysed in two (P‐gp) or three (CK7, BSEP, and BGP) of the cultures. PCLS = precision‐cut liver slices; PDMS = polydimethylsiloxane; PS = polystyrene [Colour figure can be viewed at http://wileyonlinelibrary.com ]

    Techniques Used: Expressing, Cell Culture

    6) Product Images from "Whole-transcriptome splicing profiling of E7.5 mouse primary germ layers reveals frequent alternative promoter usage during mouse early embryogenesis"

    Article Title: Whole-transcriptome splicing profiling of E7.5 mouse primary germ layers reveals frequent alternative promoter usage during mouse early embryogenesis

    Journal: Biology Open

    doi: 10.1242/bio.032508

    Whole-transcriptome profiling of E7.5 mouse primary germ layers. (A) Scatter plot showing the comparison result of the RNA-seq data with an independent Microarray analysis. (B) Venn diagram of the number of the differentially expressed genes in the three germ layers (at least one RPKM > 5, Fold Change > 2, FDR
    Figure Legend Snippet: Whole-transcriptome profiling of E7.5 mouse primary germ layers. (A) Scatter plot showing the comparison result of the RNA-seq data with an independent Microarray analysis. (B) Venn diagram of the number of the differentially expressed genes in the three germ layers (at least one RPKM > 5, Fold Change > 2, FDR

    Techniques Used: RNA Sequencing Assay, Microarray

    7) Product Images from "CD163+ tumor‐associated macrophage accumulation in breast cancer patients reflects both local differentiation signals and systemic skewing of monocytes"

    Article Title: CD163+ tumor‐associated macrophage accumulation in breast cancer patients reflects both local differentiation signals and systemic skewing of monocytes

    Journal: Clinical & Translational Immunology

    doi: 10.1002/cti2.1108

    High frequency of CD163 + TAMs is correlated with higher risk of relapse in BC patients. CD163 neg/low and CD163 high TAMs within live CD45 + CD11b + HLA‐DR + CD14 + CD64 + cells from BC suspensions were analysed by FACS (a) (control isotype in grey). Dot plots shown are representative of each TAM profile for CD163 expression (low, intermediate and high) among 93 BC patients analysed. (b) Presence of total CD14 + TAMs, CD14 + CD163 neg/low , and CD14 + CD163 high TAMs subsets among total live leucocytes ( n = 93; horizontal bars represent the mean). Values depicted were log(base2)‐transformed from percentages obtained by FACS. (c) One representative May–Grünwald–Giemsa staining for sorted CD163 neg/low and CD163 high TAMs (objective 40x) obtained for one BC patient out of two performed. (d) Different levels of CD163 + TAM infiltration detected in TMAs: 0, low infiltration and 1–2, high infiltration. (e) Analysis of the PFS of the 238 BC patients according to their high (red line, n = 129) or low (blue line, n = 109) level of CD163 + TAM infiltration. (f) Frequency of CD163 + TAMs in patients according to their BC molecular subtype.
    Figure Legend Snippet: High frequency of CD163 + TAMs is correlated with higher risk of relapse in BC patients. CD163 neg/low and CD163 high TAMs within live CD45 + CD11b + HLA‐DR + CD14 + CD64 + cells from BC suspensions were analysed by FACS (a) (control isotype in grey). Dot plots shown are representative of each TAM profile for CD163 expression (low, intermediate and high) among 93 BC patients analysed. (b) Presence of total CD14 + TAMs, CD14 + CD163 neg/low , and CD14 + CD163 high TAMs subsets among total live leucocytes ( n = 93; horizontal bars represent the mean). Values depicted were log(base2)‐transformed from percentages obtained by FACS. (c) One representative May–Grünwald–Giemsa staining for sorted CD163 neg/low and CD163 high TAMs (objective 40x) obtained for one BC patient out of two performed. (d) Different levels of CD163 + TAM infiltration detected in TMAs: 0, low infiltration and 1–2, high infiltration. (e) Analysis of the PFS of the 238 BC patients according to their high (red line, n = 129) or low (blue line, n = 109) level of CD163 + TAM infiltration. (f) Frequency of CD163 + TAMs in patients according to their BC molecular subtype.

    Techniques Used: FACS, Expressing, Transformation Assay, Staining

    8) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    9) Product Images from "Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells"

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0034778

    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Figure Legend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Techniques Used: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    10) Product Images from "Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes"

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017625

    Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.
    Figure Legend Snippet: Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Purification, Hybridization

    11) Product Images from "Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity"

    Article Title: Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2015-0095OC

    Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and
    Figure Legend Snippet: Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and

    Techniques Used:

    12) Product Images from "Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development"

    Article Title: Disrupting the three-dimensional regulatory topology of the Pitx1 locus results in overtly normal development

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.158550

    Pitx1- PDE interaction in mouse E11.5 limb buds. (Top) 4C interaction profiles of the Pitx1 promoter in wild-type forelimb (FL) and hindlimb (HL) are shown in light blue. Reproducible, nominally significant interacting regions as called by FourCSeq are shown below each track. Transcription profiles (RNA-seq) in forelimb and hindlimb are shown in light green, and H3K27ac signal and enriched regions in hindlimb are shown in dark green. The ChIA-PET interaction between Pitx1 between the Pitx1 are shown in orange. The location of the PDE (chr13:56,055,928-56,068,947 in mm9) is shown in red. (Bottom) Pitx1 promoter interaction profile and H3K27ac signal in PDE −/− E11.5 hindlimb.
    Figure Legend Snippet: Pitx1- PDE interaction in mouse E11.5 limb buds. (Top) 4C interaction profiles of the Pitx1 promoter in wild-type forelimb (FL) and hindlimb (HL) are shown in light blue. Reproducible, nominally significant interacting regions as called by FourCSeq are shown below each track. Transcription profiles (RNA-seq) in forelimb and hindlimb are shown in light green, and H3K27ac signal and enriched regions in hindlimb are shown in dark green. The ChIA-PET interaction between Pitx1 between the Pitx1 are shown in orange. The location of the PDE (chr13:56,055,928-56,068,947 in mm9) is shown in red. (Bottom) Pitx1 promoter interaction profile and H3K27ac signal in PDE −/− E11.5 hindlimb.

    Techniques Used: RNA Sequencing Assay, ChIA Pet Assay

    13) Product Images from "Highly efficient therapeutic gene editing of human hematopoietic stem cells"

    Article Title: Highly efficient therapeutic gene editing of human hematopoietic stem cells

    Journal: Nature medicine

    doi: 10.1038/s41591-019-0401-y

    Flow cytometry of CD34 + HSPCs with 24 hours of culture. Cryopreserved G-CSF mobilized CD34 + HSPCs were stained with CD34, CD38, CD90, and CD45RA antibodies (in a ), or stained with Hoechst 33342 and Pyronin Y (in b ) at 0 hours following thaw or after 24 hours in culture with SCF, TPO and FLT3-L. HSPCs were electroporated with RNP with 3x-NLS-SpCas9 with BCL11A enhancer or AAVS1 targeting sgRNA. After 2 hour recovery, cells were stained with CD34, CD38, CD90, and CD45RA or with Hoechst 33342 and Pyronin Y, and sorted according to gates as shown in c - d .
    Figure Legend Snippet: Flow cytometry of CD34 + HSPCs with 24 hours of culture. Cryopreserved G-CSF mobilized CD34 + HSPCs were stained with CD34, CD38, CD90, and CD45RA antibodies (in a ), or stained with Hoechst 33342 and Pyronin Y (in b ) at 0 hours following thaw or after 24 hours in culture with SCF, TPO and FLT3-L. HSPCs were electroporated with RNP with 3x-NLS-SpCas9 with BCL11A enhancer or AAVS1 targeting sgRNA. After 2 hour recovery, cells were stained with CD34, CD38, CD90, and CD45RA or with Hoechst 33342 and Pyronin Y, and sorted according to gates as shown in c - d .

    Techniques Used: Flow Cytometry, Cytometry, Staining

    14) Product Images from "ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice"

    Article Title: ROS-mediated iron overload injures the hematopoiesis of bone marrow by damaging hematopoietic stem/progenitor cells in mice

    Journal: Scientific Reports

    doi: 10.1038/srep10181

    Iron overload activated the NOX4/ROS/P38MAPK signaling pathways.(a–d) The levels of NOX4 and GPX1 mRNA expression are expressed as the means ± SE of fold changes compared with their respective controls. N = 3, ** P
    Figure Legend Snippet: Iron overload activated the NOX4/ROS/P38MAPK signaling pathways.(a–d) The levels of NOX4 and GPX1 mRNA expression are expressed as the means ± SE of fold changes compared with their respective controls. N = 3, ** P

    Techniques Used: Expressing

    15) Product Images from "Ponatinib (AP24534) inhibits MEKK3-KLF signaling and prevents formation and progression of cerebral cavernous malformations"

    Article Title: Ponatinib (AP24534) inhibits MEKK3-KLF signaling and prevents formation and progression of cerebral cavernous malformations

    Journal: Science Advances

    doi: 10.1126/sciadv.aau0731

    Ponatinib normalized MEKK3-induced signaling and endothelium ultrastructure in CCM mouse models. ( A to D ) Gene expression analysis of ponatinib-treated Ccm1 iECKO mice. Ponatinib treatment at P6 normalized the increased expression of Klf2 , Klf4 , eNos , and Id1 in the freshly isolated brain endothelial cells from Ccm1 iECKO mice as analyzed at P8. Error bars are shown as SEM, and significance was determined by one-way ANOVA, n = 6. “**” indicates P
    Figure Legend Snippet: Ponatinib normalized MEKK3-induced signaling and endothelium ultrastructure in CCM mouse models. ( A to D ) Gene expression analysis of ponatinib-treated Ccm1 iECKO mice. Ponatinib treatment at P6 normalized the increased expression of Klf2 , Klf4 , eNos , and Id1 in the freshly isolated brain endothelial cells from Ccm1 iECKO mice as analyzed at P8. Error bars are shown as SEM, and significance was determined by one-way ANOVA, n = 6. “**” indicates P

    Techniques Used: Expressing, Mouse Assay, Isolation

    Ponatinib directly inhibits MEKK3 kinase activity. ( A to C ) Computer modeling demonstrating that ponatinib can bind with the MEKK3 kinase domain in the ATP-binding pocket with hydrogen-bonds and π-π interaction (color) as demonstrated by a ribbon plot with full-length proteins (A) and the ribbon plot (B) and surface plot (C) of the binding pocket. ( D ) Immunoprecipitation (IP) demonstrating robust MEKK3 and MEK5 interactions. Deletion of 66 amino acids at the N terminus (MEKK3-ΔN66) abolishes the interaction. Deletion of 11 amino acids at the N terminus (MEKK3-ΔN11) or kinase-dead MEKK3 (MEKK3-KD) do not affect MEKK3-MEK5 interaction. ( E ) In vitro kinase assays showing changes in MEK5 phosphorylation following treatment with ponatinib. MEKK3-K391A, a kinase-dead mutant, was included as a negative control. The quantification of p-MEK5/MEK5 density ratios was shown between the blots. Results are representative of three independent experiments.
    Figure Legend Snippet: Ponatinib directly inhibits MEKK3 kinase activity. ( A to C ) Computer modeling demonstrating that ponatinib can bind with the MEKK3 kinase domain in the ATP-binding pocket with hydrogen-bonds and π-π interaction (color) as demonstrated by a ribbon plot with full-length proteins (A) and the ribbon plot (B) and surface plot (C) of the binding pocket. ( D ) Immunoprecipitation (IP) demonstrating robust MEKK3 and MEK5 interactions. Deletion of 66 amino acids at the N terminus (MEKK3-ΔN66) abolishes the interaction. Deletion of 11 amino acids at the N terminus (MEKK3-ΔN11) or kinase-dead MEKK3 (MEKK3-KD) do not affect MEKK3-MEK5 interaction. ( E ) In vitro kinase assays showing changes in MEK5 phosphorylation following treatment with ponatinib. MEKK3-K391A, a kinase-dead mutant, was included as a negative control. The quantification of p-MEK5/MEK5 density ratios was shown between the blots. Results are representative of three independent experiments.

    Techniques Used: Activity Assay, Binding Assay, Immunoprecipitation, In Vitro, Mutagenesis, Negative Control

    Ponatinib blocks MEKK3-induced signaling in endothelial cells. ( A to F ) Gene expression analysis of ponatinib-treated HUVECs with siRNA-induced CCM1 (si- CCM1 ) gene knockdown. Ponatinib treatment in HUVECs normalized the increased expression of KLF2 (A), eNOS (B), and AQP1 (C), as well as ADAMTS1 (D), ADAMTS4 (E), and ADAMTS9 (F), following CCM1 knockdown. ( G to H ) Western blotting analysis showing that ponatinib treatment decreased KLF2, eNOS, ADAMTS1, and ADAMTS4 expression. ( I ) Ponatinib treatment decreased the expression levels of ERK5 and p-ERK5. Error bars shown as SEM and significance determined by one-way analysis of variance (ANOVA) for multiple comparisons ( n = 4). “*” indicates P
    Figure Legend Snippet: Ponatinib blocks MEKK3-induced signaling in endothelial cells. ( A to F ) Gene expression analysis of ponatinib-treated HUVECs with siRNA-induced CCM1 (si- CCM1 ) gene knockdown. Ponatinib treatment in HUVECs normalized the increased expression of KLF2 (A), eNOS (B), and AQP1 (C), as well as ADAMTS1 (D), ADAMTS4 (E), and ADAMTS9 (F), following CCM1 knockdown. ( G to H ) Western blotting analysis showing that ponatinib treatment decreased KLF2, eNOS, ADAMTS1, and ADAMTS4 expression. ( I ) Ponatinib treatment decreased the expression levels of ERK5 and p-ERK5. Error bars shown as SEM and significance determined by one-way analysis of variance (ANOVA) for multiple comparisons ( n = 4). “*” indicates P

    Techniques Used: Expressing, Western Blot

    Ponatinib inhibits CCM lesion formation and progression in the CCM1 -deficient CCM model. ( A ) Schematic of experimental design. Neonatal pups at P1 were induced with 4-HT and treated with ponatinib at P6. Brains were collected at P13 for micro–computed tomography (CT) analysis. ( B to G ) Micro-CT imaging of CCM lesions in Ccm1 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H to J ) Quantification of micro-CT analysis shows that ponatinib treatment in the Ccm1 iECKO reduced CCM lesion burden by 72% (H) and total lesions number by 35% (I) compared with that of sham-treated controls. CCM lesions distribution analysis showing decreased number (I) and total volume (J) of medium and large lesions in ponatinib-treated Ccm1 iECKO mice, but the number and collective volume of small lesions did not change. Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P
    Figure Legend Snippet: Ponatinib inhibits CCM lesion formation and progression in the CCM1 -deficient CCM model. ( A ) Schematic of experimental design. Neonatal pups at P1 were induced with 4-HT and treated with ponatinib at P6. Brains were collected at P13 for micro–computed tomography (CT) analysis. ( B to G ) Micro-CT imaging of CCM lesions in Ccm1 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H to J ) Quantification of micro-CT analysis shows that ponatinib treatment in the Ccm1 iECKO reduced CCM lesion burden by 72% (H) and total lesions number by 35% (I) compared with that of sham-treated controls. CCM lesions distribution analysis showing decreased number (I) and total volume (J) of medium and large lesions in ponatinib-treated Ccm1 iECKO mice, but the number and collective volume of small lesions did not change. Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P

    Techniques Used: Micro-CT, Imaging, Mouse Assay

    Ponatinib inhibits CCM lesion formation and progression in the CCM2 -deficient CCM model. ( A ) Schematic of experimental design. ( B to G ) Micro-CT imaging of CCM lesions in the CCM2 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H and I ) Quantification of micro-CT analysis shows that ponatinib treatment reduced CCM lesion burden by 85% (H) and total lesions number by 36% (I) in the Ccm2 iECKO mice compared with that of sham controls. ( J ) CCM lesions distribution analysis showing that the decreased lesion burden was mainly due to the reduction in the number of large lesions. Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P
    Figure Legend Snippet: Ponatinib inhibits CCM lesion formation and progression in the CCM2 -deficient CCM model. ( A ) Schematic of experimental design. ( B to G ) Micro-CT imaging of CCM lesions in the CCM2 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H and I ) Quantification of micro-CT analysis shows that ponatinib treatment reduced CCM lesion burden by 85% (H) and total lesions number by 36% (I) in the Ccm2 iECKO mice compared with that of sham controls. ( J ) CCM lesions distribution analysis showing that the decreased lesion burden was mainly due to the reduction in the number of large lesions. Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P

    Techniques Used: Micro-CT, Imaging, Mouse Assay

    Ponatinib impairs the growth of established CCM lesions in mouse model. ( A ) Schematic of experimental design. Neonatal pups at P1 is induced with 4-HT and treated with ponatinib at P11. Brains and eyes were collected at P22 for micro-CT analysis and retina staining. ( B to G ) Micro-CT imaging of CCM lesions in brain in Ccm1 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H to J ) Quantification of micro-CT analysis shows that ponatinib treatment reduced CCM lesion volume burden by 70% (H) without changes to the total lesion number (I) in Ccm1 iECKO mice compared with that of sham controls. CCM lesion distribution analysis shows decreased number of medium and large lesions in ponatinib-treated Ccm1 iECKO mice, but the number of small lesions increased (I). Collective volume of small and medium lesions did not change, but the collective volume of large lesions is decreased (J). ( K to M ) Isolectin staining of retinal vasculature demonstrates smaller CCM lesions in the ponatinib-treated retina (M) compared with that of sham treatment (K and L). Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P
    Figure Legend Snippet: Ponatinib impairs the growth of established CCM lesions in mouse model. ( A ) Schematic of experimental design. Neonatal pups at P1 is induced with 4-HT and treated with ponatinib at P11. Brains and eyes were collected at P22 for micro-CT analysis and retina staining. ( B to G ) Micro-CT imaging of CCM lesions in brain in Ccm1 iECKO with (E to G) or without (B to D) ponatinib treatment. ( H to J ) Quantification of micro-CT analysis shows that ponatinib treatment reduced CCM lesion volume burden by 70% (H) without changes to the total lesion number (I) in Ccm1 iECKO mice compared with that of sham controls. CCM lesion distribution analysis shows decreased number of medium and large lesions in ponatinib-treated Ccm1 iECKO mice, but the number of small lesions increased (I). Collective volume of small and medium lesions did not change, but the collective volume of large lesions is decreased (J). ( K to M ) Isolectin staining of retinal vasculature demonstrates smaller CCM lesions in the ponatinib-treated retina (M) compared with that of sham treatment (K and L). Error bars are shown as SEM, and significance was determined by Student’s t test. “**” indicates P

    Techniques Used: Micro-CT, Staining, Imaging, Mouse Assay

    16) Product Images from "Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway"

    Article Title: Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060518774687

    (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.
    Figure Legend Snippet: (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

    17) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    18) Product Images from "LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair"

    Article Title: LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair

    Journal: eLife

    doi: 10.7554/eLife.30498

    Lrp1- deficient OLs are sensitive to statin treatment but not to bath applied mevalonate. ( a ) Primary OPCs were isolated by anti-PDGFRα immunopanning from Lrp1 flox/+ and Lrp1 flox/+ ;Olig2-Cre pups and cultured for 3 days in differentiation medium (D3 in DM). Cells were lysed and subjected to immunoblotting with anti-SREBP2 and anti-β-actin. ( b ) Quantification of SREBP2 protein levels in cell lysates of Lrp1 flox/+ (n = 4) and Lrp1 flox/+ ;Olig2-Cre (n = 4) cultures revealed comparable levels. This demonstrates that the presence or absence of the Olig2-Cre allele does not affect SREBP2 levels. ( c ) Cholesterol biosynthetic pathway and site of action of statins (simvastatin), which function as inhibitors of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMG-CoA), the rate controlling enzyme of the mevalonate pathway. ( d ) Timeline in days showing when growth medium (GM) and differentiation medium (DM), either containing simvastatin or mevalonate (M/S) were added to cultures and when cells were harvested (H) for immunofluorescence labeling. ( e ) Immunostaining of control and Lrp1- deficient OL cultures after 5 days in DM treated with vehicle or statin. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( f ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 4), Lrp1 control cultures treated with statin (n = 3), Lrp1 cKO OL cultures treated with vehicle (n = 4), and Lrp1 cKO OL cultures treated with statin (n = 3). ( g ) Immunostaining of control and Lrp1 deficient OL cultures after 5 days in DM treated with vehicle or mevalonate. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( h ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 3), Lrp1 control cultures treated with mevalonate (n = 3), Lrp1 cKO OL cultures treated vehicle (n = 3), and Lrp1 cKO OL cultures treated with mevalonate (n = 3). Results are shown as mean values ± SEM, *p
    Figure Legend Snippet: Lrp1- deficient OLs are sensitive to statin treatment but not to bath applied mevalonate. ( a ) Primary OPCs were isolated by anti-PDGFRα immunopanning from Lrp1 flox/+ and Lrp1 flox/+ ;Olig2-Cre pups and cultured for 3 days in differentiation medium (D3 in DM). Cells were lysed and subjected to immunoblotting with anti-SREBP2 and anti-β-actin. ( b ) Quantification of SREBP2 protein levels in cell lysates of Lrp1 flox/+ (n = 4) and Lrp1 flox/+ ;Olig2-Cre (n = 4) cultures revealed comparable levels. This demonstrates that the presence or absence of the Olig2-Cre allele does not affect SREBP2 levels. ( c ) Cholesterol biosynthetic pathway and site of action of statins (simvastatin), which function as inhibitors of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMG-CoA), the rate controlling enzyme of the mevalonate pathway. ( d ) Timeline in days showing when growth medium (GM) and differentiation medium (DM), either containing simvastatin or mevalonate (M/S) were added to cultures and when cells were harvested (H) for immunofluorescence labeling. ( e ) Immunostaining of control and Lrp1- deficient OL cultures after 5 days in DM treated with vehicle or statin. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( f ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 4), Lrp1 control cultures treated with statin (n = 3), Lrp1 cKO OL cultures treated with vehicle (n = 4), and Lrp1 cKO OL cultures treated with statin (n = 3). ( g ) Immunostaining of control and Lrp1 deficient OL cultures after 5 days in DM treated with vehicle or mevalonate. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( h ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 3), Lrp1 control cultures treated with mevalonate (n = 3), Lrp1 cKO OL cultures treated vehicle (n = 3), and Lrp1 cKO OL cultures treated with mevalonate (n = 3). Results are shown as mean values ± SEM, *p

    Techniques Used: Isolation, Cell Culture, Immunofluorescence, Labeling, Immunostaining

    Gene ontology (GO) analysis of Lrp1- deficient OPCs revealed enrichment of peroxisomal genes. Acutely isolated OPCs from Lrp1 +/+ and Lrp1 flox/flox ;Olig2-Cre mouse pups were subjected to microarray analysis. ( a ) GO structure of biological process module related to peroxisome function. Each box shows the GO term ID, p -value, GO term, and the genes from the input list associated with the GO term. The color of each box shows the level of enrichment for each GO term. Specific GO terms were queried with the Mouse Genome Informatics (MGI) GO browser. p-Values were calculated by Fisher’s exact test. The fold-enrichment was calculated by dividing the ratio of genes that are associated with each GO term from the input list by the ratio of genes that are expected in the database. ( b ) Quantification of relative expression levels of gene products that are associated with specific GO terms listed in ( a ). mRNA was prepared from acutely isolated OPCs of Lrp1 controls (n = 4) and cKO OL (n = 4) pups and analyzed with the Affymetrix mouse gene 2.1 ST array. Differentially regulated gene products include Pex2 (peroxisomal biogenesis factor 2), Pex5l (peroxisomal biogenesis factor five like), Hrasls (hRas-like suppressor), Ptgis (prostaglandin I2 synthase), Mavs (Mitochondrial antiviral signaling), and Stard10 (StAR-related lipid transfer protein 10). ( c ) Immunoblotting of lysates prepared from Lrp1 control and cKO OL OL cultures after 5 days in DM. Representative blots probed with anti-LRP1β, anti-PEX2, and anti-β-actin. ( d ) Quantification of PEX2 in Lrp1 control (n = 3) and cKO OL (n = 3) cultures. Results are shown as mean values ± SEM, *p
    Figure Legend Snippet: Gene ontology (GO) analysis of Lrp1- deficient OPCs revealed enrichment of peroxisomal genes. Acutely isolated OPCs from Lrp1 +/+ and Lrp1 flox/flox ;Olig2-Cre mouse pups were subjected to microarray analysis. ( a ) GO structure of biological process module related to peroxisome function. Each box shows the GO term ID, p -value, GO term, and the genes from the input list associated with the GO term. The color of each box shows the level of enrichment for each GO term. Specific GO terms were queried with the Mouse Genome Informatics (MGI) GO browser. p-Values were calculated by Fisher’s exact test. The fold-enrichment was calculated by dividing the ratio of genes that are associated with each GO term from the input list by the ratio of genes that are expected in the database. ( b ) Quantification of relative expression levels of gene products that are associated with specific GO terms listed in ( a ). mRNA was prepared from acutely isolated OPCs of Lrp1 controls (n = 4) and cKO OL (n = 4) pups and analyzed with the Affymetrix mouse gene 2.1 ST array. Differentially regulated gene products include Pex2 (peroxisomal biogenesis factor 2), Pex5l (peroxisomal biogenesis factor five like), Hrasls (hRas-like suppressor), Ptgis (prostaglandin I2 synthase), Mavs (Mitochondrial antiviral signaling), and Stard10 (StAR-related lipid transfer protein 10). ( c ) Immunoblotting of lysates prepared from Lrp1 control and cKO OL OL cultures after 5 days in DM. Representative blots probed with anti-LRP1β, anti-PEX2, and anti-β-actin. ( d ) Quantification of PEX2 in Lrp1 control (n = 3) and cKO OL (n = 3) cultures. Results are shown as mean values ± SEM, *p

    Techniques Used: Isolation, Microarray, Expressing

    19) Product Images from "LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair"

    Article Title: LRP1 regulates peroxisome biogenesis and cholesterol homeostasis in oligodendrocytes and is required for proper CNS myelin development and repair

    Journal: eLife

    doi: 10.7554/eLife.30498

    Lrp1- deficient OLs are sensitive to statin treatment but not to bath applied mevalonate. ( a ) Primary OPCs were isolated by anti-PDGFRα immunopanning from Lrp1 flox/+ and Lrp1 flox/+ ;Olig2-Cre pups and cultured for 3 days in differentiation medium (D3 in DM). Cells were lysed and subjected to immunoblotting with anti-SREBP2 and anti-β-actin. ( b ) Quantification of SREBP2 protein levels in cell lysates of Lrp1 flox/+ (n = 4) and Lrp1 flox/+ ;Olig2-Cre (n = 4) cultures revealed comparable levels. This demonstrates that the presence or absence of the Olig2-Cre allele does not affect SREBP2 levels. ( c ) Cholesterol biosynthetic pathway and site of action of statins (simvastatin), which function as inhibitors of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMG-CoA), the rate controlling enzyme of the mevalonate pathway. ( d ) Timeline in days showing when growth medium (GM) and differentiation medium (DM), either containing simvastatin or mevalonate (M/S) were added to cultures and when cells were harvested (H) for immunofluorescence labeling. ( e ) Immunostaining of control and Lrp1- deficient OL cultures after 5 days in DM treated with vehicle or statin. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( f ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 4), Lrp1 control cultures treated with statin (n = 3), Lrp1 cKO OL cultures treated with vehicle (n = 4), and Lrp1 cKO OL cultures treated with statin (n = 3). ( g ) Immunostaining of control and Lrp1 deficient OL cultures after 5 days in DM treated with vehicle or mevalonate. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( h ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 3), Lrp1 control cultures treated with mevalonate (n = 3), Lrp1 cKO OL cultures treated vehicle (n = 3), and Lrp1 cKO OL cultures treated with mevalonate (n = 3). Results are shown as mean values ± SEM, *p
    Figure Legend Snippet: Lrp1- deficient OLs are sensitive to statin treatment but not to bath applied mevalonate. ( a ) Primary OPCs were isolated by anti-PDGFRα immunopanning from Lrp1 flox/+ and Lrp1 flox/+ ;Olig2-Cre pups and cultured for 3 days in differentiation medium (D3 in DM). Cells were lysed and subjected to immunoblotting with anti-SREBP2 and anti-β-actin. ( b ) Quantification of SREBP2 protein levels in cell lysates of Lrp1 flox/+ (n = 4) and Lrp1 flox/+ ;Olig2-Cre (n = 4) cultures revealed comparable levels. This demonstrates that the presence or absence of the Olig2-Cre allele does not affect SREBP2 levels. ( c ) Cholesterol biosynthetic pathway and site of action of statins (simvastatin), which function as inhibitors of 3-hydroxy-3methyl-glutaryl-coenzyme A reductase (HMG-CoA), the rate controlling enzyme of the mevalonate pathway. ( d ) Timeline in days showing when growth medium (GM) and differentiation medium (DM), either containing simvastatin or mevalonate (M/S) were added to cultures and when cells were harvested (H) for immunofluorescence labeling. ( e ) Immunostaining of control and Lrp1- deficient OL cultures after 5 days in DM treated with vehicle or statin. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( f ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 4), Lrp1 control cultures treated with statin (n = 3), Lrp1 cKO OL cultures treated with vehicle (n = 4), and Lrp1 cKO OL cultures treated with statin (n = 3). ( g ) Immunostaining of control and Lrp1 deficient OL cultures after 5 days in DM treated with vehicle or mevalonate. Cell cultures were labeled with anti-MBP and Hoechst dye33342. Scale bar = 50 µm. ( h ) Quantification of MBP + cells in Lrp1 control cultures treated with vehicle (n = 3), Lrp1 control cultures treated with mevalonate (n = 3), Lrp1 cKO OL cultures treated vehicle (n = 3), and Lrp1 cKO OL cultures treated with mevalonate (n = 3). Results are shown as mean values ± SEM, *p

    Techniques Used: Isolation, Cell Culture, Immunofluorescence, Labeling, Immunostaining

    20) Product Images from "BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice"

    Article Title: BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.150136

    Activation of Klf4 in dental apical mesenchyme explants activates expression of the odontoblast marker Dspp . (A-B′) Ki67 and Klf4 immunostaining (red) of molars from P7.5 Gli1-CreER;Bmpr1 α fl/fl mice induced at P3.5. Boxes in A and B are shown magnified in A′ and B′, respectively. (C) Western blot of Klf4 in cultured dental pulp cells from P7.5 wild-type mice treated with Bmp2 or Bmp4, or mock-treated (control). (D) Co-immunoprecipitation experiment using Flag-tagged Smad1 and HA-tagged Klf4 expressed in 293T cells. Smad1 was immunoprecipitated (IP) and immunoblotted (IB) for association with Klf4. (E,E′) Klf4 immunofluorescence after treatment of dissociated wild-type apical pulp culture for 48 h with Ad-m-Klf4 (E) or ad-GFP (E′). (F) qPCR for Dspp in apical pulp cultures treated with Ad-m-Klf4 (blue bar) compared with Ad-m-GFP (gray bar). n =3. * P
    Figure Legend Snippet: Activation of Klf4 in dental apical mesenchyme explants activates expression of the odontoblast marker Dspp . (A-B′) Ki67 and Klf4 immunostaining (red) of molars from P7.5 Gli1-CreER;Bmpr1 α fl/fl mice induced at P3.5. Boxes in A and B are shown magnified in A′ and B′, respectively. (C) Western blot of Klf4 in cultured dental pulp cells from P7.5 wild-type mice treated with Bmp2 or Bmp4, or mock-treated (control). (D) Co-immunoprecipitation experiment using Flag-tagged Smad1 and HA-tagged Klf4 expressed in 293T cells. Smad1 was immunoprecipitated (IP) and immunoblotted (IB) for association with Klf4. (E,E′) Klf4 immunofluorescence after treatment of dissociated wild-type apical pulp culture for 48 h with Ad-m-Klf4 (E) or ad-GFP (E′). (F) qPCR for Dspp in apical pulp cultures treated with Ad-m-Klf4 (blue bar) compared with Ad-m-GFP (gray bar). n =3. * P

    Techniques Used: Activation Assay, Expressing, Marker, Immunostaining, Mouse Assay, Western Blot, Cell Culture, Immunoprecipitation, Immunofluorescence, Real-time Polymerase Chain Reaction

    21) Product Images from "Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo"

    Article Title: Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191279

    Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid. (A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers ( Sox9 , Otx2 , and Rpe65 ) in three biological replicates was analyzed by RT-qPCR in triplicate using Gapdh , Hprt , and Actb as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers ( Vwf and Col6a1 ) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p
    Figure Legend Snippet: Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid. (A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers ( Sox9 , Otx2 , and Rpe65 ) in three biological replicates was analyzed by RT-qPCR in triplicate using Gapdh , Hprt , and Actb as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers ( Vwf and Col6a1 ) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p

    Techniques Used: Expressing, Quantitative RT-PCR

    P-cadherin is the dominant cadherin in mouse and human RPE in situ . (A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1 , Cdh2 , and Cdh3 in mouse RPE in situ . Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1 , CDH2 , and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).
    Figure Legend Snippet: P-cadherin is the dominant cadherin in mouse and human RPE in situ . (A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1 , Cdh2 , and Cdh3 in mouse RPE in situ . Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1 , CDH2 , and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).

    Techniques Used: In Situ, Mouse Assay, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    22) Product Images from "Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells"

    Article Title: Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.02.056

    Aging Affects Gene Expression in ISCs and Niche (A) Histogram showing the downregulated process in ISCs from aged intestine. (B) Heatmap showing differential expression of Wnt genes in young and aged ISCs and Paneth cells. (C) Wnt3 expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. (D) Wnt3 expression normalized to β Actin transcript levels in young and aged Paneth cells. (E) Wnt3 expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (F) Wnt2b expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (G) β Catenin , Axin 2 , Ascl2 , and Lgr5 expression normalized to β Actin transcript levels in young and aged ISCs. (H and I) Notch1 (H) and Atoh1 (I) expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. All qRT-PCRs were performed on RNA isolated from crypts of the proximal part of mouse small intestine. n = 3–5 mice/experimental group. *p
    Figure Legend Snippet: Aging Affects Gene Expression in ISCs and Niche (A) Histogram showing the downregulated process in ISCs from aged intestine. (B) Heatmap showing differential expression of Wnt genes in young and aged ISCs and Paneth cells. (C) Wnt3 expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. (D) Wnt3 expression normalized to β Actin transcript levels in young and aged Paneth cells. (E) Wnt3 expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (F) Wnt2b expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (G) β Catenin , Axin 2 , Ascl2 , and Lgr5 expression normalized to β Actin transcript levels in young and aged ISCs. (H and I) Notch1 (H) and Atoh1 (I) expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. All qRT-PCRs were performed on RNA isolated from crypts of the proximal part of mouse small intestine. n = 3–5 mice/experimental group. *p

    Techniques Used: Expressing, Isolation, Mouse Assay

    23) Product Images from "Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells"

    Article Title: Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.02.056

    Aging Affects Gene Expression in ISCs and Niche (A) Histogram showing the downregulated process in ISCs from aged intestine. (B) Heatmap showing differential expression of Wnt genes in young and aged ISCs and Paneth cells. (C) Wnt3 expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. (D) Wnt3 expression normalized to β Actin transcript levels in young and aged Paneth cells. (E) Wnt3 expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (F) Wnt2b expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (G) β Catenin , Axin 2 , Ascl2 , and Lgr5 expression normalized to β Actin transcript levels in young and aged ISCs. (H and I) Notch1 (H) and Atoh1 (I) expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. All qRT-PCRs were performed on RNA isolated from crypts of the proximal part of mouse small intestine. n = 3–5 mice/experimental group. *p
    Figure Legend Snippet: Aging Affects Gene Expression in ISCs and Niche (A) Histogram showing the downregulated process in ISCs from aged intestine. (B) Heatmap showing differential expression of Wnt genes in young and aged ISCs and Paneth cells. (C) Wnt3 expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. (D) Wnt3 expression normalized to β Actin transcript levels in young and aged Paneth cells. (E) Wnt3 expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (F) Wnt2b expression normalized to β Actin transcript levels in young and aged mesenchyme of mouse small intestine. (G) β Catenin , Axin 2 , Ascl2 , and Lgr5 expression normalized to β Actin transcript levels in young and aged ISCs. (H and I) Notch1 (H) and Atoh1 (I) expression normalized to β Actin transcript levels in young and aged ISCs of mouse small intestine. All qRT-PCRs were performed on RNA isolated from crypts of the proximal part of mouse small intestine. n = 3–5 mice/experimental group. *p

    Techniques Used: Expressing, Isolation, Mouse Assay

    24) Product Images from "PGC-1α expression in murine AgRP neurons regulates food intake and energy balance"

    Article Title: PGC-1α expression in murine AgRP neurons regulates food intake and energy balance

    Journal: Molecular Metabolism

    doi: 10.1016/j.molmet.2016.05.008

    PGC-1α is required by AgRP but not POMC neurons to control basal metabolism . ( A and B ) Food intake, ( C and D ) oxygen consumption ( E and F ) respiratory exchange ratio and ( G and H ) spontaneous locomotion of AgRP-, POMC-PGC1α KO and Ctr mice (n = 7–8). Values and error bars represent the mean ± SEM. **p
    Figure Legend Snippet: PGC-1α is required by AgRP but not POMC neurons to control basal metabolism . ( A and B ) Food intake, ( C and D ) oxygen consumption ( E and F ) respiratory exchange ratio and ( G and H ) spontaneous locomotion of AgRP-, POMC-PGC1α KO and Ctr mice (n = 7–8). Values and error bars represent the mean ± SEM. **p

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay

    PGC-1α deletion in AgRP neurons reduces energy expenditure . ( A ) Voluntary activity measured by running wheel revolutions in AgRP-PGC1α KO and Ctr mice (n = 5–6). Values represent 2 weeks of measurements. ( B ) Basal body temperature measured during 48-h in AgRP-PGC1α KO and Ctr mice (n = 7–8) in the absence of running wheel. Values and error bars represent the mean ± SEM. *p
    Figure Legend Snippet: PGC-1α deletion in AgRP neurons reduces energy expenditure . ( A ) Voluntary activity measured by running wheel revolutions in AgRP-PGC1α KO and Ctr mice (n = 5–6). Values represent 2 weeks of measurements. ( B ) Basal body temperature measured during 48-h in AgRP-PGC1α KO and Ctr mice (n = 7–8) in the absence of running wheel. Values and error bars represent the mean ± SEM. *p

    Techniques Used: Pyrolysis Gas Chromatography, Activity Assay, Mouse Assay

    PGC-1α deletion in AgRP, but not in POMC neurons promotes fat storage . ( A and B ) Body weight curves (n = 10–14) ( C and D ) body composition (n = 7–8) and ( E and F ) blood triglycerides and glucose levels (n = 5–7) in AgRP-, POMC-PGC1α KO and Ctr mice. Values and error bars represent the mean ± SEM. *p
    Figure Legend Snippet: PGC-1α deletion in AgRP, but not in POMC neurons promotes fat storage . ( A and B ) Body weight curves (n = 10–14) ( C and D ) body composition (n = 7–8) and ( E and F ) blood triglycerides and glucose levels (n = 5–7) in AgRP-, POMC-PGC1α KO and Ctr mice. Values and error bars represent the mean ± SEM. *p

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay

    PGC-1α deletion in AgRP neurons impairs energy homeostasis and hypothalamic signaling in response to fasting . ( A ) Food intake, ( B ) respiratory exchange ratio and ( C ) spontaneous locomotion in AgRP-PGC1α KO and Ctr mice measured with CLAMS during fasting and refeeding (n = 7–8). ( D and E ) AgRP, NPY and POMC mRNA levels in the hypothalamus of AgRP-PGC1α KO and Ctr in fed and overnight fasted mice measured by qPCR (n = 4–8). Data are normalized by mRNA values of fed animals. ( F ) AgRP mRNA level in fed, 4-hours starved and 1-hour refed hypothalamic cells measured by qPCR (n = 3). Values and error bars represent the mean ± SEM. *p
    Figure Legend Snippet: PGC-1α deletion in AgRP neurons impairs energy homeostasis and hypothalamic signaling in response to fasting . ( A ) Food intake, ( B ) respiratory exchange ratio and ( C ) spontaneous locomotion in AgRP-PGC1α KO and Ctr mice measured with CLAMS during fasting and refeeding (n = 7–8). ( D and E ) AgRP, NPY and POMC mRNA levels in the hypothalamus of AgRP-PGC1α KO and Ctr in fed and overnight fasted mice measured by qPCR (n = 4–8). Data are normalized by mRNA values of fed animals. ( F ) AgRP mRNA level in fed, 4-hours starved and 1-hour refed hypothalamic cells measured by qPCR (n = 3). Values and error bars represent the mean ± SEM. *p

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Real-time Polymerase Chain Reaction

    Specific deletion of PGC-1α in the ARC nucleus of AgRP and POMC mice . ( A ) Detection of AgRP-Cre and POMC-Cre expression in punches targeting the ARC nucleus. Genotyping PCR with specific primers was used to detect the presence of the AgRP-cre or POMC-cre allele in isolated hypothalamic region. ( B ) Genotyping PCR using specific primers showing the specific deletion of PGC-1α in the ARC.
    Figure Legend Snippet: Specific deletion of PGC-1α in the ARC nucleus of AgRP and POMC mice . ( A ) Detection of AgRP-Cre and POMC-Cre expression in punches targeting the ARC nucleus. Genotyping PCR with specific primers was used to detect the presence of the AgRP-cre or POMC-cre allele in isolated hypothalamic region. ( B ) Genotyping PCR using specific primers showing the specific deletion of PGC-1α in the ARC.

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Expressing, Polymerase Chain Reaction, Isolation

    PGC-1α deletion in AgRP neurons alters response to leptin . ( A ) Food intake of AgRP-PGC1α KO and Ctr mice 1, 2 and 3 h after ghrelin or vehicle injection (n = 8). ( B and C ) Feeding response and body weight changes of 8 weeks old AgRP-PGC1α KO and Ctr individually housed-mice 16 and 24 h after leptin or vehicle injection (n = 8). A second injection was performed 16 h later. Values and error bars represent the mean ± SEM. *p
    Figure Legend Snippet: PGC-1α deletion in AgRP neurons alters response to leptin . ( A ) Food intake of AgRP-PGC1α KO and Ctr mice 1, 2 and 3 h after ghrelin or vehicle injection (n = 8). ( B and C ) Feeding response and body weight changes of 8 weeks old AgRP-PGC1α KO and Ctr individually housed-mice 16 and 24 h after leptin or vehicle injection (n = 8). A second injection was performed 16 h later. Values and error bars represent the mean ± SEM. *p

    Techniques Used: Pyrolysis Gas Chromatography, Mouse Assay, Injection

    25) Product Images from "Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells *"

    Article Title: Nonantibiotic Effects of Fluoroquinolones in Mammalian Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.671222

    FQ-dependent reduction of HIF-1α does not involve proteasomal or lysosomal degradation. Inhibition of proteasomal (MG132) ( A ) or lysosomal (leupeptin) ( B ) protein degradation does not rescue HIF1α levels in cells treated with CIPRO. C, qRT-PCR analysis of relative HIF-1α mRNA in cells treated with DFO or CIPRO. Data are the means ± S.D. representative of n ≥ 3 independent experiments.
    Figure Legend Snippet: FQ-dependent reduction of HIF-1α does not involve proteasomal or lysosomal degradation. Inhibition of proteasomal (MG132) ( A ) or lysosomal (leupeptin) ( B ) protein degradation does not rescue HIF1α levels in cells treated with CIPRO. C, qRT-PCR analysis of relative HIF-1α mRNA in cells treated with DFO or CIPRO. Data are the means ± S.D. representative of n ≥ 3 independent experiments.

    Techniques Used: Inhibition, Quantitative RT-PCR

    FQs suppress HIF-1α in HEK293 cells. A, relative HIF-1α levels assessed by Western blotting in HEK293 cells with 0.5 m m FQ treatment for 4 h in hypoxia (2% oxygen). B, HIF-1α levels in HEK293 cells treated with CIPRO in hypoxia. HIF-1α status in HEK293 cells with co-treatment of CIPRO and DFO ( C ) or CoCl 2 for 4 h in hypoxia ( D ) is shown. E, HIF-2α levels in HEK293 cells with co-treatment of CIPRO and DEF or CoCl 2 in hypoxia for 4 h. 100 μ m DFO (positive control) or CoCl 2 (positive control) was used in the co-treatment experiments. NT (diluent only).
    Figure Legend Snippet: FQs suppress HIF-1α in HEK293 cells. A, relative HIF-1α levels assessed by Western blotting in HEK293 cells with 0.5 m m FQ treatment for 4 h in hypoxia (2% oxygen). B, HIF-1α levels in HEK293 cells treated with CIPRO in hypoxia. HIF-1α status in HEK293 cells with co-treatment of CIPRO and DFO ( C ) or CoCl 2 for 4 h in hypoxia ( D ) is shown. E, HIF-2α levels in HEK293 cells with co-treatment of CIPRO and DEF or CoCl 2 in hypoxia for 4 h. 100 μ m DFO (positive control) or CoCl 2 (positive control) was used in the co-treatment experiments. NT (diluent only).

    Techniques Used: Western Blot, Positive Control

    HIF-1α mRNA translation is repressed in CIPRO-treated cells. HIF-1α and actin immunoprecipitation after metabolic labeling with [ 35 S]Met. Cell cultures were treated with 1 m m CIPRO or 100 μ m DFO after methionine starvation, and then nascent proteins were radiolabeled and immunoprecipitated for further processing and imaging.
    Figure Legend Snippet: HIF-1α mRNA translation is repressed in CIPRO-treated cells. HIF-1α and actin immunoprecipitation after metabolic labeling with [ 35 S]Met. Cell cultures were treated with 1 m m CIPRO or 100 μ m DFO after methionine starvation, and then nascent proteins were radiolabeled and immunoprecipitated for further processing and imaging.

    Techniques Used: Immunoprecipitation, Labeling, Imaging

    FQs are potent iron chelators. A, iron chelation as determined by CAS competition assay. Iron binding stoichiometries were determined for DFO (positive control) ( B ) and CIPRO with iron at indicated concentrations (higher than K d ) ( C ). Data are representative of n ≥ 3 independent experiments. NT , not treated (diluent only).
    Figure Legend Snippet: FQs are potent iron chelators. A, iron chelation as determined by CAS competition assay. Iron binding stoichiometries were determined for DFO (positive control) ( B ) and CIPRO with iron at indicated concentrations (higher than K d ) ( C ). Data are representative of n ≥ 3 independent experiments. NT , not treated (diluent only).

    Techniques Used: Competitive Binding Assay, Binding Assay, Positive Control

    FQ treatment inhibits collagen proline hydroxylation. HEK293 cells were co-treated with 50 μg/ml ascorbate and either NT, DFO, or CIPRO for 72 h as follows: 0 h, 100 μ m ; 24 h, 150 μ m; and 48 h, 250 μ m . At 72 h, cells were harvested and processed for quantification of hydroxyproline ( HyP ) in total collagen. Similarly, 300 μ m ferric citrate was added to NT, DFO, or CIPRO cell cultures, and hydroxyproline levels were assessed accordingly. Data are normalized to cell number and represent at least three independent experiments. Data are reported as means ± S.D. Statistical analysis was performed using paired t test (compared with NT). Significant difference (***, p ≤ 0.005) is shown. NT (diluent only).
    Figure Legend Snippet: FQ treatment inhibits collagen proline hydroxylation. HEK293 cells were co-treated with 50 μg/ml ascorbate and either NT, DFO, or CIPRO for 72 h as follows: 0 h, 100 μ m ; 24 h, 150 μ m; and 48 h, 250 μ m . At 72 h, cells were harvested and processed for quantification of hydroxyproline ( HyP ) in total collagen. Similarly, 300 μ m ferric citrate was added to NT, DFO, or CIPRO cell cultures, and hydroxyproline levels were assessed accordingly. Data are normalized to cell number and represent at least three independent experiments. Data are reported as means ± S.D. Statistical analysis was performed using paired t test (compared with NT). Significant difference (***, p ≤ 0.005) is shown. NT (diluent only).

    Techniques Used:

    26) Product Images from "A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons"

    Article Title: A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03538-9

    Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01
    Figure Legend Snippet: Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01

    Techniques Used: Purification, Expressing, Mouse Assay, In Situ Hybridization, FACS, RNA Sequencing Assay, Construct, Multiple Displacement Amplification, Two Tailed Test

    27) Product Images from "Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin"

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-04-0228

    Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.
    Figure Legend Snippet: Surface vimentin is localized along the periphery of HMEC-1. (A–D) Cells were stained for vimentin using the rabbit anti-vimentin H-84 antibody. For negative controls, cells were stained with secondary anti-rabbit IgG antibody alone. Representative phase image of cells (left column), image of the nuclei (middle column), and H-84 anti-vimentin antibody fluorescence (right column) are shown for (A) permeabilized HMEC-1 strained for intracellular vimentin; (B) permeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls; (C) nonpermeabilized HMEC-1 stained for surface vimentin; (D) nonpermeabilized HMEC-1 incubated with anti-rabbit IgG alone as negative controls. Scale bar shown in white is 20 μm. White arrows point at the localization of surface vimentin at cell–cell junctions.

    Techniques Used: Staining, Fluorescence, Incubation

    Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (
    Figure Legend Snippet: Infection of HMEC-1 by Lm is in part mediated by InlB in a manner independent of matrix stiffness. (A) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: wild type (circle); Δ inlA (square); Δ inlB (cross); Δ inlF (diamond; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (B) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black circles); Δ actA/ Δ inlB (gray squares; actAp::mTagRFP). The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. (C) Boxplots of percentage of HMEC-1 infected with Lm as a function of substrate stiffness ( N = 5–6 replicates). HMEC-1 were infected with the indicated Lm strains: Δ actA (gray); Δ actA/ Δ inlB (black; actAp::mTagRFP) at an MOI of 20. Infection was analyzed by flow cytometry 7–8 h after infection. Representative data come from one of three independent experiments. One or two asterisks denote statistically significant differences between the medians of two distributions (

    Techniques Used: Infection, Flow Cytometry, Cytometry

    Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
    Figure Legend Snippet: Uptake of Lm by HMEC-1 depends on the stiffness of the matrix on which cells reside. HMEC-1 residing on PA hydrogels of varying stiffness coated with collagen I were infected with Δ actA Lm (actAp::mTagRFP). Infection was analyzed by flow cytometry 7–8 h postinfection. Bacteria were added at a multiplicity of infection (MOI) between 30 and 50 bacteria per host cell. (A–D) Histograms of the logarithm of bacterial fluorescence intensity per cell for HMEC-1 plated on 0.6-kPa (A), 3-kPa (B), 20-kPa (C), and 70-kPa (D) PA hydrogels. Histograms for N = 5 replicates are shown in different colors. The histogram of control uninfected cells is shown in purple. Based on the autofluorescence of the control group, a gate is defined (see black and red lines) showing what is considered uninfected (left, black line) and infected (right, red line). (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm vs. hydrogel stiffness for the data shown in panels A–D. Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Techniques Used: Infection, Flow Cytometry, Cytometry, Fluorescence, MANN-WHITNEY

    Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (
    Figure Legend Snippet: Lm adhesion, but not invasion efficiency, is increased when HMEC-1 reside on stiff hydrogels. HMEC-1 residing on soft (3-kPa) or stiff (70-kPa) PA hydrogels and treated with vehicle control or 2 μM PF537228 FAK inhibitor were infected with Lm (constitutively expressing GFP) at an MOI between 1.5 and 15. At 30 min postinfection, samples were fixed and immunostained, and infection was analyzed by microscopy followed by image processing. Boxplots show (A) total bacteria per cell; (B) internalized bacteria per cell; (C) invasion efficiency (ratio of internalized bacteria to total bacteria); (D) cells in the field of view. Representative data come from one of three independent experiments. N = 800–1000 cells were analyzed for each condition. Two asterisks denote statistically significant differences between the medians of two distributions (

    Techniques Used: Infection, Expressing, Microscopy

    Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).
    Figure Legend Snippet: Lower FAK activity leads to reduced amount of cell surface vimentin. (A, B) 2D-PAGE gels of plasma membrane proteins of HMEC-1 grown on TC polystyrene substrates treated for 1 h with vehicle control (A) or 2 µM PF537228 FAK inhibitor (B). pH increases from left to right. Gels were silver-stained and one isoelectric point marker (tropomyosin), added to each sample as an internal standard, is marked with a black arrow. The one spot that differed consistently between three independent experiments is indicated with a black circle and corresponds to vimentin (55 kDa).

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Marker

    Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (
    Figure Legend Snippet: Blocking HMEC-1 with anti-vimentin antibody reduces Li adhesion onto HMEC-1 but not uptake of beads. (A) Boxplots showing the number of bacteria per cell, for HMEC-1 residing on glass substrates and treated with vehicle control, 2 μM PF537228 FAK inhibitor, or 80 μg/ml H-84 anti-vimentin antibody prior to infection. Cells were infected with Lm or Li at an MOI of 4. At 30 min postinfection, samples were fixed and immunostained and adhesion of bacteria was analyzed by microscopy followed by image processing. For each condition, 2300–2600 cells were analyzed in total and data refer to one of two independent experiments. Two asterisks denote statistically significant differences between the median values of control cells vs. all other groups (

    Techniques Used: Blocking Assay, Infection, Microscopy

    FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
    Figure Legend Snippet: FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Techniques Used: Activity Assay, Western Blot, Expressing, Inhibition, Infection, Concentration Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p
    Figure Legend Snippet: Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p

    Techniques Used: Blocking Assay, Infection, Concentration Assay, Flow Cytometry, Cytometry

    28) Product Images from "Characterization of Lgr6+ Cells as an Enriched Population of Hair Cell Progenitors Compared to Lgr5+ Cells for Hair Cell Generation in the Neonatal Mouse Cochlea"

    Article Title: Characterization of Lgr6+ Cells as an Enriched Population of Hair Cell Progenitors Compared to Lgr5+ Cells for Hair Cell Generation in the Neonatal Mouse Cochlea

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00147

    Re-sort analysis, immunostaining, and q-PCR of flow-sorted Lgr5+ and Lgr6+ cells from the postnatal cochlea. (A) At P3, Lgr5 was expressed in the third row of Deiters’ cells (DC3), the inner pillar cells (IPs), the inner phalangeal cells (IPCs), and the lateral GER, while Lgr6 was only expressed in the IPs. (B) Cryosection showed that Lgr5 was expressed in DC3s, IPs, IPCs and the GER, and Lgr6 was only expressed in a subset of IPs in the P3 organ of Corti. (C) GFP+ cells and GFP– cells were isolated using flow cytometry. Re-sort analysis of GFP+ cells demonstrated > 90% purity. (D) Immunostaining of Lgr5+ cells and Lgr6+ cells from the cochlea showed a high percentage of Sox2+ (95.4% and 95.2%, respectively) and GFP+ (95.8% and 96.6%, respectively) cells, and no Myo7a+ cells, among the sorted cells. (E,F) q-PCR showed that isolated Lgr5+ cells and Lgr6+ cells had significantly higher Lgr5 and Lgr6 expression, slightly higher Sox2 expression, and significantly lower Brn3.1 expression compared to the Lgr5- cells and Lgr6– cells, respectively. Scale bars are 20 μm. ∗∗ p
    Figure Legend Snippet: Re-sort analysis, immunostaining, and q-PCR of flow-sorted Lgr5+ and Lgr6+ cells from the postnatal cochlea. (A) At P3, Lgr5 was expressed in the third row of Deiters’ cells (DC3), the inner pillar cells (IPs), the inner phalangeal cells (IPCs), and the lateral GER, while Lgr6 was only expressed in the IPs. (B) Cryosection showed that Lgr5 was expressed in DC3s, IPs, IPCs and the GER, and Lgr6 was only expressed in a subset of IPs in the P3 organ of Corti. (C) GFP+ cells and GFP– cells were isolated using flow cytometry. Re-sort analysis of GFP+ cells demonstrated > 90% purity. (D) Immunostaining of Lgr5+ cells and Lgr6+ cells from the cochlea showed a high percentage of Sox2+ (95.4% and 95.2%, respectively) and GFP+ (95.8% and 96.6%, respectively) cells, and no Myo7a+ cells, among the sorted cells. (E,F) q-PCR showed that isolated Lgr5+ cells and Lgr6+ cells had significantly higher Lgr5 and Lgr6 expression, slightly higher Sox2 expression, and significantly lower Brn3.1 expression compared to the Lgr5- cells and Lgr6– cells, respectively. Scale bars are 20 μm. ∗∗ p

    Techniques Used: Immunostaining, Polymerase Chain Reaction, Flow Cytometry, Isolation, Cytometry, Expressing

    29) Product Images from "Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo"

    Article Title: Cadherins in the retinal pigment epithelium (RPE) revisited: P-cadherin is the highly dominant cadherin expressed in human and mouse RPE in vivo

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0191279

    Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid. (A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers ( Sox9 , Otx2 , and Rpe65 ) in three biological replicates was analyzed by RT-qPCR in triplicate using Gapdh , Hprt , and Actb as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers ( Vwf and Col6a1 ) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p
    Figure Legend Snippet: Cadherin subtypes show distinct preferential expression patterns in mouse RPE and choroid. (A) A method for extracting RNA individually from mouse RPE and choroid was established, and RNA samples were tested for cross-contamination. The expression of RPE markers ( Sox9 , Otx2 , and Rpe65 ) in three biological replicates was analyzed by RT-qPCR in triplicate using Gapdh , Hprt , and Actb as reference genes. Relative RNA quantity was calculated as a ratio to the expression level in mouse RPE samples. The values represent the means and SEM (bar). (B) The same RNA samples were tested for cross-contamination using choroid markers ( Vwf and Col6a1 ) by RT-qPCR in the same manner as in A. Relative RNA quantity was calculated as a ratio to the expression level in mouse choroid samples. The values represent the means and SEM (bar). (C) Total RNA from mouse RPE and choroid was prepared individually using the newly established method, and the mRNA expression of three cadherins was tested. RT-qPCR analysis was performed for Cdh1 (gene for E-cadherin), Cdh2 (N-cadherin), and Cdh3 (P-cadherin) in the same manner as described in A. Relative expression was calculated as a ratio to the expression level in mouse RPE. The values represent the means and SEM (bar). Statistical significance is shown by * (p

    Techniques Used: Expressing, Quantitative RT-PCR

    P-cadherin is the dominant cadherin in mouse and human RPE in situ . (A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1 , Cdh2 , and Cdh3 in mouse RPE in situ . Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1 , CDH2 , and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).
    Figure Legend Snippet: P-cadherin is the dominant cadherin in mouse and human RPE in situ . (A) Absolute quantification of cDNA to assess the mRNA quantity of Cdh1 , Cdh2 , and Cdh3 in mouse RPE in situ . Total RNA was prepared from the RPE of 2 week-old and 2 month-old mice, and RT-qPCR was performed, along with gel-purified PCR products to create standard curves ranging from 1 attomole (amole) to 0.1 zeptomole (zmole). Based on Ct values of the standard curves, the quantity of cDNA for each gene was calculated for 200 ng total RNA used for cDNA synthesis. Three biological replicates were analyzed in triplicate for each sample. The values represent the means and SEM (bar). (B) Absolute quantification of cDNA to assess the mRNA quantity of CDH1 , CDH2 , and CDH3 in human RPE. Total RNA was prepared from the RPE of two donor eyes (RPE-1 and RPE-2) and human RPE primary cells (M1), and RT-qPCR was performed in triplicate in the same manner as described in A, along with gel-purified PCR products to create standard curves. Based on Ct values, the quantity of cDNA for each gene was calculated for 200 ng total RNA. The values represent the means and SEM (bar).

    Techniques Used: In Situ, Mouse Assay, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    30) Product Images from "Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit"

    Article Title: Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2018.09.017

    Molecular mechanisms of palatal shelf elevation (A-F) EdU incoorporation assay was used to identify proliferative cells at E13.5 in the palatal mesenchyme four hours after EdU injection. The ratio of EdU+ cells to total cells (Hoechst+) does not differ significantly in the anterior (A-C) or posterior (D-F) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (G-L) Immunostaining for cleaved caspase 3 was used to identify apoptotic cells at E13.5 in the palatal mesenchyme. The ratio of cleaved caspase3+ cells to total cells does not differ significantly in the anterior (G-I) or posterior (J-L) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (M) Quantitative RT-PCR analysis was performed to determine expression of genes known to regulate palate development. Osr2 mRNA levels were markedly reduced in Tbx3- deficient mice at E13.5 (p = 0.0079). Expression levels are relative to mean expression in Tbx3 controls. Error bar = SEM. (N=5 of each genotype). Scale bar =100 μm.
    Figure Legend Snippet: Molecular mechanisms of palatal shelf elevation (A-F) EdU incoorporation assay was used to identify proliferative cells at E13.5 in the palatal mesenchyme four hours after EdU injection. The ratio of EdU+ cells to total cells (Hoechst+) does not differ significantly in the anterior (A-C) or posterior (D-F) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (G-L) Immunostaining for cleaved caspase 3 was used to identify apoptotic cells at E13.5 in the palatal mesenchyme. The ratio of cleaved caspase3+ cells to total cells does not differ significantly in the anterior (G-I) or posterior (J-L) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (M) Quantitative RT-PCR analysis was performed to determine expression of genes known to regulate palate development. Osr2 mRNA levels were markedly reduced in Tbx3- deficient mice at E13.5 (p = 0.0079). Expression levels are relative to mean expression in Tbx3 controls. Error bar = SEM. (N=5 of each genotype). Scale bar =100 μm.

    Techniques Used: Injection, Mouse Assay, Immunostaining, Quantitative RT-PCR, Expressing

    Enteric neural crest-derived precursors colonize the bowel normally in Tbx3 fl/fl; Wnt1-Cre mice at E13.5 (A-B) At E 13.5 the colon is colonized by TuJ1+ ENCDC that have nearly completed their rostro-caudal migration from the vagal neural tube down to the distal colon in both Tbx3 fl/fl; Wnt1-Cre mice and control littermates. (C) Measurements of the percent of colon that is colonized by TuJ1+ fibers that accompany migrating ENCDC. (D) Mean absolute length of colon in Tbx3 fl/fl; Wnt1-Cre mice and control littermate is comparable. (Student’s t-test, p > 0.05, N=7 control, N=4 mutant). Error bar = SEM. Scale bar = 500 μm. Scale bar in B also applies to A.
    Figure Legend Snippet: Enteric neural crest-derived precursors colonize the bowel normally in Tbx3 fl/fl; Wnt1-Cre mice at E13.5 (A-B) At E 13.5 the colon is colonized by TuJ1+ ENCDC that have nearly completed their rostro-caudal migration from the vagal neural tube down to the distal colon in both Tbx3 fl/fl; Wnt1-Cre mice and control littermates. (C) Measurements of the percent of colon that is colonized by TuJ1+ fibers that accompany migrating ENCDC. (D) Mean absolute length of colon in Tbx3 fl/fl; Wnt1-Cre mice and control littermate is comparable. (Student’s t-test, p > 0.05, N=7 control, N=4 mutant). Error bar = SEM. Scale bar = 500 μm. Scale bar in B also applies to A.

    Techniques Used: Derivative Assay, Mouse Assay, Migration, Mutagenesis

    31) Product Images from "Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes"

    Article Title: Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes

    Journal: Diabetes

    doi: 10.2337/db16-0846

    IFN-γ exposure during activation reduces the cytotoxicity of human β-cell–reactive CD8 +  T cells.  A : Specific lysis of BL5 human β-cell line target cells coincubated at different E:T ratios with HLA-A*02-01–restricted IGRP-specific CD8 +  T cells transduced in the presence or absence of 1,000 U/mL IFN-γ.  B : Specific lysis of BL5 target cells coincubated with nondiabetogenic MART-1–specific CD8 +  T cells transduced in the presence or absence of IFN-γ. BL5 cells were pre-exposed to 1,000 U/mL IFN-γ and washed before they were used in the cell-mediated lympholysis assays.  P  values calculated using a paired  t  test.
    Figure Legend Snippet: IFN-γ exposure during activation reduces the cytotoxicity of human β-cell–reactive CD8 + T cells. A : Specific lysis of BL5 human β-cell line target cells coincubated at different E:T ratios with HLA-A*02-01–restricted IGRP-specific CD8 + T cells transduced in the presence or absence of 1,000 U/mL IFN-γ. B : Specific lysis of BL5 target cells coincubated with nondiabetogenic MART-1–specific CD8 + T cells transduced in the presence or absence of IFN-γ. BL5 cells were pre-exposed to 1,000 U/mL IFN-γ and washed before they were used in the cell-mediated lympholysis assays. P values calculated using a paired t test.

    Techniques Used: Activation Assay, Lysis

    IFN-γ–deficient but not standard NOD mice develop AI4 T cell–induced T1D.  A : Diabetes development in female NOD and NOD. IFN-γ null  mice injected i.v. at 6 weeks of age with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes. Survival curves compared by log-rank test.  B  and  C : In vivo proliferation and activation of CFSE-labeled NOD. Rag1 null .AI4  T cells in PLNs of NOD and NOD. IFN-γ null  mice.  B : CFSE dilution of AI4 T cells in PLNs of NOD and NOD. IFN-γ null  mice at 3 days posttransfer. Representative histograms are shown in the left panel, and mean fluorescence intensity (MFI) of CFSE staining of AI4 T cells is shown in the right panel.  C : The frequency of AI4 CD8 +  T cells among live PLN cells at 3 days posttransfer.  D : CFSE dilution and activation of AI4 T cells in spleens of NOD and NOD. IFN-γ null  mice at 8 days posttransfer. Results for each quadrant represent the mean ± SE of three mice per treatment.  B – D  represent results from a single experiment. * P
    Figure Legend Snippet: IFN-γ–deficient but not standard NOD mice develop AI4 T cell–induced T1D. A : Diabetes development in female NOD and NOD. IFN-γ null mice injected i.v. at 6 weeks of age with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes. Survival curves compared by log-rank test. B and C : In vivo proliferation and activation of CFSE-labeled NOD. Rag1 null .AI4 T cells in PLNs of NOD and NOD. IFN-γ null mice. B : CFSE dilution of AI4 T cells in PLNs of NOD and NOD. IFN-γ null mice at 3 days posttransfer. Representative histograms are shown in the left panel, and mean fluorescence intensity (MFI) of CFSE staining of AI4 T cells is shown in the right panel. C : The frequency of AI4 CD8 + T cells among live PLN cells at 3 days posttransfer. D : CFSE dilution and activation of AI4 T cells in spleens of NOD and NOD. IFN-γ null mice at 8 days posttransfer. Results for each quadrant represent the mean ± SE of three mice per treatment. B – D represent results from a single experiment. * P

    Techniques Used: Mouse Assay, Injection, In Vivo, Activation Assay, Labeling, Fluorescence, Staining

    32) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    33) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    34) Product Images from "Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines"

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8436

    Lineage characterizations of PLum-AD and PLum-AI cell lines ( A ) Representative bright-field images of PLum-AD and PLum-AI cells. Scale bar = 100 μm. ( B ) Representative immunofluorescent images of PLum-AD and PLum-AI cells stained for the indicated antibodies and the nuclear counterstain Dapi are shown. Scale bars = 20 μm. ( C ) Expression of different prostate epithelial lineages and mesenchymal cell markers determined using qRT-PCR analysis. Values were normalized to GAPDH and the data were plotted relative to PLum-AD. The data are reported as mean ± SD (** P
    Figure Legend Snippet: Lineage characterizations of PLum-AD and PLum-AI cell lines ( A ) Representative bright-field images of PLum-AD and PLum-AI cells. Scale bar = 100 μm. ( B ) Representative immunofluorescent images of PLum-AD and PLum-AI cells stained for the indicated antibodies and the nuclear counterstain Dapi are shown. Scale bars = 20 μm. ( C ) Expression of different prostate epithelial lineages and mesenchymal cell markers determined using qRT-PCR analysis. Values were normalized to GAPDH and the data were plotted relative to PLum-AD. The data are reported as mean ± SD (** P

    Techniques Used: Staining, Expressing, Quantitative RT-PCR

    35) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    36) Product Images from "Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes"

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017625

    Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.
    Figure Legend Snippet: Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Purification, Hybridization

    37) Product Images from "Ionic currents in intimal cultured synoviocytes from the rabbit"

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit

    Journal: American Journal of Physiology - Cell Physiology

    doi: 10.1152/ajpcell.00028.2010

    Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.
    Figure Legend Snippet: Cells from passage 6 (the passage used for electrophysiological studies) were subjected to total RNA extraction using the RNeasy Micro Kit. Total RNA was also prepared from freshly microdissected synovium using the TRIzol method. A : the transcription product was amplified with primers specific for hyaluronan synthase 2 (HAS2), and the resulting DNA bands are shown. HAS2 message was evident in both the passage 6 -cultured synoviocytes (Cult Syn P6) and in intact synovium at dilutions of 1:1 and 1:5 but absent from the nontemplate control (NTC). B, bottom : fixed erythrocyte exclusion test. Under normal conditions the synoviocytes were surrounded by a clear area from which erythrocytes were excluded. This clear area disappeared after hyaluronidase addition, suggesting that it was due to hyaluronan secretion by the synoviocyte. Rab, rabbit; P4H, prolyl 4-hydroxylase. Black calibration bar represents 20 μm in each case.

    Techniques Used: RNA Extraction, Amplification, Cell Culture

    38) Product Images from "Gene Expression Analysis of In Vivo Fluorescent Cells"

    Article Title: Gene Expression Analysis of In Vivo Fluorescent Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001151

    RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.
    Figure Legend Snippet: RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.

    Techniques Used: Isolation, Amplification, Marker

    39) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    40) Product Images from "The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction"

    Article Title: The CUL3-KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

    Journal: Biochemical Journal

    doi: 10.1042/BJ20121903

    Evidence that CUL3 knockdown enhances WNK1 expression and activity HeLa cells were treated with 50 nM scrambled siRNA or CUL3-directed siRNA (probe 1) for 5 days and cells were lysed. ( A ) Cell lysates were subjected to immunoblotting with the indicated antibody. Each lane contains cell extract from an independent dish of cells from an identical experiment undertaken on the same day. ( B – D ) Quantitative LI-COR immunoblot analysis was undertaken and the ratio of CUL3 ( B ), KLHL3 ( C ) and WNK1 ( D ) to GAPDH was quantified. Results are means±S.E.M. for two independent samples each assayed in duplicates with P values indicated. ( E ) Total mRNA was isolated from cells and WNK1 mRNA levels were determined by qRT-PCR. Data were normalized to internal GAPDH and RPL13A controls as described in the Materials and methods section. Results are means±S.D. for three independent samples each assayed in triplicate. ( F ) WNK1 was immunoprecipitated from 0.25 mg of cell extracts and assayed for its ability to phosphorylate kinase inactive SPAK expressed in E. coli cells. The top panel displays activity in 32 P radioactivity incorporated into kinase inactive SPAK (c.p.m.) as mean±S.D. The lower panels display representative immunoblot analysis of WNK1, autorad of phosphorylated SPAK and Coomassie Blue staining of kinase-inactive SPAK employed in the kinase assay. Similar findings were made with an independent CUL3-directed siRNA (probe 2); see data in Supplementary Figure S1 (at http://www.biochemj.org/bj/451/bj4510111add.htm ). Cont, control; KD, kinase-dead; IP, immunoprecipitation; RNAi, RNA interference.
    Figure Legend Snippet: Evidence that CUL3 knockdown enhances WNK1 expression and activity HeLa cells were treated with 50 nM scrambled siRNA or CUL3-directed siRNA (probe 1) for 5 days and cells were lysed. ( A ) Cell lysates were subjected to immunoblotting with the indicated antibody. Each lane contains cell extract from an independent dish of cells from an identical experiment undertaken on the same day. ( B – D ) Quantitative LI-COR immunoblot analysis was undertaken and the ratio of CUL3 ( B ), KLHL3 ( C ) and WNK1 ( D ) to GAPDH was quantified. Results are means±S.E.M. for two independent samples each assayed in duplicates with P values indicated. ( E ) Total mRNA was isolated from cells and WNK1 mRNA levels were determined by qRT-PCR. Data were normalized to internal GAPDH and RPL13A controls as described in the Materials and methods section. Results are means±S.D. for three independent samples each assayed in triplicate. ( F ) WNK1 was immunoprecipitated from 0.25 mg of cell extracts and assayed for its ability to phosphorylate kinase inactive SPAK expressed in E. coli cells. The top panel displays activity in 32 P radioactivity incorporated into kinase inactive SPAK (c.p.m.) as mean±S.D. The lower panels display representative immunoblot analysis of WNK1, autorad of phosphorylated SPAK and Coomassie Blue staining of kinase-inactive SPAK employed in the kinase assay. Similar findings were made with an independent CUL3-directed siRNA (probe 2); see data in Supplementary Figure S1 (at http://www.biochemj.org/bj/451/bj4510111add.htm ). Cont, control; KD, kinase-dead; IP, immunoprecipitation; RNAi, RNA interference.

    Techniques Used: Expressing, Activity Assay, Isolation, Quantitative RT-PCR, Immunoprecipitation, Radioactivity, Staining, Kinase Assay

    41) Product Images from "Activation of Akt1 accelerates carcinogen-induced tumorigenesis in mammary gland of virgin and post-lactating transgenic mice"

    Article Title: Activation of Akt1 accelerates carcinogen-induced tumorigenesis in mammary gland of virgin and post-lactating transgenic mice

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-266

    Estradiol level and total AKT expression in wild-type (WT) and transgenic mice. (A) Blood samples were collected from virgin, lactating, and post-lactating mice and serum estradiol levels were measured by ELISA assay according to the manufacturer’s instructions. The line graph indicates trend of estradiol levels (mean of seven samples plus SD) in WT (n = 21) and transgenic mice (n = 21) from virgin to post-lactating stage. (B) RNA was collected from the mammary glands of WT and transgenic mice and RT-PCR was performed with AKT primers and quantified with β-actin. The lines indicate the trends of mean levels of total AKT adjusted for β-actin in the mammary glands of WT (n = 9) and transgenic mice (n = 9) at pre-puberty virgin (5 weeks olde, n = 3), puberty virgin (9 weeks olde, n = 3), lactating (14 weeks old, n = 3) and post-lactating (16 weeks old, n = 3) stages. (C) mRNA levels of total AKT from different organs in WT (top) and transgenic mice (bottom) at indicated stages were determined by RT-PCR. The mean total AKT levels are adjusted for β-actin was shown in the bar graphs. Each bar indicates mean and SD from four mice. Each sample was triplicated and “*” indicates p
    Figure Legend Snippet: Estradiol level and total AKT expression in wild-type (WT) and transgenic mice. (A) Blood samples were collected from virgin, lactating, and post-lactating mice and serum estradiol levels were measured by ELISA assay according to the manufacturer’s instructions. The line graph indicates trend of estradiol levels (mean of seven samples plus SD) in WT (n = 21) and transgenic mice (n = 21) from virgin to post-lactating stage. (B) RNA was collected from the mammary glands of WT and transgenic mice and RT-PCR was performed with AKT primers and quantified with β-actin. The lines indicate the trends of mean levels of total AKT adjusted for β-actin in the mammary glands of WT (n = 9) and transgenic mice (n = 9) at pre-puberty virgin (5 weeks olde, n = 3), puberty virgin (9 weeks olde, n = 3), lactating (14 weeks old, n = 3) and post-lactating (16 weeks old, n = 3) stages. (C) mRNA levels of total AKT from different organs in WT (top) and transgenic mice (bottom) at indicated stages were determined by RT-PCR. The mean total AKT levels are adjusted for β-actin was shown in the bar graphs. Each bar indicates mean and SD from four mice. Each sample was triplicated and “*” indicates p

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction

    Characterization of malignant mammary tumors. (A) H E staining (a and e) and immunohistochemical analysis were used to determine the level of phospho-Akt1 (pAkt ser473) (b and f), ERα (c and g), and EGFR (d and h) expression in mammary tumors from wild-type mouse (N2) and MMTVmyr-Akt1 transgenic mouse (P1). The arrows indicate positive staining. (B) Protein was extracted from the mammary tumors and western blot analysis was used to determine the indicate protein expression. (C) RNA was extracted from mammary carcinomas from four transgenic mice and two wild-type mice. RT-Q-PCR was performed with the indicated primers. The bar graph indicates relative expression levels of the indicated genes adjusted for 18S from the four transgenic and two wild-type mice. Each bar indicates mean and SD of three individual tests. “*” indicates p
    Figure Legend Snippet: Characterization of malignant mammary tumors. (A) H E staining (a and e) and immunohistochemical analysis were used to determine the level of phospho-Akt1 (pAkt ser473) (b and f), ERα (c and g), and EGFR (d and h) expression in mammary tumors from wild-type mouse (N2) and MMTVmyr-Akt1 transgenic mouse (P1). The arrows indicate positive staining. (B) Protein was extracted from the mammary tumors and western blot analysis was used to determine the indicate protein expression. (C) RNA was extracted from mammary carcinomas from four transgenic mice and two wild-type mice. RT-Q-PCR was performed with the indicated primers. The bar graph indicates relative expression levels of the indicated genes adjusted for 18S from the four transgenic and two wild-type mice. Each bar indicates mean and SD of three individual tests. “*” indicates p

    Techniques Used: Staining, Immunohistochemistry, Expressing, Transgenic Assay, Western Blot, Mouse Assay, Polymerase Chain Reaction

    Expression of activated Akt1 in transgenic mice at different development stages. (A) Total RNA was extracted from the indicated organs of virgin (9 week old) and post-lactating (16 week old) transgenic mice. RT-PCR was performed with primers for myr-Akt1 and β-actin. Three mice per genotype, per age group were used for analysis. Myr-Akt1 was detected in the lungs, heart, uterus, and mammary glands of the virgin and post-lactating mice. (B) The mRNA levels of myr-Akt1 were quantified in different organs of the transgenic mice at different developmental states. Myr-Akt1 levels were adjusted for the levels of β-actin. Data are presented as bar graphs. The bars indicate mean levels and SD from three mice and “*” indicates p
    Figure Legend Snippet: Expression of activated Akt1 in transgenic mice at different development stages. (A) Total RNA was extracted from the indicated organs of virgin (9 week old) and post-lactating (16 week old) transgenic mice. RT-PCR was performed with primers for myr-Akt1 and β-actin. Three mice per genotype, per age group were used for analysis. Myr-Akt1 was detected in the lungs, heart, uterus, and mammary glands of the virgin and post-lactating mice. (B) The mRNA levels of myr-Akt1 were quantified in different organs of the transgenic mice at different developmental states. Myr-Akt1 levels were adjusted for the levels of β-actin. Data are presented as bar graphs. The bars indicate mean levels and SD from three mice and “*” indicates p

    Techniques Used: Expressing, Transgenic Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    42) Product Images from "Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning"

    Article Title: Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    Journal: Neural Plasticity

    doi: 10.1155/2016/1752176

    Distribution of cannula placements and RAG1 antisense oligonucleotide diffusion within the amygdala. After behavioral treatments with RAG1 antisense or random oligonucleotides, animals were microinfused the next day with thionine to verify cannulae injectors' placement. Another set of animals was used to observe FITC-labeled RAG1 antisense diffusion. (a) Schematic representation of the amygdala at different rostrocaudal planes illustrating the position of cannulae injectors determined by thionine microinfusion. Injector tips for each cannula are represented by dark spots. (b) FITC- RAG1 antisense diffusion within the amygdalar complex; arrow indicates the injector's tip. (c) Schemes of coronal sections showing the diffusion of FITC- RAG1 antisense diffusion into the amygdala of animals decapitated 3 h after fluorescent oligonucleotide infusion. FITC- RAG1 antisense diffusion is represented by green shading from anterior to posterior areas of the amygdalar complex. The numbers in (a) and (c) indicate the distance from bregma in millimeters. A total of 4 mice were used in these studies. (d) Photomicrograph at higher magnification of FITC- RAG1 antisense diffusion showed clearly incorporation into the cells (depicted by the arrows) within amygdalar regions.
    Figure Legend Snippet: Distribution of cannula placements and RAG1 antisense oligonucleotide diffusion within the amygdala. After behavioral treatments with RAG1 antisense or random oligonucleotides, animals were microinfused the next day with thionine to verify cannulae injectors' placement. Another set of animals was used to observe FITC-labeled RAG1 antisense diffusion. (a) Schematic representation of the amygdala at different rostrocaudal planes illustrating the position of cannulae injectors determined by thionine microinfusion. Injector tips for each cannula are represented by dark spots. (b) FITC- RAG1 antisense diffusion within the amygdalar complex; arrow indicates the injector's tip. (c) Schemes of coronal sections showing the diffusion of FITC- RAG1 antisense diffusion into the amygdala of animals decapitated 3 h after fluorescent oligonucleotide infusion. FITC- RAG1 antisense diffusion is represented by green shading from anterior to posterior areas of the amygdalar complex. The numbers in (a) and (c) indicate the distance from bregma in millimeters. A total of 4 mice were used in these studies. (d) Photomicrograph at higher magnification of FITC- RAG1 antisense diffusion showed clearly incorporation into the cells (depicted by the arrows) within amygdalar regions.

    Techniques Used: Diffusion-based Assay, Labeling, Mouse Assay

    RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P

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

    RAG1 antisense amygdalar treatment does not interfere with reconsolidation of context fear conditioning. To test the effects of RAG1 gapmer antisense treatment on memory reconsolidation of context fear conditioning, another set of animals was bilaterally implanted with cannulas to target the amygdala. Top panel: diagram depicting the experimental design. On day 1, mice were trained in context fear conditioning and immediately returned to their home cages. Antisense or random oligonucleotides were microinfused into the amygdala 1 h before memory reactivation on day 2. The effect of antisense or random oligonucleotide treatment on LTM reconsolidation was assessed on day 3, 48 h after conditioning. (a) On day 1, mice were microinfused with saline 1 h before training and returned to their home cages immediately after conditioning. Two-Way RM ANOVA and Bonferroni posttesting demonstrated that the infusions did not impair the animals' response in developing and expressing fear during the conditioning experience (Treatment Assignment Factor: F (1,8.194) = 3.979, P > 0.05; Training Factor F (3,3134) = 1725, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment does not interfere with reconsolidation of context fear conditioning. To test the effects of RAG1 gapmer antisense treatment on memory reconsolidation of context fear conditioning, another set of animals was bilaterally implanted with cannulas to target the amygdala. Top panel: diagram depicting the experimental design. On day 1, mice were trained in context fear conditioning and immediately returned to their home cages. Antisense or random oligonucleotides were microinfused into the amygdala 1 h before memory reactivation on day 2. The effect of antisense or random oligonucleotide treatment on LTM reconsolidation was assessed on day 3, 48 h after conditioning. (a) On day 1, mice were microinfused with saline 1 h before training and returned to their home cages immediately after conditioning. Two-Way RM ANOVA and Bonferroni posttesting demonstrated that the infusions did not impair the animals' response in developing and expressing fear during the conditioning experience (Treatment Assignment Factor: F (1,8.194) = 3.979, P > 0.05; Training Factor F (3,3134) = 1725, ∗∗∗ P

    Techniques Used: Mouse Assay, Expressing

    RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).
    Figure Legend Snippet: RAG1 protein expression in amygdalar neuronal cells. Amygdalar coronal sections of context fear conditioning-trained mice, perfused 1 h after conditioning, were used for immunofluorescence and analyzed by confocal microscopy. Antibodies from immunofluorescence were validated by Western blot analysis. (a) Amygdalar area representative images of a double immunostaining using RAG1 antibody labeled with Alexa Fluor 488, green channel signal, and NeuN antibody labeled with Alexa Fluor 568, red channel signal. The left panel shows the NeuN positive neuronal nuclei, while the middle panel depicts RAG1 immunopositive cells. The right panel is the merge image showing colocalization of the NeuN neuronal nuclei marker and RAG1. Arrows point to some of the RAG1 immunopositive neurons. These immunofluorescent images revealed colocalization of RAG1 protein expressing cells with those expressing NeuN, suggesting the presence of RAG1 in neurons, although not all neurons expressed RAG1. (b) Tissue punches from amygdala (Amy) were obtained 1 h after context fear conditioning and analyzed in Western blot by comparative comigration with a standard molecular weight (MW) marker and protein extracts from bone marrow (BM) ((b)-1) and thymus (Thy) ((b)-2). Both sets of experiments consistently showed comigration between the tissues with a band corresponding to ~120 KD of RAG1 protein (green channel corresponding to RAG1 and red channel corresponding to beta-actin, ~42 KD); prestained molecular weight (MW) marker (ladder) was included in all the Western blots. ((b)-3) Additionally, tissue protein extracts from leg muscle (Mus) (negative control) were analyzed compared to amygdalar extracts with respect to RAG1 expression. As expected, RAG1 was not expressed in muscle compared to amygdala ((b)-3), bone marrow ((b)-1), and thymus ((b)-2). ((b)-4) RAG1 antibody preabsorption assays, either with muscle or with bone marrow extracts, showed that only bone marrow extracts, which express RAG1 as opposed to muscle, were able to block the ~120 KD band from amygdalar protein extracts in the Western blots, indicating that RAG1 antibody was preabsorbed (blocked) only by RAG1 protein expressing tissue (bone marrow).

    Techniques Used: Expressing, Mouse Assay, Immunofluorescence, Confocal Microscopy, Western Blot, Double Immunostaining, Labeling, Marker, Molecular Weight, Negative Control, Blocking Assay

    43) Product Images from "Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning"

    Article Title: Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

    Journal: Neural Plasticity

    doi: 10.1155/2016/1752176

    RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P
    Figure Legend Snippet: RAG1 antisense amygdalar treatment impaired consolidation of context fear conditioning. Top panel: diagram depicting the experimental design of these experiments for pretraining or posttraining amygdalar antisense or random oligonucleotide microinfusion experiments. In the pretraining microinfusion experiments, mice received RAG1 antisense or random bilateral oligonucleotide microinfusions directed at the amygdala 1 h before conditioning followed by either LTM testing or molecular evaluation. LTM was tested 24 h after conditioning. For molecular evaluation of antisense treatment effectiveness, another group of mice was sacrificed 30 min after conditioning and amygdalar RNA was used for real-time PCR. In the posttraining microinfusion experiments, mice were conditioned, returned to their home cages, and received microinfusions of antisense or random oligonucleotides 5 h after training and returned to their home cages until next day. Nineteen (19) hours later (24 h after conditioning), mice were reexposed to the conditioning chamber without any shocks in order to test LTM. (a) Mice receiving either RAG1 antisense or random oligonucleotide treatment displayed no significant differences during memory acquisition measured as the progressive enhancement of freezing behavior (Two-Way ANOVA, Treatment Factor: F (1,0.8457) = 0.01015, P > 0.9; Training Factor F (3,7863) = 94.37, ∗∗∗ P

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

    44) Product Images from "Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes"

    Article Title: Endothelial Cells Use a Formin-Dependent Phagocytosis-Like Process to Internalize the Bacterium Listeria monocytogenes

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1005603

    Formin activity is necessary for L . monocytogenes invasion in HUVEC. HUVEC were infected with JAT983. ( A, B ) Histograms of bacterial fluorescence intensity per cell. Infection was analyzed by flow cytometry 7–8 hours after infection. In each trace, a minor peak of higher bacterial fluorescence indicates infected HUVEC. ( A ) Effect of formin inhibition on bacterial uptake. SMIFH2 or vehicle control (DMSO) was present during invasion at the concentration indicated, and washed out with the addition of gentamicin. ( B ) Effect of formin inhibition on cell-to-cell spread. SMIFH2 or vehicle control was added with gentamicin and therefore was present only after invasion. ( C ) Effects of formin knockdown on bacterial uptake. HUVEC were treated with siRNAs targeting all 15 mammalian formins (gray bars) or non-targeting siRNAs (red bars) and analyzed by flow cytometry 7–8 hours after infection. The value for each sample is the average fold change (from 3 independent experiments with 4 biological replicates per experiment), relative to the mean percent infected among control siRNA wells (black line). Red vertical lines represent two SD from the mean. Names are listed for formins that were subsequently confirmed to be significantly different from controls. ( D ) Frequency of infected HUVEC (mean +/- SD, n = 4 biological replicates), for cells treated with siRNAs targeting FMNL3 , FHOD1 , GRID2IP , INF2 , or non-targeting control siRNA, and analyzed by microscopy 8 hours after infection. P-values for each siRNA condition relative to control (unpaired two-sided, two-sample t-test, Benjamini-Hochberg correction): 0.0086 (siFMNL3), 1.0670 *10 −5 (siFHOD1), 1.0670 * 10 −5 (siGRID2IP), 0.0011 (siINF2).
    Figure Legend Snippet: Formin activity is necessary for L . monocytogenes invasion in HUVEC. HUVEC were infected with JAT983. ( A, B ) Histograms of bacterial fluorescence intensity per cell. Infection was analyzed by flow cytometry 7–8 hours after infection. In each trace, a minor peak of higher bacterial fluorescence indicates infected HUVEC. ( A ) Effect of formin inhibition on bacterial uptake. SMIFH2 or vehicle control (DMSO) was present during invasion at the concentration indicated, and washed out with the addition of gentamicin. ( B ) Effect of formin inhibition on cell-to-cell spread. SMIFH2 or vehicle control was added with gentamicin and therefore was present only after invasion. ( C ) Effects of formin knockdown on bacterial uptake. HUVEC were treated with siRNAs targeting all 15 mammalian formins (gray bars) or non-targeting siRNAs (red bars) and analyzed by flow cytometry 7–8 hours after infection. The value for each sample is the average fold change (from 3 independent experiments with 4 biological replicates per experiment), relative to the mean percent infected among control siRNA wells (black line). Red vertical lines represent two SD from the mean. Names are listed for formins that were subsequently confirmed to be significantly different from controls. ( D ) Frequency of infected HUVEC (mean +/- SD, n = 4 biological replicates), for cells treated with siRNAs targeting FMNL3 , FHOD1 , GRID2IP , INF2 , or non-targeting control siRNA, and analyzed by microscopy 8 hours after infection. P-values for each siRNA condition relative to control (unpaired two-sided, two-sample t-test, Benjamini-Hochberg correction): 0.0086 (siFMNL3), 1.0670 *10 −5 (siFHOD1), 1.0670 * 10 −5 (siGRID2IP), 0.0011 (siINF2).

    Techniques Used: Activity Assay, Infection, Fluorescence, Flow Cytometry, Cytometry, Inhibition, Concentration Assay, Microscopy

    FAK regulates uptake of bacteria by modulating the activity of Rho and Rho kinase. ( A ) Effect of FAK inhibition after siRNA treatment targeting ARHGAP5 (p190RhoGAP), ARHGEF2 (GEF-H1) or non-targeting siRNA controls. DMSO (vehicle control), 10μM FAK-14, or 10μM PF573228 was added 40 minutes prior to infection with JAT983. Infection was analyzed by microscopy (mean +/- SD, n = 4 biological replicates) 8 hours after infection. ( B ) Frequency of infected HUVEC as a function of Y27632 concentration (n = 2 biological replicates). Y27632 was added 30 minutes prior to infection with JAT1045; infection was analyzed by flow cytometry 7–8 hours after infection. Representative data from 1 of 2 independent experiments.
    Figure Legend Snippet: FAK regulates uptake of bacteria by modulating the activity of Rho and Rho kinase. ( A ) Effect of FAK inhibition after siRNA treatment targeting ARHGAP5 (p190RhoGAP), ARHGEF2 (GEF-H1) or non-targeting siRNA controls. DMSO (vehicle control), 10μM FAK-14, or 10μM PF573228 was added 40 minutes prior to infection with JAT983. Infection was analyzed by microscopy (mean +/- SD, n = 4 biological replicates) 8 hours after infection. ( B ) Frequency of infected HUVEC as a function of Y27632 concentration (n = 2 biological replicates). Y27632 was added 30 minutes prior to infection with JAT1045; infection was analyzed by flow cytometry 7–8 hours after infection. Representative data from 1 of 2 independent experiments.

    Techniques Used: Activity Assay, Inhibition, Infection, Microscopy, Concentration Assay, Flow Cytometry, Cytometry

    45) Product Images from "Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling"

    Article Title: Discrete somatic niches coordinate proliferation and migration of primordial germ cells via Wnt signaling

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201511061

    Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2 Y324C . (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-catenin GOF/+ ; Pou5f1 +/+ ) and βcat GOF ( β-catenin GOF/+ ; Pou5f1 Cre-ER/+ ) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcat GOF embryos at different ages. n = 13 Cnt embryos and 15 βcat GOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcat GOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP + PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcat GOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P
    Figure Legend Snippet: Overactivation of β-catenin in PGCs leads to an increase in proliferation and misregulation of genes in common with Ror2 Y324C . (A) Schematic of breeding and tamoxifen exposure to generate control (Cnt, β-catenin GOF/+ ; Pou5f1 +/+ ) and βcat GOF ( β-catenin GOF/+ ; Pou5f1 Cre-ER/+ ) embryos in vivo and cells ex vivo. (B) Mean number of PGCs counted in histological sections of Cnt and βcat GOF embryos at different ages. n = 13 Cnt embryos and 15 βcat GOF embryos. P-values by Student’s t test for section counts. Estimates for total numbers of Cnt and βcat GOF PGCs per embryo were calculated using the multiplier of 100× for E9.5, 125× for E10.5, and 150× for E11.5 based on cell counts reported in the literature. (C) Oct4-ΔPE-GFP + PGCs (gray) in E9.5 littermates. Bar, 100 µm. (D) Rate of in vitro EdU incorporation in Cnt and βcat GOF PGCs cultured for 10–22 h with 4-OHT. n = 4 litters; 794–818 cells; *, P

    Techniques Used: In Vivo, Ex Vivo, In Vitro, Cell Culture

    Ror2 Y324C downstream targets overlap with cell cycle–associated genes. (A) Schematic of experimental design. Single embryos were collected at E9.5, and Oct4-ΔPE-GFP + PGCs were isolated by FACS. RNA was extracted from WT ( Ror2 +/+ ) and Ror2 Y324C PGCs, converted to cDNA, amplified, and hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Age and number of PGCs collected per embryo are shown in the table. (B) Microarray data from Ror2 Y324C PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (dChip). Each dot represents a single probe. Purple dots show genes with a P
    Figure Legend Snippet: Ror2 Y324C downstream targets overlap with cell cycle–associated genes. (A) Schematic of experimental design. Single embryos were collected at E9.5, and Oct4-ΔPE-GFP + PGCs were isolated by FACS. RNA was extracted from WT ( Ror2 +/+ ) and Ror2 Y324C PGCs, converted to cDNA, amplified, and hybridized to the GeneChip Mouse Gene 1.0 ST Array (Affymetrix). Age and number of PGCs collected per embryo are shown in the table. (B) Microarray data from Ror2 Y324C PGCs compared with WT shown as log fold change versus log p-values for all annotated microarray probes (dChip). Each dot represents a single probe. Purple dots show genes with a P

    Techniques Used: Isolation, FACS, Amplification, Microarray

    Increased nuclear β-catenin levels in PGCs during their migratory progression and perturbation in Ror2 Y324C PGCs. (A) Nuclear β-catenin (n-βcat, red) immunofluorescence in transverse histological sections of an E9.75 WT embryo treated with Ficin enzyme to disrupt E-cadherin/β-catenin membrane staining. The hindgut (hg), neural tube (nt), and mesonephric duct (mes) are indicated. Bar, 30 µm. (i) PGCs are identified by expression of Oct4-ΔPE-GFP (green; white arrows in A). Bar, 30 µm. (ii) Pseudocoloring (rainbow) indicates individually selected Oct4-ΔPE-GFP + PGCs for quantitative measurement of n-βcat. (iii) Pseudocoloring (rainbow) indicates DAPI-selected nuclei of all cells in the field ( > 600 counted) used to obtain the mean intensity of n-βcat. (iv) Inset from box in A and i to exemplify differences in n-βcat levels in PGCs (dashed white lines) relative to the mean n-βcat levels in all nuclei in the field. Fold differences in staining intensity are indicated. Bar, 10 µm. (B) Quantification of n-βcat in all E9.5 Ror2 Y324C PGCs relative to WT/het littermates shows an increase in accumulation of n-βcat. Each dot represents a single PGC; bars indicate the mean and boxes denote the middle 50% of data points. n = 100–134 cells from two embryos per group; **, P
    Figure Legend Snippet: Increased nuclear β-catenin levels in PGCs during their migratory progression and perturbation in Ror2 Y324C PGCs. (A) Nuclear β-catenin (n-βcat, red) immunofluorescence in transverse histological sections of an E9.75 WT embryo treated with Ficin enzyme to disrupt E-cadherin/β-catenin membrane staining. The hindgut (hg), neural tube (nt), and mesonephric duct (mes) are indicated. Bar, 30 µm. (i) PGCs are identified by expression of Oct4-ΔPE-GFP (green; white arrows in A). Bar, 30 µm. (ii) Pseudocoloring (rainbow) indicates individually selected Oct4-ΔPE-GFP + PGCs for quantitative measurement of n-βcat. (iii) Pseudocoloring (rainbow) indicates DAPI-selected nuclei of all cells in the field ( > 600 counted) used to obtain the mean intensity of n-βcat. (iv) Inset from box in A and i to exemplify differences in n-βcat levels in PGCs (dashed white lines) relative to the mean n-βcat levels in all nuclei in the field. Fold differences in staining intensity are indicated. Bar, 10 µm. (B) Quantification of n-βcat in all E9.5 Ror2 Y324C PGCs relative to WT/het littermates shows an increase in accumulation of n-βcat. Each dot represents a single PGC; bars indicate the mean and boxes denote the middle 50% of data points. n = 100–134 cells from two embryos per group; **, P

    Techniques Used: Immunofluorescence, Staining, Expressing, Pyrolysis Gas Chromatography

    46) Product Images from "A novel redox regulator, MnTnBuOE-2-PyP5+, enhances normal hematopoietic stem/progenitor cell function"

    Article Title: A novel redox regulator, MnTnBuOE-2-PyP5+, enhances normal hematopoietic stem/progenitor cell function

    Journal: Redox Biology

    doi: 10.1016/j.redox.2017.02.005

    MnP increases Nrf2/ARE and ETS transcription activities. A. Oxidative transcription profiling analysis. BMNCs were isolated, treated with vehicle or 20 μM MnP for 1 h. Cells were harvested and immunostained. Nuclear extracts were isolated from flow cytometry sorted LK (lin-, ckit+) cells. Equal amounts of nuclear extracts were used for TF assay (n=3). B. Quantitative PCR on some of the Nrf2 target genes was performed on LSK cells treated with vehicle or 20 μM MnP for 1 h. C. Quantitative PCR on some of the Nrf2 target genes on LSK cells treated with vehicle or 20 μM MnP for 16 h. D. Western analysis of the protein expression of some of the Nrf2 targets. The left panel shows the representative Western blots. The right panel shows the quantitation of repeats of the experiments. The data presented are the fold changes of MnP/vehicle treated with vehicle treated as value 1. Bar graphs represent the mean + SD of at least three independent experiments except for the TF assay.
    Figure Legend Snippet: MnP increases Nrf2/ARE and ETS transcription activities. A. Oxidative transcription profiling analysis. BMNCs were isolated, treated with vehicle or 20 μM MnP for 1 h. Cells were harvested and immunostained. Nuclear extracts were isolated from flow cytometry sorted LK (lin-, ckit+) cells. Equal amounts of nuclear extracts were used for TF assay (n=3). B. Quantitative PCR on some of the Nrf2 target genes was performed on LSK cells treated with vehicle or 20 μM MnP for 1 h. C. Quantitative PCR on some of the Nrf2 target genes on LSK cells treated with vehicle or 20 μM MnP for 16 h. D. Western analysis of the protein expression of some of the Nrf2 targets. The left panel shows the representative Western blots. The right panel shows the quantitation of repeats of the experiments. The data presented are the fold changes of MnP/vehicle treated with vehicle treated as value 1. Bar graphs represent the mean + SD of at least three independent experiments except for the TF assay.

    Techniques Used: Isolation, Flow Cytometry, Cytometry, Transcription Factor Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Quantitation Assay

    47) Product Images from "A novel organotypic 3D sweat gland model with physiological functionality"

    Article Title: A novel organotypic 3D sweat gland model with physiological functionality

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182752

    Histological analysis of human eccrine sweat glands, monolayer cells and 3D cell culture models in vitro . (A) H E and azan staining of paraffin axillary skin tissue sections visualizing the morphology of native eccrine SG’s with secretory coil portions (sc) and ductal portions (du) of the gland. (B) Bright-field microscopy of primary SG cells in 2D culture with their typical ‘cobblestone’ morphology is shown. (C) Bright-field microscopy and H E-/azan-stained sections of a differentiated SG 3D model with irregularly arranged, multilayered cells and less dense cells in the center of the spheroid. Collagen in blue could not be detected in azan staining, but acidic structures like nuclei or matrix were stained in red or dark red. Scale bars 100 μm.
    Figure Legend Snippet: Histological analysis of human eccrine sweat glands, monolayer cells and 3D cell culture models in vitro . (A) H E and azan staining of paraffin axillary skin tissue sections visualizing the morphology of native eccrine SG’s with secretory coil portions (sc) and ductal portions (du) of the gland. (B) Bright-field microscopy of primary SG cells in 2D culture with their typical ‘cobblestone’ morphology is shown. (C) Bright-field microscopy and H E-/azan-stained sections of a differentiated SG 3D model with irregularly arranged, multilayered cells and less dense cells in the center of the spheroid. Collagen in blue could not be detected in azan staining, but acidic structures like nuclei or matrix were stained in red or dark red. Scale bars 100 μm.

    Techniques Used: Cell Culture, In Vitro, Staining, Microscopy

    48) Product Images from "Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes"

    Article Title: Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes

    Journal: Diabetes

    doi: 10.2337/db16-0846

    IFN-γ exposure during activation reduces the cytotoxicity of human β-cell–reactive CD8 +  T cells.  A : Specific lysis of BL5 human β-cell line target cells coincubated at different E:T ratios with HLA-A*02-01–restricted IGRP-specific CD8 +  T cells transduced in the presence or absence of 1,000 U/mL IFN-γ.  B : Specific lysis of BL5 target cells coincubated with nondiabetogenic MART-1–specific CD8 +  T cells transduced in the presence or absence of IFN-γ. BL5 cells were pre-exposed to 1,000 U/mL IFN-γ and washed before they were used in the cell-mediated lympholysis assays.  P  values calculated using a paired  t  test.
    Figure Legend Snippet: IFN-γ exposure during activation reduces the cytotoxicity of human β-cell–reactive CD8 + T cells. A : Specific lysis of BL5 human β-cell line target cells coincubated at different E:T ratios with HLA-A*02-01–restricted IGRP-specific CD8 + T cells transduced in the presence or absence of 1,000 U/mL IFN-γ. B : Specific lysis of BL5 target cells coincubated with nondiabetogenic MART-1–specific CD8 + T cells transduced in the presence or absence of IFN-γ. BL5 cells were pre-exposed to 1,000 U/mL IFN-γ and washed before they were used in the cell-mediated lympholysis assays. P values calculated using a paired t test.

    Techniques Used: Activation Assay, Lysis

    IFN-γ–producing CD4 +  T cells suppress diabetogenic CD8 +  T cells through mechanisms that do not involve quantitative or functional variations in Tregs.  A : Quantitative PCR analysis of IFN-γ mRNA expression by host-type CD4 +  and CD8 +  (Vα8 − ) T cells and B cells purified from spleens of NOD mice 3 days postadoptive transfer with 2 × 10 7  NOD. Rag1 null .AI4  splenocytes (post-AT) or untreated NOD mice (untreated). Results represent the mean ± SE of three samples per treatment.  B : Diabetes incidence for female NOD. scid  mice injected at 6–8 weeks of age with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes in the presence or absence of 3 × 10 6  CD4 +  T cells purified from NOD or NOD. IFN-γ null  donors.  C : Frequencies, numbers, and mean fluorescence intensity (MFI) of FoxP3 antibody staining of splenic CD4 + CD25 + FoxP3 +  Tregs in NOD and NOD. IFN-γ null  mice. Results represent the mean ± SE of five mice per treatment.  D : Crisscross cultures were established to assess the ability of CD4 + CD25 +  Tregs from NOD and NOD. IFN-γ null  mice to suppress the anti-CD3–stimulated proliferation of CD4 + CD25 −  effectors from both strains (assessed by flow cytometic detection of CFSE dilution).  E : Beginning at 6 weeks of age, NOD female mice received three biweekly i.p. injections with the Treg-depleting CD25-specific PC61 antibody or a rat IgG1 isotype control. One week after the first treatment, mice in both groups were injected i.v. with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes and subsequently monitored for diabetes. Survival curves compared by log-rank test. * P
    Figure Legend Snippet: IFN-γ–producing CD4 + T cells suppress diabetogenic CD8 + T cells through mechanisms that do not involve quantitative or functional variations in Tregs. A : Quantitative PCR analysis of IFN-γ mRNA expression by host-type CD4 + and CD8 + (Vα8 − ) T cells and B cells purified from spleens of NOD mice 3 days postadoptive transfer with 2 × 10 7 NOD. Rag1 null .AI4 splenocytes (post-AT) or untreated NOD mice (untreated). Results represent the mean ± SE of three samples per treatment. B : Diabetes incidence for female NOD. scid mice injected at 6–8 weeks of age with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes in the presence or absence of 3 × 10 6 CD4 + T cells purified from NOD or NOD. IFN-γ null donors. C : Frequencies, numbers, and mean fluorescence intensity (MFI) of FoxP3 antibody staining of splenic CD4 + CD25 + FoxP3 + Tregs in NOD and NOD. IFN-γ null mice. Results represent the mean ± SE of five mice per treatment. D : Crisscross cultures were established to assess the ability of CD4 + CD25 + Tregs from NOD and NOD. IFN-γ null mice to suppress the anti-CD3–stimulated proliferation of CD4 + CD25 − effectors from both strains (assessed by flow cytometic detection of CFSE dilution). E : Beginning at 6 weeks of age, NOD female mice received three biweekly i.p. injections with the Treg-depleting CD25-specific PC61 antibody or a rat IgG1 isotype control. One week after the first treatment, mice in both groups were injected i.v. with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes and subsequently monitored for diabetes. Survival curves compared by log-rank test. * P

    Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Purification, Mouse Assay, Injection, Fluorescence, Staining, Flow Cytometry

    Higher STAT1 but not STAT4 expression by transferred AI4 T cells is associated with the lesser ability of such effectors to induce diabetes in NOD and NOD. IFN-γR null  than NOD. IFN-γ null  recipients.  A – C : Otherwise unmanipulated NOD or NOD. IFN-γ null  recipients were injected with 1 × 10 6  AI4 T cells and analyzed for total STAT1 or STAT4 levels 2–7 days after transfer.  A : Representative pattern (from day 4) of transferred CD8 +  tetramer +  AI4 T cells from the indicated recipients showing STAT1 (left) or STAT4 (right) compared with fluorescence minus one (FMO) control stains.  B : Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8 +  T cells after transfer into the indicated recipients.  C : Endogenous CD8 +  tetramer −  T cells from the indicated recipients were analyzed for mean fluorescence intensity (MFI) of STAT1 (left) or STAT4 (right) staining after transfer of AI4 CD8 +  T cells.  B  and  C  display combined data for days 2–7 posttransfer of AI4 T cells.  D : Comparison of STAT1 expression by tetramer +  AI4 donor T cells before and 2 days after transfer into otherwise unmanipulated NOD. IFN-γ null  recipients.  E : Otherwise unmanipulated NOD or NOD. IFN-γR null  recipients were injected with 1 × 10 6  AI4 T cells and analyzed for STAT1 or STAT4 levels 2 days after transfer. Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8 +  T cells 2 days after transfer into the indicated recipient.  F : Endogenous CD8 +  tetramer −  T cells from the indicated recipients were analyzed for MFI of STAT1 (left) or STAT4 (right) staining 2 days after transfer of AI4 CD8 +  T cells.  D–F  display data from a single experiment.  G  and  H : NOD, NOD. IFN-γ null , or NOD. IFN-γR null  mice were irradiated (600 cGy) and injected with 1 × 10 6  AI4 T cells. Two days posttransfer, AI4 T cells were analyzed for STAT1 or STAT4 levels. Left panels: Histograms showing STAT1 ( G ) or STAT4 ( H ) expression of AI4 T cells from the indicated recipients compared with an FMO control. Right panels: Quantification of MFI of STAT1 ( G ) or STAT4 ( H ) staining from one of two experiments showing  n  ≥ 3 per group.  P  values calculated using Mann–Whitney analysis.
    Figure Legend Snippet: Higher STAT1 but not STAT4 expression by transferred AI4 T cells is associated with the lesser ability of such effectors to induce diabetes in NOD and NOD. IFN-γR null than NOD. IFN-γ null recipients. A – C : Otherwise unmanipulated NOD or NOD. IFN-γ null recipients were injected with 1 × 10 6 AI4 T cells and analyzed for total STAT1 or STAT4 levels 2–7 days after transfer. A : Representative pattern (from day 4) of transferred CD8 + tetramer + AI4 T cells from the indicated recipients showing STAT1 (left) or STAT4 (right) compared with fluorescence minus one (FMO) control stains. B : Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8 + T cells after transfer into the indicated recipients. C : Endogenous CD8 + tetramer − T cells from the indicated recipients were analyzed for mean fluorescence intensity (MFI) of STAT1 (left) or STAT4 (right) staining after transfer of AI4 CD8 + T cells. B and C display combined data for days 2–7 posttransfer of AI4 T cells. D : Comparison of STAT1 expression by tetramer + AI4 donor T cells before and 2 days after transfer into otherwise unmanipulated NOD. IFN-γ null recipients. E : Otherwise unmanipulated NOD or NOD. IFN-γR null recipients were injected with 1 × 10 6 AI4 T cells and analyzed for STAT1 or STAT4 levels 2 days after transfer. Quantification of STAT1 (left) or STAT4 (right) staining of splenic AI4 CD8 + T cells 2 days after transfer into the indicated recipient. F : Endogenous CD8 + tetramer − T cells from the indicated recipients were analyzed for MFI of STAT1 (left) or STAT4 (right) staining 2 days after transfer of AI4 CD8 + T cells. D–F display data from a single experiment. G and H : NOD, NOD. IFN-γ null , or NOD. IFN-γR null mice were irradiated (600 cGy) and injected with 1 × 10 6 AI4 T cells. Two days posttransfer, AI4 T cells were analyzed for STAT1 or STAT4 levels. Left panels: Histograms showing STAT1 ( G ) or STAT4 ( H ) expression of AI4 T cells from the indicated recipients compared with an FMO control. Right panels: Quantification of MFI of STAT1 ( G ) or STAT4 ( H ) staining from one of two experiments showing n ≥ 3 per group. P values calculated using Mann–Whitney analysis.

    Techniques Used: Expressing, Injection, Fluorescence, Staining, Mouse Assay, Irradiation, MANN-WHITNEY

    IFN-γ–deficient but not standard NOD mice develop AI4 T cell–induced T1D.  A : Diabetes development in female NOD and NOD. IFN-γ null  mice injected i.v. at 6 weeks of age with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes. Survival curves compared by log-rank test.  B  and  C : In vivo proliferation and activation of CFSE-labeled NOD. Rag1 null .AI4  T cells in PLNs of NOD and NOD. IFN-γ null  mice.  B : CFSE dilution of AI4 T cells in PLNs of NOD and NOD. IFN-γ null  mice at 3 days posttransfer. Representative histograms are shown in the left panel, and mean fluorescence intensity (MFI) of CFSE staining of AI4 T cells is shown in the right panel.  C : The frequency of AI4 CD8 +  T cells among live PLN cells at 3 days posttransfer.  D : CFSE dilution and activation of AI4 T cells in spleens of NOD and NOD. IFN-γ null  mice at 8 days posttransfer. Results for each quadrant represent the mean ± SE of three mice per treatment.  B – D  represent results from a single experiment. * P
    Figure Legend Snippet: IFN-γ–deficient but not standard NOD mice develop AI4 T cell–induced T1D. A : Diabetes development in female NOD and NOD. IFN-γ null mice injected i.v. at 6 weeks of age with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes. Survival curves compared by log-rank test. B and C : In vivo proliferation and activation of CFSE-labeled NOD. Rag1 null .AI4 T cells in PLNs of NOD and NOD. IFN-γ null mice. B : CFSE dilution of AI4 T cells in PLNs of NOD and NOD. IFN-γ null mice at 3 days posttransfer. Representative histograms are shown in the left panel, and mean fluorescence intensity (MFI) of CFSE staining of AI4 T cells is shown in the right panel. C : The frequency of AI4 CD8 + T cells among live PLN cells at 3 days posttransfer. D : CFSE dilution and activation of AI4 T cells in spleens of NOD and NOD. IFN-γ null mice at 8 days posttransfer. Results for each quadrant represent the mean ± SE of three mice per treatment. B – D represent results from a single experiment. * P

    Techniques Used: Mouse Assay, Injection, In Vivo, Activation Assay, Labeling, Fluorescence, Staining

    Human CD8+  T-Cell Transduction and Cell-Mediated Lysis Assays
    Figure Legend Snippet: Human CD8+ T-Cell Transduction and Cell-Mediated Lysis Assays

    Techniques Used: Transduction, Lysis

    49) Product Images from "Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin"

    Article Title: Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E18-04-0228

    FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (
    Figure Legend Snippet: FAK activity of HMEC-1 residing on soft PA hydrogels is decreased, as is Lm uptake. (A) Western blots from whole HMEC-1 lysates showing expression of phosphorylated FAK (Tyr397) and total FAK for cells residing on soft gels (3 kPa), stiff gels (70 kPa), and TC polystyrene substrates with or without 2 μM PF537228 FAK inhibitor. In each Western blot, equal quantities of protein were loaded and equal loading was confirmed in relation to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. In each case, the Western blots shown are representative of three independent experiments. (B, C) Normalized ratios of FAK/GAPDH (B) and pFAK (Tyr397)/GAPDH (C) for HMEC-1 residing on varying-stiffness substrates and treated or not with 2 μM PF537228 FAK inhibitor. Different color circles correspond to data from three independent experiments. Black bars represent the means of the three independent experiments. For each experiment, values have been normalized relative to the ratio for cells residing on polystyrene substrates. (D) Inhibition of bacterial uptake by FAK inhibitors. FAK-14, PF573228, or vehicle control was added 1 h before addition of bacteria to HMEC-1 residing on polystyrene substrates. Percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of inhibitor concentration (mean ± SD, N = 4 replicates). x = 0 corresponds to cells treated with vehicle control. Inset shows the same data with concentration on a log scale. Infection was analyzed by flow cytometry, 7–8 h after infection. MOI is 80. Representative data come from one of three independent experiments. (E) Boxplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or FAK siRNA (siFAK) (means ± SD, three independent experiments and N = 6 replicates per experiment). MOI is 60 (gray) or 20 (green). Circles represent outliers, and the boxplots’ notched sections show the 95% confidence interval around the median (Wilcoxon–Mann–Whitney test; for details about boxplots see Materials and Methods ). One or two asterisks denote statistically significant differences between the medians of two distributions (

    Techniques Used: Activity Assay, Western Blot, Expressing, Inhibition, Infection, Concentration Assay, Flow Cytometry, Cytometry, MANN-WHITNEY

    Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p
    Figure Legend Snippet: Surface vimentin of HMEC-1 is implicated in Lm uptake. (A) Decrease in bacterial uptake after blocking HMEC-1 with anti-vimentin antibody H-84. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of antibody concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry, 7–8 h after infection. (B) Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) for cells treated either with nontargeting siRNA (siNT) or with vimentin siRNA (siVIM) (means ± SD, and N = 6 replicates per experiment). Representative data come from one of three independent experiments. MOI is 50 (black barplots) and 17 (gray barplots). (C) Decreased uptake of Lm when HMEC-1 are treated with withaferin that captures soluble vimentin 30 min prior to infection. Barplots of percentage of HMEC-1 infected with Δ actA Lm (actAp::mTagRFP) as a function of withaferin concentration (means ± SD and N = 6 replicates per experiment). Representative data come from one of three independent experiments. Infection was analyzed by flow cytometry 7–8 h after infection. (D) Percentage of HMEC-1 infected with Lm as a function of the logarithm of MOI (mean ± SD, N = 4 replicates). HMEC-1 were infected with the indicated strains: Δ actA (black), Δ actA/ Δ inlB (gray; actAp::mTagRFP), and HMEC-1 were treated with vehicle control (circle) or withaferin (diamond) for 30 min prior to infection. The frequency of infected HMEC-1 was determined by flow cytometry 7–8 h postinfection. Representative data come from one of three independent experiments. MOI ranged from 50 to120. Two asterisks denote statistically significant differences between the medians of infection fraction of control vs. all other groups ( p

    Techniques Used: Blocking Assay, Infection, Concentration Assay, Flow Cytometry, Cytometry

    50) Product Images from "T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1"

    Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

    Journal: eLife

    doi: 10.7554/eLife.38314

    Cells in the HAVCR2 high subgroup are in a quiescent cell cycle state. ( A ) Left panel: GSEA analysis shows signaling pathways enriched in the HAVCR2 high and blast subpopulations. Right panel: Percentage of cells in each phase of the cell cycle based on single-cell RNA-seq; ( B ) Intracellular FACS analyses of MYC levels in the HAVCR2 high ,HAVCR2 mid , HAVCR2 low and blast subgroups. Gray line: isotype control.
    Figure Legend Snippet: Cells in the HAVCR2 high subgroup are in a quiescent cell cycle state. ( A ) Left panel: GSEA analysis shows signaling pathways enriched in the HAVCR2 high and blast subpopulations. Right panel: Percentage of cells in each phase of the cell cycle based on single-cell RNA-seq; ( B ) Intracellular FACS analyses of MYC levels in the HAVCR2 high ,HAVCR2 mid , HAVCR2 low and blast subgroups. Gray line: isotype control.

    Techniques Used: RNA Sequencing Assay, FACS

    51) Product Images from "Increased Levels of the FoxM1 Transcription Factor Accelerate Development and Progression of Prostate Carcinomas in both TRAMP and LADY Transgenic Mice"

    Article Title: Increased Levels of the FoxM1 Transcription Factor Accelerate Development and Progression of Prostate Carcinomas in both TRAMP and LADY Transgenic Mice

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-05-3138

    FoxM1 is essential for growth and proliferation of PC-3, LNCaP and DU-145 prostate cancer cell lines. We transfected siRNA duplexes specific to either FoxM1 (siFoxM1 #2) or p27 Kip1 (siP27) into PC-3, LNCaP and DU-145 prostate cancer cell lines and at 48 hours after transfection they were used for growth or flow cytometry analysis and at 72 hours after transfection they were used to prepare total RNA. (A) Transfection of FoxM1 siRNA inhibits expression of FoxM1 in PC-3, LNCaP and DU-145 cells. Total RNA was prepared from prostate cancer cell lines PC-3, LNCaP and DU-145 at 48 hours after transfection with either siRNA duplexes specific to FoxM1 (siFoxM1 #2) or p27Kip1 (siP27) or left untransfected and then was analyzed for expression levels of FoxM1 and Cyclophilin by quantitative Real-Time RT-PCR (QRT-PCR) as described in Material and Methods. FoxM1 mRNA levels in each individual sample were normalized to its corresponding Cyclophilin mRNA level. (B) Transfection of FoxM1 siRNAs into prostate cancer cell lines decreases their growth in culture. Prostate cancer cell lines PC-3, LNCaP and DU-145 were transfected with either siFoxM1 #2 or siP27 duplexes or left untransfected and were then re-plated 48 hours after siRNA transfection and cell numbers were counted at day 3, day 4 or day 5 after transfection. A statistically significant decrease in the growth of FoxM1 depleted prostate cancer cells compared to untransfected cells. (C) Flow cytometry analysis of FoxM1 depleted prostate cancer cell lines shows decreased S-phase progression. The indicated prostate cancer cell lines were transfected with siFoxM1 #2 or siP27 duplexes or left untransfected and then subjected to flow cytometry analysis at 72 hours post transfection after staining with propidium iodide. Graphically shown is the percentage of cells accumulating in G1, S, and G2/M (4N) in FoxM1 or p27 Kip1 depleted prostate cancer cells compared to untransfected prostate cancer cells ± SD in triplicate. The asterisks indicate statistically significant increases with P values calculated by Student T Test: *P
    Figure Legend Snippet: FoxM1 is essential for growth and proliferation of PC-3, LNCaP and DU-145 prostate cancer cell lines. We transfected siRNA duplexes specific to either FoxM1 (siFoxM1 #2) or p27 Kip1 (siP27) into PC-3, LNCaP and DU-145 prostate cancer cell lines and at 48 hours after transfection they were used for growth or flow cytometry analysis and at 72 hours after transfection they were used to prepare total RNA. (A) Transfection of FoxM1 siRNA inhibits expression of FoxM1 in PC-3, LNCaP and DU-145 cells. Total RNA was prepared from prostate cancer cell lines PC-3, LNCaP and DU-145 at 48 hours after transfection with either siRNA duplexes specific to FoxM1 (siFoxM1 #2) or p27Kip1 (siP27) or left untransfected and then was analyzed for expression levels of FoxM1 and Cyclophilin by quantitative Real-Time RT-PCR (QRT-PCR) as described in Material and Methods. FoxM1 mRNA levels in each individual sample were normalized to its corresponding Cyclophilin mRNA level. (B) Transfection of FoxM1 siRNAs into prostate cancer cell lines decreases their growth in culture. Prostate cancer cell lines PC-3, LNCaP and DU-145 were transfected with either siFoxM1 #2 or siP27 duplexes or left untransfected and were then re-plated 48 hours after siRNA transfection and cell numbers were counted at day 3, day 4 or day 5 after transfection. A statistically significant decrease in the growth of FoxM1 depleted prostate cancer cells compared to untransfected cells. (C) Flow cytometry analysis of FoxM1 depleted prostate cancer cell lines shows decreased S-phase progression. The indicated prostate cancer cell lines were transfected with siFoxM1 #2 or siP27 duplexes or left untransfected and then subjected to flow cytometry analysis at 72 hours post transfection after staining with propidium iodide. Graphically shown is the percentage of cells accumulating in G1, S, and G2/M (4N) in FoxM1 or p27 Kip1 depleted prostate cancer cells compared to untransfected prostate cancer cells ± SD in triplicate. The asterisks indicate statistically significant increases with P values calculated by Student T Test: *P

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Staining

    52) Product Images from "Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway"

    Article Title: Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060518774687

    Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.
    Figure Legend Snippet: Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Mutagenesis, Staining, In Situ Hybridization

    (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P
    Figure Legend Snippet: (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P

    Techniques Used: Staining, Mouse Assay

    (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.
    Figure Legend Snippet: (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

    (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.
    Figure Legend Snippet: (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.

    Techniques Used: Mouse Assay

    53) Product Images from "A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons"

    Article Title: A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03538-9

    Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01
    Figure Legend Snippet: Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01

    Techniques Used: Purification, Expressing, Mouse Assay, In Situ Hybridization, FACS, RNA Sequencing Assay, Construct, Multiple Displacement Amplification, Two Tailed Test

    54) Product Images from "Chemokines modulate the tumour microenvironment in pituitary neuroendocrine tumours"

    Article Title: Chemokines modulate the tumour microenvironment in pituitary neuroendocrine tumours

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-019-0830-3

    a  Morphological evaluation of GH3 cells after treatment for 72 h with complete medium and RAW 264.7 macrophage-CM, either from untreated macrophages (−PMA_Raw.CM) or macrophages treated with PMA (+PMA_Raw.CM). Data are shown as mean ± standard error of the mean (SEM) for the 6 morphological parameters evaluated by Image J: cell area (μm 2 ), Feret’s diameter (μm), solidity (0–1), perimeter (μm), roundness (0–1) and circularity (0–1). Seventy-five cells were analysed per experiment, with a minimum of 3 experiments per treatment condition. Scale bar 25 μm. ***,
    Figure Legend Snippet: a Morphological evaluation of GH3 cells after treatment for 72 h with complete medium and RAW 264.7 macrophage-CM, either from untreated macrophages (−PMA_Raw.CM) or macrophages treated with PMA (+PMA_Raw.CM). Data are shown as mean ± standard error of the mean (SEM) for the 6 morphological parameters evaluated by Image J: cell area (μm 2 ), Feret’s diameter (μm), solidity (0–1), perimeter (μm), roundness (0–1) and circularity (0–1). Seventy-five cells were analysed per experiment, with a minimum of 3 experiments per treatment condition. Scale bar 25 μm. ***,

    Techniques Used:

    a  Transwell chemotaxis assay performed on RAW 264.7 macrophages towards complete medium, GH3-CM and recombinant CX3CL1 (rCX3CL1) at concentration 100 ng/mL for 72 h. Data are shown as mean ± standard error of the mean (SEM) for the ratio of migrated RAW 264.7 macrophages towards GH3-CM or rCX3CL1 in relation to migrated macrophages in complete medium.  n  = 6. ***,
    Figure Legend Snippet: a Transwell chemotaxis assay performed on RAW 264.7 macrophages towards complete medium, GH3-CM and recombinant CX3CL1 (rCX3CL1) at concentration 100 ng/mL for 72 h. Data are shown as mean ± standard error of the mean (SEM) for the ratio of migrated RAW 264.7 macrophages towards GH3-CM or rCX3CL1 in relation to migrated macrophages in complete medium. n  = 6. ***,

    Techniques Used: Chemotaxis Assay, Recombinant, Concentration Assay

    55) Product Images from "Ankyrin-G regulated epithelial phenotype is required for mouse lens morphogenesis and growth"

    Article Title: Ankyrin-G regulated epithelial phenotype is required for mouse lens morphogenesis and growth

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2018.12.016

    ), and below it is a schematic of the membrane binding domain (MBD), spectrin binding domain (SBD), variable region (VR) and C-term domain (CTD) of AnkG protein. The double arrows in the bottom panel indicate two closely migrating, distinct DNA products). E  F). Immunoblotting analysis of distribution of AnkG in the mouse lens epithelium and fiber fractions derived from three independent pooled specimens of P1 lenses and its relative levels in these fractions. G). Immunofluorescence analysis of distribution of AnkG in the developing lenses from E12.5 and E16.5 embryonic and P21 mice (arrows indicate discrete distribution of AnkG to the presumptive lens epithelium), and H). Immunofluorescence analysis of the colocalization of AnkG with E-cadherin, β-catenin, Zo-1, ezrin, β-spectrin and β-actin in the lens epithelium of P1 mouse. GAPDH was immunoblotted as a loading control in data shown in panels B    E. Where indicated scale bars represent image magnification, with the molecular mass of proteins reported in kDa units and nucleotide base pairs of amplified DNA product reported as kb units. EPF: Elongating primary fibers.
    Figure Legend Snippet: ), and below it is a schematic of the membrane binding domain (MBD), spectrin binding domain (SBD), variable region (VR) and C-term domain (CTD) of AnkG protein. The double arrows in the bottom panel indicate two closely migrating, distinct DNA products). E F). Immunoblotting analysis of distribution of AnkG in the mouse lens epithelium and fiber fractions derived from three independent pooled specimens of P1 lenses and its relative levels in these fractions. G). Immunofluorescence analysis of distribution of AnkG in the developing lenses from E12.5 and E16.5 embryonic and P21 mice (arrows indicate discrete distribution of AnkG to the presumptive lens epithelium), and H). Immunofluorescence analysis of the colocalization of AnkG with E-cadherin, β-catenin, Zo-1, ezrin, β-spectrin and β-actin in the lens epithelium of P1 mouse. GAPDH was immunoblotted as a loading control in data shown in panels B E. Where indicated scale bars represent image magnification, with the molecular mass of proteins reported in kDa units and nucleotide base pairs of amplified DNA product reported as kb units. EPF: Elongating primary fibers.

    Techniques Used: Binding Assay, Derivative Assay, Immunofluorescence, Mouse Assay, Amplification

    56) Product Images from "Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease"

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease

    Journal: Kidney international

    doi: 10.1038/ki.2010.106

    RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.
    Figure Legend Snippet: RNA extracted from whole urine cells and debris has a different RNA profile from that of tissue and urinary microvesicles ( a ) Analysis of RNA isolated from whole urine (exclusive of microvesicles that are not captured by the isolation technique) showed that a large yield of nucleic acids can be isolated (see the red profile). Processing of the isolated nucleic acids using the RNeasy Plus Micro kit (which removes gDNA) reveals that the majority of nucleic acids isolated using the ZR urine RNA isolation kit is DNA and the remaining RNA lacks rRNA peaks found in tissue and urinary exosomes. Red — nucleic acids isolated from whole urine without gDNA removal, blue — nucleic acids isolated from whole urine post gDNA removal using the RNeasy Plus Micro kit. ( b ) Isolation of microvesicles from the same urine sample revealed that the microvesicles retained a normal total RNA profile suggesting that RNA within whole cells may be less stable than that contained in urinary microvesicles. Red — without removal of gDNA, blue — sample processed using the RNeasy Plus Micro kit to remove contaminating gDNA. ( c ) Isolation of nucleic acids from the pellet formed during the 300 g spin revealed that the nucleic acid profile was different from that of microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 300 g pellet without gDNA removal, blue — nucleic acid isolated from the 300 g pellet post gDNA removal using the RNeasy Plus Micro kit. ( d ) Isolation of nucleic acids from pellets formed during the 17,000 g spin revealed that the nucleic acid profile was different to microvesicles and that it contained a large amount of gDNA following processing using the RNeasy Plus Micro kit. Red — nucleic acids isolated from the 17,000 g pellet without gDNA removal, blue — nucleic acids isolated from the 17,000 g pellet post gDNA removal using the RNeasy Plus Micro kit.

    Techniques Used: Isolation

    Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.
    Figure Legend Snippet: Analysis of nucleic acids associated with urinary microvesicles using the Agilent Bioanalyzer ( a ) Plot showing that microvesicles may co-isolate with extraneous DNA that can be removed by DNase digestion of the microvesicle pellet prior to lysis and nucleic acid extraction. Red — profile without DNase digestion, blue — profile with DNase digestion. ( b ) Plot showing that microvesicles do not co-isolate with detectable levels of extraneous RNA. Red — without RNase digestion, blue — with RNase digestion. ( c ) RNA isolated from rat kidney (red) and microvesicles (blue) exhibited a very similar profile, including the presence of 18S and 28S rRNA peaks. Both samples underwent processing using the RNeasy Plus Micro kit to remove genomic DNA (gDNA) contamination. ( d ) Urinary microvesicles contain a prominent ‘small RNA’ peak between 25–200 nt when miRNA isolation techniques are used. Red — kidney RNA isolated using RNeasy Plus Micro kit using the miRNA extraction method, blue — microvesicle RNA isolated with RNeasy Plus Micro kit using the miRNA extraction method. ( e ) Nucleic acids were isolated from microvesicles that had undergone RNase and DNase digestion on the outside before microvesicle lysis. During RNA extraction using the RNeasy Micro kit, half of the samples underwent on-column RNase digestion (see Materials and methods) while the other half underwent the same on-column incubation without the presence of RNase. Results revealed that RNase digestion was able to remove the majority of the profile, suggesting that RNA is the major nucleic acid within urinary microvesicles. Red — nucleic acid profile without intra-microvesicular RNase digestion, blue — nucleic acid profile with intra-microvesicular RNase digestion. ( f ) Further digestion with DNase following RNase digestion revealed that the remaining peak could be further reduced, suggesting that some material prone to DNase digestion remained in the sample potentially representing intra-exosomal DNA. Red — nucleic acid profile following intra-microvesicular on-column RNase digestion alone, blue — nucleic acid profile following both intra-microvesicular on-column RNase and DNase digestion. 18S and 28S rRNA peaks are indicated in ( a ). The peak at 25 nt represents an internal standard.

    Techniques Used: Lysis, Isolation, RNA Extraction, Incubation

    57) Product Images from "Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate"

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    Journal: EMBO Molecular Medicine

    doi: 10.15252/emmm.201606382

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.
    Figure Legend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Techniques Used: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification

    58) Product Images from "High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos"

    Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172171

    Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.
    Figure Legend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

    Techniques Used: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

    Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.
    Figure Legend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

    Techniques Used: RNA Extraction, Migration, Isolation

    59) Product Images from "Expression profiling of genes regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary fibrosis"

    Article Title: Expression profiling of genes regulated by Fra-1/AP-1 transcription factor during bleomycin-induced pulmonary fibrosis

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-381

    Analysis of some gene expression between Fra-1 +/+ and Fra-1 Δ/Δ mice at different time points after bleomycin treatment. Lung mRNA abundance was determined by quantitative real-time RT-PCR. The graphs represent the fold change over vehicle treated Fra-1 +/+ controls after normalization with the expression of GAPDH. Results are mean ± SD for 3-4 mice in each group. * p
    Figure Legend Snippet: Analysis of some gene expression between Fra-1 +/+ and Fra-1 Δ/Δ mice at different time points after bleomycin treatment. Lung mRNA abundance was determined by quantitative real-time RT-PCR. The graphs represent the fold change over vehicle treated Fra-1 +/+ controls after normalization with the expression of GAPDH. Results are mean ± SD for 3-4 mice in each group. * p

    Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR

    Validation of the differences in mRNA expression between Fra-1 +/+ and Fra-1 Δ/Δ mice for selected microarray genes at different time points after bleomycin treatment. Lung mRNA abundance was determined by quantitative real-time RT-PCR. The graphs represent the fold change over vehicle treated Fra-1 +/+ controls after normalization with the expression of GAPDH. Results are mean ± SD for 3-4 mice in each group. * p
    Figure Legend Snippet: Validation of the differences in mRNA expression between Fra-1 +/+ and Fra-1 Δ/Δ mice for selected microarray genes at different time points after bleomycin treatment. Lung mRNA abundance was determined by quantitative real-time RT-PCR. The graphs represent the fold change over vehicle treated Fra-1 +/+ controls after normalization with the expression of GAPDH. Results are mean ± SD for 3-4 mice in each group. * p

    Techniques Used: Expressing, Mouse Assay, Microarray, Quantitative RT-PCR

    60) Product Images from "Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit"

    Article Title: Loss of Tbx3 in murine neural crest reduces enteric glia and causes cleft palate, but does not influence heart development or bowel transit

    Journal: Developmental biology

    doi: 10.1016/j.ydbio.2018.09.017

    Molecular mechanisms of palatal shelf elevation (A-F) EdU incoorporation assay was used to identify proliferative cells at E13.5 in the palatal mesenchyme four hours after EdU injection. The ratio of EdU+ cells to total cells (Hoechst+) does not differ significantly in the anterior (A-C) or posterior (D-F) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (G-L) Immunostaining for cleaved caspase 3 was used to identify apoptotic cells at E13.5 in the palatal mesenchyme. The ratio of cleaved caspase3+ cells to total cells does not differ significantly in the anterior (G-I) or posterior (J-L) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (M) Quantitative RT-PCR analysis was performed to determine expression of genes known to regulate palate development. Osr2 mRNA levels were markedly reduced in Tbx3- deficient mice at E13.5 (p = 0.0079). Expression levels are relative to mean expression in Tbx3 controls. Error bar = SEM. (N=5 of each genotype). Scale bar =100 μm.
    Figure Legend Snippet: Molecular mechanisms of palatal shelf elevation (A-F) EdU incoorporation assay was used to identify proliferative cells at E13.5 in the palatal mesenchyme four hours after EdU injection. The ratio of EdU+ cells to total cells (Hoechst+) does not differ significantly in the anterior (A-C) or posterior (D-F) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (G-L) Immunostaining for cleaved caspase 3 was used to identify apoptotic cells at E13.5 in the palatal mesenchyme. The ratio of cleaved caspase3+ cells to total cells does not differ significantly in the anterior (G-I) or posterior (J-L) region of the palatal shelf in Tbx3 fl/fl; Wnt1-Cre mice. (Student’s t-test, p > 0.05, N=3 of each genotype). (M) Quantitative RT-PCR analysis was performed to determine expression of genes known to regulate palate development. Osr2 mRNA levels were markedly reduced in Tbx3- deficient mice at E13.5 (p = 0.0079). Expression levels are relative to mean expression in Tbx3 controls. Error bar = SEM. (N=5 of each genotype). Scale bar =100 μm.

    Techniques Used: Injection, Mouse Assay, Immunostaining, Quantitative RT-PCR, Expressing

    Enteric neural crest-derived precursors colonize the bowel normally in Tbx3 fl/fl; Wnt1-Cre mice at E13.5 (A-B) At E 13.5 the colon is colonized by TuJ1+ ENCDC that have nearly completed their rostro-caudal migration from the vagal neural tube down to the distal colon in both Tbx3 fl/fl; Wnt1-Cre mice and control littermates. (C) Measurements of the percent of colon that is colonized by TuJ1+ fibers that accompany migrating ENCDC. (D) Mean absolute length of colon in Tbx3 fl/fl; Wnt1-Cre mice and control littermate is comparable. (Student’s t-test, p > 0.05, N=7 control, N=4 mutant). Error bar = SEM. Scale bar = 500 μm. Scale bar in B also applies to A.
    Figure Legend Snippet: Enteric neural crest-derived precursors colonize the bowel normally in Tbx3 fl/fl; Wnt1-Cre mice at E13.5 (A-B) At E 13.5 the colon is colonized by TuJ1+ ENCDC that have nearly completed their rostro-caudal migration from the vagal neural tube down to the distal colon in both Tbx3 fl/fl; Wnt1-Cre mice and control littermates. (C) Measurements of the percent of colon that is colonized by TuJ1+ fibers that accompany migrating ENCDC. (D) Mean absolute length of colon in Tbx3 fl/fl; Wnt1-Cre mice and control littermate is comparable. (Student’s t-test, p > 0.05, N=7 control, N=4 mutant). Error bar = SEM. Scale bar = 500 μm. Scale bar in B also applies to A.

    Techniques Used: Derivative Assay, Mouse Assay, Migration, Mutagenesis

    61) Product Images from "Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes"

    Article Title: Interferon-γ Limits Diabetogenic CD8+ T-Cell Effector Responses in Type 1 Diabetes

    Journal: Diabetes

    doi: 10.2337/db16-0846

    IFN-γ–producing CD4 +  T cells suppress diabetogenic CD8 +  T cells through mechanisms that do not involve quantitative or functional variations in Tregs.  A : Quantitative PCR analysis of IFN-γ mRNA expression by host-type CD4 +  and CD8 +  (Vα8 − ) T cells and B cells purified from spleens of NOD mice 3 days postadoptive transfer with 2 × 10 7  NOD. Rag1 null .AI4  splenocytes (post-AT) or untreated NOD mice (untreated). Results represent the mean ± SE of three samples per treatment.  B : Diabetes incidence for female NOD. scid  mice injected at 6–8 weeks of age with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes in the presence or absence of 3 × 10 6  CD4 +  T cells purified from NOD or NOD. IFN-γ null  donors.  C : Frequencies, numbers, and mean fluorescence intensity (MFI) of FoxP3 antibody staining of splenic CD4 + CD25 + FoxP3 +  Tregs in NOD and NOD. IFN-γ null  mice. Results represent the mean ± SE of five mice per treatment.  D : Crisscross cultures were established to assess the ability of CD4 + CD25 +  Tregs from NOD and NOD. IFN-γ null  mice to suppress the anti-CD3–stimulated proliferation of CD4 + CD25 −  effectors from both strains (assessed by flow cytometic detection of CFSE dilution).  E : Beginning at 6 weeks of age, NOD female mice received three biweekly i.p. injections with the Treg-depleting CD25-specific PC61 antibody or a rat IgG1 isotype control. One week after the first treatment, mice in both groups were injected i.v. with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes and subsequently monitored for diabetes. Survival curves compared by log-rank test. * P
    Figure Legend Snippet: IFN-γ–producing CD4 + T cells suppress diabetogenic CD8 + T cells through mechanisms that do not involve quantitative or functional variations in Tregs. A : Quantitative PCR analysis of IFN-γ mRNA expression by host-type CD4 + and CD8 + (Vα8 − ) T cells and B cells purified from spleens of NOD mice 3 days postadoptive transfer with 2 × 10 7 NOD. Rag1 null .AI4 splenocytes (post-AT) or untreated NOD mice (untreated). Results represent the mean ± SE of three samples per treatment. B : Diabetes incidence for female NOD. scid mice injected at 6–8 weeks of age with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes in the presence or absence of 3 × 10 6 CD4 + T cells purified from NOD or NOD. IFN-γ null donors. C : Frequencies, numbers, and mean fluorescence intensity (MFI) of FoxP3 antibody staining of splenic CD4 + CD25 + FoxP3 + Tregs in NOD and NOD. IFN-γ null mice. Results represent the mean ± SE of five mice per treatment. D : Crisscross cultures were established to assess the ability of CD4 + CD25 + Tregs from NOD and NOD. IFN-γ null mice to suppress the anti-CD3–stimulated proliferation of CD4 + CD25 − effectors from both strains (assessed by flow cytometic detection of CFSE dilution). E : Beginning at 6 weeks of age, NOD female mice received three biweekly i.p. injections with the Treg-depleting CD25-specific PC61 antibody or a rat IgG1 isotype control. One week after the first treatment, mice in both groups were injected i.v. with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes and subsequently monitored for diabetes. Survival curves compared by log-rank test. * P

    Techniques Used: Functional Assay, Real-time Polymerase Chain Reaction, Expressing, Purification, Mouse Assay, Injection, Fluorescence, Staining, Flow Cytometry

    IFN-γ produced by T or B cells suppresses AI4 T cells through both direct and indirect mechanisms.  A : Diabetes incidence in female NOD and NOD. IFN- γ R null  recipient mice injected i.v. at 6 weeks of age with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes.  B : Diabetes incidence in female NOD, NOD. IFN- γ R null , and NOD. IFN- γ null  recipient mice injected i.v. at 6 weeks of age with 1 × 10 6  purified AI4 T cells.  C : Diabetes incidence in female NOD. Rag1 null  and NOD. Rag1 null .IFN- γ R null  recipient mice injected i.v. with 1 × 10 6  purified AI4 T cells and 2 × 10 6  purified NOD splenic CD4 +  T cells.  D : Diabetes incidence in female NOD. Rag1 null  recipient mice injected i.v. with 1 × 10 7  NOD. Rag1 null .AI4  splenocytes and 2 × 10 6  purified CD4 +  splenic T cells from NOD or NOD. IFN- γ R null  donors. Survival curves compared by log-rank test.
    Figure Legend Snippet: IFN-γ produced by T or B cells suppresses AI4 T cells through both direct and indirect mechanisms. A : Diabetes incidence in female NOD and NOD. IFN- γ R null recipient mice injected i.v. at 6 weeks of age with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes. B : Diabetes incidence in female NOD, NOD. IFN- γ R null , and NOD. IFN- γ null recipient mice injected i.v. at 6 weeks of age with 1 × 10 6 purified AI4 T cells. C : Diabetes incidence in female NOD. Rag1 null and NOD. Rag1 null .IFN- γ R null recipient mice injected i.v. with 1 × 10 6 purified AI4 T cells and 2 × 10 6 purified NOD splenic CD4 + T cells. D : Diabetes incidence in female NOD. Rag1 null recipient mice injected i.v. with 1 × 10 7 NOD. Rag1 null .AI4 splenocytes and 2 × 10 6 purified CD4 + splenic T cells from NOD or NOD. IFN- γ R null donors. Survival curves compared by log-rank test.

    Techniques Used: Produced, Mouse Assay, Injection, Purification

    62) Product Images from "A novel organotypic 3D sweat gland model with physiological functionality"

    Article Title: A novel organotypic 3D sweat gland model with physiological functionality

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0182752

    Gene expression profile analysis via qRT-PCR of in vitro cultures of the eccrine sweat gland. (A) In multicellular spheroids, expression of CEACAM5 as a differentiation-related marker was significantly induced compared to 2D cultures. Further, gene expression in 3D was on a similar level to native sweat glands (One way ANOVA; n≥8, mean±SEM, p
    Figure Legend Snippet: Gene expression profile analysis via qRT-PCR of in vitro cultures of the eccrine sweat gland. (A) In multicellular spheroids, expression of CEACAM5 as a differentiation-related marker was significantly induced compared to 2D cultures. Further, gene expression in 3D was on a similar level to native sweat glands (One way ANOVA; n≥8, mean±SEM, p

    Techniques Used: Expressing, Quantitative RT-PCR, In Vitro, Marker

    63) Product Images from "Neuropilin 1 sequestration by neuropathogenic mutant glycyl-tRNA synthetase is permissive to vascular homeostasis"

    Article Title: Neuropilin 1 sequestration by neuropathogenic mutant glycyl-tRNA synthetase is permissive to vascular homeostasis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10005-w

    Pathways integral to vascular homeostasis appear unperturbed in mutant Gars muscle. ( A ) Representative Nrp1 (white) and VEGFR2 (red) staining in one month old wholemount wild-type and Gars C201R /+ TVA muscles. Nrp1 and VEGFR2 colocalise with the endothelial marker IB4 (green) in both genotypes, and show no obvious differences. A similar pattern was seen at three months and in the lumbrical muscles at both time points (data not shown). Both panels are single plane images. Scale bars = 200 μm. ( B,C ) qPCR analysis of a series of genes integral to the maintenance of blood vessels indicates very little perturbation in gene expression in one month Gars C201R /+ tibialis anterior muscles. This was confirmed using either Gapdh (top graphs) or endothelium-specific Pecam1 (bottom graphs) as the reference gene. All individual gene data sets were separately analysed with unpaired, two-tailed t -tests. n = 6 wild-type and 4 Gars C201R /+ .
    Figure Legend Snippet: Pathways integral to vascular homeostasis appear unperturbed in mutant Gars muscle. ( A ) Representative Nrp1 (white) and VEGFR2 (red) staining in one month old wholemount wild-type and Gars C201R /+ TVA muscles. Nrp1 and VEGFR2 colocalise with the endothelial marker IB4 (green) in both genotypes, and show no obvious differences. A similar pattern was seen at three months and in the lumbrical muscles at both time points (data not shown). Both panels are single plane images. Scale bars = 200 μm. ( B,C ) qPCR analysis of a series of genes integral to the maintenance of blood vessels indicates very little perturbation in gene expression in one month Gars C201R /+ tibialis anterior muscles. This was confirmed using either Gapdh (top graphs) or endothelium-specific Pecam1 (bottom graphs) as the reference gene. All individual gene data sets were separately analysed with unpaired, two-tailed t -tests. n = 6 wild-type and 4 Gars C201R /+ .

    Techniques Used: Mutagenesis, Staining, Marker, Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test

    Capillaries appear unaffected in Gars C201R /+ skeletal muscles. ( A,B ) Representative single plane (A, top) and collapsed Z-stack ( A , bottom and B ) images of IB4 (green) staining in one month lumbrical ( A ) and TVA muscles ( B ). Scale bars = 1 mm ( A , top) and 20 μm ( A , bottom and B ). ( C,E,G ) No defects in capillary diameter (C, age, P = 0.007; genotype, P = 0.090; interaction, P = 0.508), density ( E , age, P
    Figure Legend Snippet: Capillaries appear unaffected in Gars C201R /+ skeletal muscles. ( A,B ) Representative single plane (A, top) and collapsed Z-stack ( A , bottom and B ) images of IB4 (green) staining in one month lumbrical ( A ) and TVA muscles ( B ). Scale bars = 1 mm ( A , top) and 20 μm ( A , bottom and B ). ( C,E,G ) No defects in capillary diameter (C, age, P = 0.007; genotype, P = 0.090; interaction, P = 0.508), density ( E , age, P

    Techniques Used: Staining

    The blood vessel network is unperturbed in Gars C201R /+ retinas. ( A ) Representative single plane (top) and collapsed Z-stack (bottom) images of IB4 (green) staining in one month retina. Scale bars = 1 mm (top) and 20 μm (bottom). ( B–E ) No defects in capillary diameter ( B , age, P = 0.823; genotype, P = 0.112; interaction, P = 0.105), density ( C , age, P = 0.878; genotype, P = 0.638; interaction, P = 0.504), branching density ( D , age, P = 0.042; genotype, P = 0.548; interaction, P = 0.345), or major radial branch (arteries and veins, arrow in A ) number (E, age, P = 0.541; genotype, P = 0.395; interaction, P = 0.188) were observed in Gars C201R /+ mouse retinas at one or three months. n = 5. ( F ) Representative single plane images of IB4 (i) and αSMA (red, ii) staining in P6-7 retina. Letters a and b (i) indicate how vascular extension was assessed ( a / a + b ) and the arrowhead (ii) highlights an αSMA + artery. Scale bars = 1 mm. ( G,H ) There is no difference in IB4 + vascular extension ( G , P = 0.818) or αSMA + artery number ( H , P = 0.967) between wild-type and Gars C201R /+ P6-7 retinas. n = 3 wild-type and 5 Gars C201R /+ . Data sets were analysed with either a two-way ANOVA ( B – E ) or an unpaired, two-tailed t -test ( G , H ). See also Fig. S2 .
    Figure Legend Snippet: The blood vessel network is unperturbed in Gars C201R /+ retinas. ( A ) Representative single plane (top) and collapsed Z-stack (bottom) images of IB4 (green) staining in one month retina. Scale bars = 1 mm (top) and 20 μm (bottom). ( B–E ) No defects in capillary diameter ( B , age, P = 0.823; genotype, P = 0.112; interaction, P = 0.105), density ( C , age, P = 0.878; genotype, P = 0.638; interaction, P = 0.504), branching density ( D , age, P = 0.042; genotype, P = 0.548; interaction, P = 0.345), or major radial branch (arteries and veins, arrow in A ) number (E, age, P = 0.541; genotype, P = 0.395; interaction, P = 0.188) were observed in Gars C201R /+ mouse retinas at one or three months. n = 5. ( F ) Representative single plane images of IB4 (i) and αSMA (red, ii) staining in P6-7 retina. Letters a and b (i) indicate how vascular extension was assessed ( a / a + b ) and the arrowhead (ii) highlights an αSMA + artery. Scale bars = 1 mm. ( G,H ) There is no difference in IB4 + vascular extension ( G , P = 0.818) or αSMA + artery number ( H , P = 0.967) between wild-type and Gars C201R /+ P6-7 retinas. n = 3 wild-type and 5 Gars C201R /+ . Data sets were analysed with either a two-way ANOVA ( B – E ) or an unpaired, two-tailed t -test ( G , H ). See also Fig. S2 .

    Techniques Used: Staining, Two Tailed Test

    Mutant GlyRS aberrantly binds to the transmembrane receptor neuropilin 1. ( A ) Schematic of Nrp1 and one of its principal co-receptors, VEGFR2, which together bind to the secreted glycoprotein VEGF-A 165 (on the left) 11 . VEGF-A functions in vasculogenesis, angiogenesis, and arteriogenesis, with the latter two processes occurring through Nrp1 and VEGFR2 signalling 17 , 18 . VEGF-A is also critical for nervous system development and maintenance 15 . VEGF-A 165 binding to Nrp1 is mainly dependent by the b1 domain 12 . CMT2D-associated mutations in GARS cause a conformational opening of GlyRS, allowing the aberrant binding of mutant GlyRS to the b1 domain of Nrp1 (on the right) 10 . This competitively antagonises Nrp1/VEGF-A signalling, which contributes to motor deficits observed in CMT2D mice 10 . Schematics are not drawn to scale and adapted from Plein et al . 18 and He et al . 10 . ( B ) Co-immunoprecipitation of endogenous Nrp1 showing aberrant interaction with P234KY, L129P, and C157R GlyRS (ectopically expressed with a V5-tag) in NSC-34 cells. Wild-type GlyRS shows no significant binding to Nrp1. The aberrant interaction is weaker with C157R than with P234KY GlyRS, which correlates with the severity of CMT phenotypes in Gars C201R /+ and Gars Nmf249 /+ mice, respectively.
    Figure Legend Snippet: Mutant GlyRS aberrantly binds to the transmembrane receptor neuropilin 1. ( A ) Schematic of Nrp1 and one of its principal co-receptors, VEGFR2, which together bind to the secreted glycoprotein VEGF-A 165 (on the left) 11 . VEGF-A functions in vasculogenesis, angiogenesis, and arteriogenesis, with the latter two processes occurring through Nrp1 and VEGFR2 signalling 17 , 18 . VEGF-A is also critical for nervous system development and maintenance 15 . VEGF-A 165 binding to Nrp1 is mainly dependent by the b1 domain 12 . CMT2D-associated mutations in GARS cause a conformational opening of GlyRS, allowing the aberrant binding of mutant GlyRS to the b1 domain of Nrp1 (on the right) 10 . This competitively antagonises Nrp1/VEGF-A signalling, which contributes to motor deficits observed in CMT2D mice 10 . Schematics are not drawn to scale and adapted from Plein et al . 18 and He et al . 10 . ( B ) Co-immunoprecipitation of endogenous Nrp1 showing aberrant interaction with P234KY, L129P, and C157R GlyRS (ectopically expressed with a V5-tag) in NSC-34 cells. Wild-type GlyRS shows no significant binding to Nrp1. The aberrant interaction is weaker with C157R than with P234KY GlyRS, which correlates with the severity of CMT phenotypes in Gars C201R /+ and Gars Nmf249 /+ mice, respectively.

    Techniques Used: Mutagenesis, Binding Assay, Mouse Assay, Immunoprecipitation

    Gars mutant embryonic hindbrains and sciatic nerves show no vascular defects. ( A,B ) Representative single plane ( A , top and B ) and collapsed Z-stack ( A , bottom) images of IB4 and Pecam1 staining (green) in E13.5 hindbrain ( A ) and one month sciatic nerve ( B ), respectively. Scale bars = 1 mm ( A , top) and 20 μm ( A , bottom, and B ). ( C–E ) Capillary diameter ( C , P = 0.593), density ( D , P = 0.456), and branching density ( E , P = 0.278) are all unimpaired in Gars C201R /+ E13.5 hindbrains. n = 9 wild-type and 5 Gars C201R /+ . ( F ) Mutant sciatic nerves show no difference in capillary density at one month ( P = 0.111). n = 4 wild-type and 5 Gars C201R /+ . All data were analysed with unpaired, two-tailed t -tests. See also Fig. S2 .
    Figure Legend Snippet: Gars mutant embryonic hindbrains and sciatic nerves show no vascular defects. ( A,B ) Representative single plane ( A , top and B ) and collapsed Z-stack ( A , bottom) images of IB4 and Pecam1 staining (green) in E13.5 hindbrain ( A ) and one month sciatic nerve ( B ), respectively. Scale bars = 1 mm ( A , top) and 20 μm ( A , bottom, and B ). ( C–E ) Capillary diameter ( C , P = 0.593), density ( D , P = 0.456), and branching density ( E , P = 0.278) are all unimpaired in Gars C201R /+ E13.5 hindbrains. n = 9 wild-type and 5 Gars C201R /+ . ( F ) Mutant sciatic nerves show no difference in capillary density at one month ( P = 0.111). n = 4 wild-type and 5 Gars C201R /+ . All data were analysed with unpaired, two-tailed t -tests. See also Fig. S2 .

    Techniques Used: Mutagenesis, Staining, Two Tailed Test

    64) Product Images from "Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway"

    Article Title: Lens-specific deletion of the Msx2 gene increased apoptosis by enhancing the caspase-3/caspase-8 signaling pathway

    Journal: The Journal of International Medical Research

    doi: 10.1177/0300060518774687

    Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.
    Figure Legend Snippet: Generation of Msx2 CKO mice. (a) Mating scheme. (b) Genotyping performed by PCR. The 350 bp band represents Le-Cre; the 347 bp band represents the wild-type; and the 381 bp band represents the mutant. (c) The recombination pattern of Le-Cre was detected by β-galactosidase whole mount staining at E10.5 and E14.5. (d) Whole mount in situ hybridization showing the absence of Msx2 mRNA in the Msx2 CKO lens vesicle at E10.5 and E11.5. (e) Msx2 CKO mice lacked eyelashes and hair on the surface of their eye lids and in a stripe running from the temporal to the nasal side of the eye (n≥3 per genotype) at P21. Msx2 CKO mice showing reduced lens size and microphthalmia at P2. Histological sections of the Msx2 CKO eyeballs showing reduction in the lens size, displacement of lens fiber nuclei toward the anterior and posterior of the lens, and vacuolation of cortical fiber cells at P21.

    Techniques Used: Mouse Assay, Polymerase Chain Reaction, Mutagenesis, Staining, In Situ Hybridization

    (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P
    Figure Legend Snippet: (a) HE staining. At E9.5, the optic vesicle (OV) is in contact with the lens placode and no structural differences are observed between Msx2 CKO and Msx2 WT embryos. At E10.5, the lens vesicle that had invaginated into the optic cup is considerably larger in the developing eyes of Msx2 WT mice. At E11.5, the lens vesicle has closed in the eyes of both Msx2 WT and Msx2 CKO mice, but appears smaller in Msx2 CKO embryo. At E12.5, the epithelium of the lens vesicle has completely separated from the surface ectoderm in Msx2 WT mice, but remains adherent in Msx2 CKO mice. Black arrow: lens stalk. Re, Retina; Le, Lens; Co, Cornea. OV, optic vesicle. (b) Column diagram of the horizontal lens diameter (left) and anteroposterior diameter (right) of Msx2 CKO and Msx2 WT mice between E9 and E12.5. Significant differences from E10.5 to E12.5 were observed between the two groups ( P

    Techniques Used: Staining, Mouse Assay

    (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.
    Figure Legend Snippet: (a) RNA microarray and differentially expressed gene analysis. Scatter plot showing upregulated genes (red) and downregulated genes (green) ( > 2 fold). Casp3 and Casp8 are shown. (b) Real-time quantitative PCR was used to detect Casp3 and Casp8 expression in the lens at P1. (c, d) Whole mount in situ hybridization of FoxE3 expression in embryonic development. FoxE3 mRNA expression was dramatically reduced in the Msx2 CKO lens vesicles compared with Msx2 WT lens vesicles at E10.5 and E11.5.

    Techniques Used: Microarray, Real-time Polymerase Chain Reaction, Expressing, In Situ Hybridization

    (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.
    Figure Legend Snippet: (a) At E10.5, a significantly higher level of lens epithelial cell apoptosis was observed that was mainly concentrated near the lens stalk in Msx2 CKO embryos. (b, c) From E12.5 to E16.5, very few apoptotic lens epithelial cells were observed in Msx2 WT mice, but the absolute number of lens epithelial cells undergoing apoptosis was significantly increased in Msx2 CKO mice compared with Msx2 WT mice.

    Techniques Used: Mouse Assay

    65) Product Images from "T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1"

    Article Title: T-ALL leukemia stem cell 'stemness' is epigenetically controlled by the master regulator SPI1

    Journal: eLife

    doi: 10.7554/eLife.38314

    HAVCR2 redefines a heterogeneous LSC-enriched subpopulation at single-cell resolution ( A ) WGCNA analysis for the bulk RNA-seq of LSC-enriched and leukemic blast subpopulations. The yellow module contains 220 genes that are preferentially expressed in the LSC-enriched subpopulation (LSC high -Blast 0 ); ( B ) Gene Ontology (GO) analysis of LSC-enriched genes in the yellow module; ( C ) Havcr2 and Itgax are specifically expressed in LSC-enriched (red) but not in leukemic blast (blue) subpopulations isolated from the indicated hematopoietic organs of M1-M4 Pten -null T-ALL mice; ( D ) Upper panel: FACS plots are overlaid to show the differential expression of HAVCR2 and ITGAX in the LSC and blast subpopulations. The previously defined Lin - CD3 + KIT mid LSC-enriched subpopulation (in the red box in the left panel) can be further separated into several subgroups based on the expression of the cell-surface markers HAVCR2 and ITGAX. The Lin - CD3 + KIT - leukemic blast subpopulation (in the blue box in the left panel) does not express HAVCR2 or ITGAX. Lower panel: Quantitative measurement of the HAVCR2 high , HAVCR2 mid and HAVCR2 low subgroups in different hematopoietic organs from Pten -null T-ALL mice (n = 5; *, p
    Figure Legend Snippet: HAVCR2 redefines a heterogeneous LSC-enriched subpopulation at single-cell resolution ( A ) WGCNA analysis for the bulk RNA-seq of LSC-enriched and leukemic blast subpopulations. The yellow module contains 220 genes that are preferentially expressed in the LSC-enriched subpopulation (LSC high -Blast 0 ); ( B ) Gene Ontology (GO) analysis of LSC-enriched genes in the yellow module; ( C ) Havcr2 and Itgax are specifically expressed in LSC-enriched (red) but not in leukemic blast (blue) subpopulations isolated from the indicated hematopoietic organs of M1-M4 Pten -null T-ALL mice; ( D ) Upper panel: FACS plots are overlaid to show the differential expression of HAVCR2 and ITGAX in the LSC and blast subpopulations. The previously defined Lin - CD3 + KIT mid LSC-enriched subpopulation (in the red box in the left panel) can be further separated into several subgroups based on the expression of the cell-surface markers HAVCR2 and ITGAX. The Lin - CD3 + KIT - leukemic blast subpopulation (in the blue box in the left panel) does not express HAVCR2 or ITGAX. Lower panel: Quantitative measurement of the HAVCR2 high , HAVCR2 mid and HAVCR2 low subgroups in different hematopoietic organs from Pten -null T-ALL mice (n = 5; *, p

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

    66) Product Images from "Negative Evidence for a Functional Role of Neuronal DNMT3a in Persistent Pain"

    Article Title: Negative Evidence for a Functional Role of Neuronal DNMT3a in Persistent Pain

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00332

    Dnmt3a knockdown after tamoxifen administration to AdvCre ERT2 -Dnmt3a fl/fl /Dnmt3b fl/fl mice. DNMT expression measured by qRT-PCR in whole L3-L5 DRG taken from sensory neuron specific Dnmt3a and Dnmt3b knockout mice. While Dnmt3b was absent, Dnmt3a knockout was significant in both male ( A , two-tailed independent samples t -test, p = 0.01, n = 3) and female mice ( B , two-tailed independent samples t -test, p = 0.03, n = 3 and 4). There appeared to be no compensatory shift in Dnmt1 expression as a result of the genetic manipulation. Values are expressed as 2 −ΔCT with respect to three housekeeping genes. Each data point (circles for Dnmt1, squares for Dnmt3a and diamonds for Dnmt3b) represents one biological replicate. Means and their standard errors are also displayed.
    Figure Legend Snippet: Dnmt3a knockdown after tamoxifen administration to AdvCre ERT2 -Dnmt3a fl/fl /Dnmt3b fl/fl mice. DNMT expression measured by qRT-PCR in whole L3-L5 DRG taken from sensory neuron specific Dnmt3a and Dnmt3b knockout mice. While Dnmt3b was absent, Dnmt3a knockout was significant in both male ( A , two-tailed independent samples t -test, p = 0.01, n = 3) and female mice ( B , two-tailed independent samples t -test, p = 0.03, n = 3 and 4). There appeared to be no compensatory shift in Dnmt1 expression as a result of the genetic manipulation. Values are expressed as 2 −ΔCT with respect to three housekeeping genes. Each data point (circles for Dnmt1, squares for Dnmt3a and diamonds for Dnmt3b) represents one biological replicate. Means and their standard errors are also displayed.

    Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Knock-Out, Two Tailed Test

    67) Product Images from "Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4"

    Article Title: Syngeneic animal models of tobacco-associated oral cancer reveal the activity of in situ anti-CTLA-4

    Journal: Nature Communications

    doi: 10.1038/s41467-019-13471-0

    Efficacy of intratumoral delivery of immune oncology agents. a Left panel, C57Bl/6 mice were implanted with 1 × 10 6 of 4MOSC1 cells into the tongue. After tumors reached ~30 mm 3 , mice were either treated IP or by intratumoral (IT) delivery of PBS, IP with 10 mg/kg or IT with 5 mg/kg anti-PD-1. Shown is the average volume of each tumor ( n = 4 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). Right panel, representative pictures of tongues from mice in a with tumors depicted with a dotted line. b Distribution of anti-PD-1 antibody in mice with 4MOSC1 tumors using IP or IT delivery of the treatment. Staining for anti-hamster IgG showed the localization of anti-PD-1 antibody in the tongue, lymph nodes, and spleen of treated mice ( n = 4 mice per group). c RNA from each tumor was isolated and comprehensive immune profiling was analyzed using the NanoString nCounter PanCancer Mouse Immune Profiling gene expression platform. The advanced analysis module of the nSolver software was used to analyze genes associated with listed immune cells and given a score. Shown is the Z -score of each cell profile score ( n = 3 mice per group). d Absolute number of live CD45 + CD3 + CD8 + T cells infiltrating 4MOSC1 tumors with or without anti-PD-1 or anti-CTLA-4 treatment. Shown is the average of the number of live CD8 T cells infiltrating per mm 3 of tumor ( n = 3 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). e Frequency of live CD45 + CD3 + CD4 + FoxP3 + Tregs infiltrating 4MOSC1 tumors with or without anti-PD-1 or anti-CTLA-4 treatment. Left panel, a representative flow cytometry plot from one mouse showing the frequency of Tregs (CD4 + FoxP3 + ) out of CD4 + cells is shown. Right panel, the frequency of Tregs out of CD4 + cells was quantified following treatment with anti-PD-1 or anti-CTLA-4 ( n = 5 mice per group; two-sided Student’s t test; data are represented as mean ± SEM).
    Figure Legend Snippet: Efficacy of intratumoral delivery of immune oncology agents. a Left panel, C57Bl/6 mice were implanted with 1 × 10 6 of 4MOSC1 cells into the tongue. After tumors reached ~30 mm 3 , mice were either treated IP or by intratumoral (IT) delivery of PBS, IP with 10 mg/kg or IT with 5 mg/kg anti-PD-1. Shown is the average volume of each tumor ( n = 4 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). Right panel, representative pictures of tongues from mice in a with tumors depicted with a dotted line. b Distribution of anti-PD-1 antibody in mice with 4MOSC1 tumors using IP or IT delivery of the treatment. Staining for anti-hamster IgG showed the localization of anti-PD-1 antibody in the tongue, lymph nodes, and spleen of treated mice ( n = 4 mice per group). c RNA from each tumor was isolated and comprehensive immune profiling was analyzed using the NanoString nCounter PanCancer Mouse Immune Profiling gene expression platform. The advanced analysis module of the nSolver software was used to analyze genes associated with listed immune cells and given a score. Shown is the Z -score of each cell profile score ( n = 3 mice per group). d Absolute number of live CD45 + CD3 + CD8 + T cells infiltrating 4MOSC1 tumors with or without anti-PD-1 or anti-CTLA-4 treatment. Shown is the average of the number of live CD8 T cells infiltrating per mm 3 of tumor ( n = 3 mice per group; two-sided Student’s t test; data are represented as mean ± SEM). e Frequency of live CD45 + CD3 + CD4 + FoxP3 + Tregs infiltrating 4MOSC1 tumors with or without anti-PD-1 or anti-CTLA-4 treatment. Left panel, a representative flow cytometry plot from one mouse showing the frequency of Tregs (CD4 + FoxP3 + ) out of CD4 + cells is shown. Right panel, the frequency of Tregs out of CD4 + cells was quantified following treatment with anti-PD-1 or anti-CTLA-4 ( n = 5 mice per group; two-sided Student’s t test; data are represented as mean ± SEM).

    Techniques Used: Mouse Assay, Staining, Isolation, Expressing, Software, Flow Cytometry, Cytometry

    68) Product Images from "Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2"

    Article Title: Overexpression of Colligin 2 in Glioma Vasculature is Associated with Overexpression of Heat Shock Factor 2

    Journal: Gene Regulation and Systems Biology

    doi: 10.4137/GRSB.S4546

    mRNA expression of colligin 2, HSF1, 2 and 3, collagen 1, CD31 and NG2 in low- and high-grade glioma and normal control brain. Data in this figure are the average ± SD of one representative experiment with 4 tissues in each group. Expression data are presented relative to the average mRNA expression levels measured in total RNA isolated from normal brain tissues (n = 4). Prior to isolation, all tissues were assessed by a qualified pathologist to ensure the origin and quality of the tissues. Total RNA was isolated with the RNeasy Micro kit (Qiagen BV, Venlo, The Netherlands). cDNA was prepared by use of the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). PCRs were performed in a 20 μL reaction volume in an Applied BioSystems 7900HT Fast Real-Time PCR system. Negative controls included minus RT and H 2 O-only samples, which showed to be negative in all cases. The most stable mRNA set for our 4 tissue groups were calculated with NormFinder 19 with the Datan Framework GenEx Pro package version 4.3.2 and was shown to be a combination of GUSB, HMBS, HPRT1 and NOXA1 . Expression of GUSB , HMBS , HPRT1 and NOXA1 was therefore used as a reference to control sample loading and RNA quality, as described previously. 20 Differences in mRNA concentrations were determined by the non parametric Kruskal-Wallis test with P
    Figure Legend Snippet: mRNA expression of colligin 2, HSF1, 2 and 3, collagen 1, CD31 and NG2 in low- and high-grade glioma and normal control brain. Data in this figure are the average ± SD of one representative experiment with 4 tissues in each group. Expression data are presented relative to the average mRNA expression levels measured in total RNA isolated from normal brain tissues (n = 4). Prior to isolation, all tissues were assessed by a qualified pathologist to ensure the origin and quality of the tissues. Total RNA was isolated with the RNeasy Micro kit (Qiagen BV, Venlo, The Netherlands). cDNA was prepared by use of the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany). The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix (Applied Biosystems, Nieuwerkerk a/d IJssel, The Netherlands). PCRs were performed in a 20 μL reaction volume in an Applied BioSystems 7900HT Fast Real-Time PCR system. Negative controls included minus RT and H 2 O-only samples, which showed to be negative in all cases. The most stable mRNA set for our 4 tissue groups were calculated with NormFinder 19 with the Datan Framework GenEx Pro package version 4.3.2 and was shown to be a combination of GUSB, HMBS, HPRT1 and NOXA1 . Expression of GUSB , HMBS , HPRT1 and NOXA1 was therefore used as a reference to control sample loading and RNA quality, as described previously. 20 Differences in mRNA concentrations were determined by the non parametric Kruskal-Wallis test with P

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

    69) Product Images from "Gene Expression Analysis of In Vivo Fluorescent Cells"

    Article Title: Gene Expression Analysis of In Vivo Fluorescent Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001151

    RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.
    Figure Legend Snippet: RNA analysis by the Bioanalyzer 2100. ( A ) RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method described here. ( B ) RNA isolated from frozen tissue by the optimized proteinase K/acid phenol method. ( C ) RNA isolated from fixed tissue by TRIzol method. ( D ) RNA isolated from fixed tissue by RNeasy Micro Kit. (E) One round of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( F ) One round of amplification of Ambion Control RNA. ( G ) Two rounds of amplification of RNA isolated from fixed tissue by the optimized proteinase K/acid phenol method. ( H ) RNA ladder: first peak is RNA marker, next mark 200, 500, 1000, 2000, 4000 and 6000 nt.

    Techniques Used: Isolation, Amplification, Marker

    70) Product Images from "High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos"

    Article Title: High-quality RNA extraction from the sea urchin Paracentrotus lividus embryos

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0172171

    Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.
    Figure Legend Snippet: Bioanalyzer Agilent electrophoresis runs. Examples of representative Bioanalyzer Agilent electrophoresis runs for the five different methods applied for RNA extractions from P . lividus embryos: TRIzol, GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), RNAqueous® Micro Kit (Ambion from Life Technologies), RNeasy® Micro Kit (Qiagen) and Aurum™ Total RNA Mini Kit (Biorad). Four different numerical amounts of embryos were used for RNA extraction: 500, 1000, 2500 and 5000 embryos. The ladder (L) is reported in the first lane of each run. The green band at the bottom of each panel is the RNA 6000 Nano Marker (Agilent RNA 6000 Nano Kit, Agilent Technologies, Inc.). Red box in the 500 lane of TRIzol indicates that the Bioanalyzer software cannot calculate RIN values (reported as N/A in the Table 1 ) for this sample, because of very low concentration and high level of degradation of the RNA.

    Techniques Used: Electrophoresis, RNA Extraction, Marker, Software, Concentration Assay

    Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.
    Figure Legend Snippet: Agilent Bioanlyzer electropherograms. Examples of representative Agilent Bioanlyzer electropherograms of P . lividus RNA: for TRIzol, GenElute and RNAqueous RNA extraction from 5000 embryos extraction; for RNeasy and Aurum RNA extraction from 2500 embryos (see also Table 1 ). Relative Fluorescent Unit (FU) and seconds of migration (s) of RNA samples isolated according to the five different extraction methods are reported. RIN values are also reported.

    Techniques Used: RNA Extraction, Migration, Isolation

    71) Product Images from "Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq"

    Article Title: Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2017.00185

    Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p
    Figure Legend Snippet: Comparison of RNA quality using different LCM methods. (A) Graph comparing RNA quality (RIN) from LCM RNA samples captured using the MMI CellCut or Arcturus PixCell Instrument and extracted with either the Arcturus PicoPure Isolation kit or QIAGEN Micro RNeasy kit. An overall significant effect was found for both conditions using a two-way analyses of variance (ANOVA; CellCut vs. PixCell F (1,119) = 114.6; PicoPure vs. QIAGEN F (1,119) = 732.5). Although, it is important to note that two groups (Pixcell PicoPure and CellCut QIAGEN) were solely represented by one tissue type (see Experimental Summary in Table 1 ). There was also a significant interaction between the two conditions (Interaction F (1,119) = 9.177, p = 0.003). (B) The same data shown in A plotted by tissue type. Each tissue (Hippocampus, Midbrain and Liver) showed a significant increase in RIN with the QIAGEN kits vs. PicoPure kits using Sidak’s multiple comparisons post hoc test. All data were normally distributed (passed KS normality test) and had similar variances as tested by Brown-Forsythe test. (C,D) Representative Bioanalyzer gel (top) and electropherogram traces (bottom) from PixCell LCM RNA samples extracted using either the (C) Arcturus PicoPure Isolation kit or (D) QIAGEN Micro RNeasy kit. Note that these LCM samples were acquired simultaneously from different brain regions (CA1 vs. CA2) on the same sections from three mouse brains (#2, #4 or #6). Graphs are plotted min to max with a line at the mean. Numbers in parentheses indicate technical replicates. #### Overall group effect; **** post hoc result p

    Techniques Used: Laser Capture Microdissection, Isolation

    72) Product Images from "BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice"

    Article Title: BMP signaling orchestrates a transcriptional network to control the fate of mesenchymal stem cells in mice

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.150136

    Activation of Klf4 in dental apical mesenchyme explants activates expression of the odontoblast marker Dspp . (A-B′) Ki67 and Klf4 immunostaining (red) of molars from P7.5 Gli1-CreER;Bmpr1 α fl/fl mice induced at P3.5. Boxes in A and B are shown magnified in A′ and B′, respectively. (C) Western blot of Klf4 in cultured dental pulp cells from P7.5 wild-type mice treated with Bmp2 or Bmp4, or mock-treated (control). (D) Co-immunoprecipitation experiment using Flag-tagged Smad1 and HA-tagged Klf4 expressed in 293T cells. Smad1 was immunoprecipitated (IP) and immunoblotted (IB) for association with Klf4. (E,E′) Klf4 immunofluorescence after treatment of dissociated wild-type apical pulp culture for 48 h with Ad-m-Klf4 (E) or ad-GFP (E′). (F) qPCR for Dspp in apical pulp cultures treated with Ad-m-Klf4 (blue bar) compared with Ad-m-GFP (gray bar). n =3. * P
    Figure Legend Snippet: Activation of Klf4 in dental apical mesenchyme explants activates expression of the odontoblast marker Dspp . (A-B′) Ki67 and Klf4 immunostaining (red) of molars from P7.5 Gli1-CreER;Bmpr1 α fl/fl mice induced at P3.5. Boxes in A and B are shown magnified in A′ and B′, respectively. (C) Western blot of Klf4 in cultured dental pulp cells from P7.5 wild-type mice treated with Bmp2 or Bmp4, or mock-treated (control). (D) Co-immunoprecipitation experiment using Flag-tagged Smad1 and HA-tagged Klf4 expressed in 293T cells. Smad1 was immunoprecipitated (IP) and immunoblotted (IB) for association with Klf4. (E,E′) Klf4 immunofluorescence after treatment of dissociated wild-type apical pulp culture for 48 h with Ad-m-Klf4 (E) or ad-GFP (E′). (F) qPCR for Dspp in apical pulp cultures treated with Ad-m-Klf4 (blue bar) compared with Ad-m-GFP (gray bar). n =3. * P

    Techniques Used: Activation Assay, Expressing, Marker, Immunostaining, Mouse Assay, Western Blot, Cell Culture, Immunoprecipitation, Immunofluorescence, Real-time Polymerase Chain Reaction

    73) Product Images from "A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons"

    Article Title: A comprehensive map coupling histone modifications with gene regulation in adult dopaminergic and serotonergic neurons

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03538-9

    Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01
    Figure Legend Snippet: Purification of adult neuronal and neural progenitor nuclei. a Rpl10a-mCherry expression (red) overlaps TH (green) expression in the ventral midbrain in DatCreER T2 - Rpl10a - mCherry mice. 5x and 20x magnification. To the left is an in situ hybridization for Slc6a3 taken from the Allen Brain Atlas (mouse.brain-map.org, image credit: Allen Institute), indicating the position of the images. Scale bars: 200 μm (upper panels), 50 μm (lower panels). b FACS plots showing a population of nuclei, indicated by “P5,” occurring in DatCreER T2 - Rpl10a - mCherry mice but not in wild-type mice. Single-nuclei RNA-seq: c PCA-plots constructed from normalized single-nuclei log-expression values of correlated HVGs. Each nucleus is colored according to the expression of Slc6a3 , Th , or Ret . Arrows indicate three outlier nuclei identified as originating from non-mDA neurons. A total of 1000 nuclei bulk RNA-seq: d Heatmap showing sample-to-sample distances between hierarchically clustered mDA, SER, and NPC RNA-seq libraries. e Expression of dopaminergic neuron markers is restricted to mDA nuclei: bars indicate average RPKM ± SD for Slc6a3 and Th as RPKMs in mDA ( n = 4 mice), SER ( n = 3 mice), and NPC nuclei ( n = 3 embryos). f Expression of serotonergic markers is restricted to SER nuclei: bars indicate average RPKM ± SD for Slc6a4 and Tph2 as RPKMs in mDA, SER, and NPC nuclei. g Expression of neural progenitor markers is restricted to NPC nuclei: bars indicate average RPKM ± SD for Sox2 and Nes as RPKMs in mDA, SER, and NPC nuclei. Significance for e – g according to two-tailed Student’s t -test assuming equal variances. *** p ≤ 0.001, ** p ≤ 0.01

    Techniques Used: Purification, Expressing, Mouse Assay, In Situ Hybridization, FACS, RNA Sequencing Assay, Construct, Multiple Displacement Amplification, Two Tailed Test

    74) Product Images from "Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity"

    Article Title: Diversity of Interstitial Lung Fibroblasts Is Regulated by Platelet-Derived Growth Factor Receptor α Kinase Activity

    Journal: American Journal of Respiratory Cell and Molecular Biology

    doi: 10.1165/rcmb.2015-0095OC

    Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and
    Figure Legend Snippet: Immunophenotyping of PDGFRα + and PDGFRα ⁻ fibroblasts in adult mouse lungs. Lin ⁻ (CD45 ⁻ CD326 ⁻ CD31 ⁻ ) stromal cells were gated for CD140α + ( green ) or CD140α ( red ) subpopulations, and

    Techniques Used:

    75) Product Images from "Increased Levels of the FoxM1 Transcription Factor Accelerate Development and Progression of Prostate Carcinomas in both TRAMP and LADY Transgenic Mice"

    Article Title: Increased Levels of the FoxM1 Transcription Factor Accelerate Development and Progression of Prostate Carcinomas in both TRAMP and LADY Transgenic Mice

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-05-3138

    FoxM1 is essential for growth and proliferation of PC-3, LNCaP and DU-145 prostate cancer cell lines. We transfected siRNA duplexes specific to either FoxM1 (siFoxM1 #2) or p27 Kip1 (siP27) into PC-3, LNCaP and DU-145 prostate cancer cell lines and at 48 hours after transfection they were used for growth or flow cytometry analysis and at 72 hours after transfection they were used to prepare total RNA. (A) Transfection of FoxM1 siRNA inhibits expression of FoxM1 in PC-3, LNCaP and DU-145 cells. Total RNA was prepared from prostate cancer cell lines PC-3, LNCaP and DU-145 at 48 hours after transfection with either siRNA duplexes specific to FoxM1 (siFoxM1 #2) or p27Kip1 (siP27) or left untransfected and then was analyzed for expression levels of FoxM1 and Cyclophilin by quantitative Real-Time RT-PCR (QRT-PCR) as described in Material and Methods. FoxM1 mRNA levels in each individual sample were normalized to its corresponding Cyclophilin mRNA level. (B) Transfection of FoxM1 siRNAs into prostate cancer cell lines decreases their growth in culture. Prostate cancer cell lines PC-3, LNCaP and DU-145 were transfected with either siFoxM1 #2 or siP27 duplexes or left untransfected and were then re-plated 48 hours after siRNA transfection and cell numbers were counted at day 3, day 4 or day 5 after transfection. A statistically significant decrease in the growth of FoxM1 depleted prostate cancer cells compared to untransfected cells. (C) Flow cytometry analysis of FoxM1 depleted prostate cancer cell lines shows decreased S-phase progression. The indicated prostate cancer cell lines were transfected with siFoxM1 #2 or siP27 duplexes or left untransfected and then subjected to flow cytometry analysis at 72 hours post transfection after staining with propidium iodide. Graphically shown is the percentage of cells accumulating in G1, S, and G2/M (4N) in FoxM1 or p27 Kip1 depleted prostate cancer cells compared to untransfected prostate cancer cells ± SD in triplicate. The asterisks indicate statistically significant increases with P values calculated by Student T Test: *P
    Figure Legend Snippet: FoxM1 is essential for growth and proliferation of PC-3, LNCaP and DU-145 prostate cancer cell lines. We transfected siRNA duplexes specific to either FoxM1 (siFoxM1 #2) or p27 Kip1 (siP27) into PC-3, LNCaP and DU-145 prostate cancer cell lines and at 48 hours after transfection they were used for growth or flow cytometry analysis and at 72 hours after transfection they were used to prepare total RNA. (A) Transfection of FoxM1 siRNA inhibits expression of FoxM1 in PC-3, LNCaP and DU-145 cells. Total RNA was prepared from prostate cancer cell lines PC-3, LNCaP and DU-145 at 48 hours after transfection with either siRNA duplexes specific to FoxM1 (siFoxM1 #2) or p27Kip1 (siP27) or left untransfected and then was analyzed for expression levels of FoxM1 and Cyclophilin by quantitative Real-Time RT-PCR (QRT-PCR) as described in Material and Methods. FoxM1 mRNA levels in each individual sample were normalized to its corresponding Cyclophilin mRNA level. (B) Transfection of FoxM1 siRNAs into prostate cancer cell lines decreases their growth in culture. Prostate cancer cell lines PC-3, LNCaP and DU-145 were transfected with either siFoxM1 #2 or siP27 duplexes or left untransfected and were then re-plated 48 hours after siRNA transfection and cell numbers were counted at day 3, day 4 or day 5 after transfection. A statistically significant decrease in the growth of FoxM1 depleted prostate cancer cells compared to untransfected cells. (C) Flow cytometry analysis of FoxM1 depleted prostate cancer cell lines shows decreased S-phase progression. The indicated prostate cancer cell lines were transfected with siFoxM1 #2 or siP27 duplexes or left untransfected and then subjected to flow cytometry analysis at 72 hours post transfection after staining with propidium iodide. Graphically shown is the percentage of cells accumulating in G1, S, and G2/M (4N) in FoxM1 or p27 Kip1 depleted prostate cancer cells compared to untransfected prostate cancer cells ± SD in triplicate. The asterisks indicate statistically significant increases with P values calculated by Student T Test: *P

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Expressing, Quantitative RT-PCR, Staining

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    Article Snippet: .. For small RNA isolation using the RNeasy Micro kit or RNeasy Plus Micro kit, the miRNA isolation method was followed according to the manufacturer’s instructions. .. RNA was isolated from whole urine using the ZR urine RNA isolation kit (Zymo Research, Orange, CA, USA) according to the manufacturer’s instructions.

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: .. Ten days post-differentiation, EBs in the supernatant were harvested by centrifugation (BeckmanAllegra-6R, 1000 rpm, 5 min) and RNA was isolated using the RNeasy Micro Kit (Qiagen). .. Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 and used as template in subsequent PCR with Taq DNA Polymerase.

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes
    Article Snippet: .. Total RNA from minimal cell numbers was also extracted using the RNeasy Micro RNA isolation kit with the following modifications (process B) ( ). ..

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: .. Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above. .. Total protein extracts from control and LC snap‐frozen tissue samples were analyzed by Western blotting following standard protocols (Singh et al , ) and using antibodies specific for CD63 (ab8219, Abcam), TSG101 (sc‐7964, Santa Cruz), and ACTB (ab6276, Abcam).

    Article Title: Gene Expression Analysis of In Vivo Fluorescent Cells
    Article Snippet: .. Conventional methods RNA was isolated either by the TRIzol (Invitrogen, Germany) method or by use of an RNeasy Micro Kit (QIAGEN, Germany) according to manufacturers' recommendations. .. RNA amplification Total RNA (2–3 ng) was amplified using the MessageAmp II aRNA Amplification Kit (Ambion, USA) according to manufacturer's recommendations.

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: .. Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). .. Complementary DNA (cDNA) was synthetized using the High Capacity cDNA Reverse Transcription kit (Applied Biosystem) with 0.5–0.7 μg (EBC) or 1 μg (FFPE sample) total RNA.

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Paragraph title: Isolation and characterization of exosomes from EBCs ... Exosome pellets were lysed in 350 μl RLT Plus Buffer (RNeasy Micro Kit, Qiagen), and 200 ng of 16S‐ and 23S‐ribosomal Spike‐In RNA (Roche) was added to the lysate.

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes
    Article Snippet: .. RNA isolation from vascular endothelial biopsies was also carried out using the RNeasy Micro RNA isolation kit (Qiagen) following process B ( ). ..

    Negative Control:

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). .. RT reaction without adding enzyme was used as negative control. qRT–PCRs were performed using SYBR® Green on the Step One plus Real‐time PCR system (Applied Biosystems) using the primers specified in the .

    Cell Culture:

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit
    Article Snippet: RNA isolated from cultured synoviocyte cells (P6) and freshly dissected synovium was assessed for HAS2 mRNA, since HAS2 is highly expressed in intimal FLS cells ( , ). .. Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen).

    Mouse Assay:

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). .. Briefly, 1× concentration of the SYBR Green master mix, 250 nM each forward and reverse primer, and 3.5 μl (EBC) or 1 μl (cell lines, mice and human lung cancer tissue) from a sixfold diluted RT reaction were used for the gene‐specific qPCR.

    SYBR Green Assay:

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines
    Article Snippet: RNA extraction and Quantitative real time PCR (qRT-PCR) RNeasy Micro Kit (Qiagen) was used to extract total RNA from 1 × 106 PLum-AD and PLum-AI cells. .. The amplification step in quantitative real time PCR was carried out using SYBR green PCR master mix (Applied Biosystems, Bedford, MA).

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). .. RT reaction without adding enzyme was used as negative control. qRT–PCRs were performed using SYBR® Green on the Step One plus Real‐time PCR system (Applied Biosystems) using the primers specified in the .

    Concentration Assay:

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen). .. Briefly, 1× concentration of the SYBR Green master mix, 250 nM each forward and reverse primer, and 3.5 μl (EBC) or 1 μl (cell lines, mice and human lung cancer tissue) from a sixfold diluted RT reaction were used for the gene‐specific qPCR.

    Incubation:

    Article Title: Nucleic acids within urinary exosomes/microvesicles are potential biomarkers for renal disease
    Article Snippet: Digestion of nucleic acids within microvesicles was carried out using on-column RNase A (Fermentas) or DNase I (Qiagen) digestion in conjunction with the RNeasy Micro kit (Qiagen). .. RNA removal was carried out using the same steps as for DNA digestion except that 700 µl of RNase A (1 µl per ml RW1 wash buffer) was incubated on-column for 1 h at 37 °C.

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Exosomes were precipitated by 6 h incubation at 4°C and centrifugation at 1,500 × g for 30 min at 4°C. .. Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above.

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes
    Article Snippet: DNase treatment was performed by pipetting 80 µl of DNase I incubation mix directly on to the RNeasy Mini Spin column membrane and incubating at room temperature for 15 minutes. .. RNA isolation from vascular endothelial biopsies was also carried out using the RNeasy Micro RNA isolation kit (Qiagen) following process B ( ).

    Formalin-fixed Paraffin-Embedded:

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Human lung tissue samples were obtained as formalin‐fixed paraffin‐embedded (FFPE) tissues, and eight sections of 10‐μm thickness were used for total RNA isolation using the RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE (Ambion). .. Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen).

    Knock-Out:

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: EB formation Human iPS cells were harvested by cell scraper and plated on Ultra low adhesion plate (STEMCELL Technologies) in DMEM/F12 (Gibco) consisting of 15% fetal bovine serum (FBS; Atlanta Biologicals), 15% knockout serum replacement (Invitrogen), 0.1 mM nonessential amino acids and 0.5% penicillin and streptomycin. .. Ten days post-differentiation, EBs in the supernatant were harvested by centrifugation (BeckmanAllegra-6R, 1000 rpm, 5 min) and RNA was isolated using the RNeasy Micro Kit (Qiagen).

    Expressing:

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit
    Article Snippet: Paragraph title: Total RNA isolation and RT-PCR for HAS2 and Kv1 expression. ... Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen).

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: .. Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above. .. Total protein extracts from control and LC snap‐frozen tissue samples were analyzed by Western blotting following standard protocols (Singh et al , ) and using antibodies specific for CD63 (ab8219, Abcam), TSG101 (sc‐7964, Santa Cruz), and ACTB (ab6276, Abcam).

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Paragraph title: Gene expression analysis by qRT–PCR ... Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen).

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Exosome pellets were lysed in 350 μl RLT Plus Buffer (RNeasy Micro Kit, Qiagen), and 200 ng of 16S‐ and 23S‐ribosomal Spike‐In RNA (Roche) was added to the lysate. .. Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above.

    Lysis:

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit
    Article Snippet: Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen). .. Briefly, cells were grown to confluence in T25-cm2 culture flasks and subsequently washed and collected in cell lysis buffer.

    Polymerase Chain Reaction:

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit
    Article Snippet: Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen). .. The cDNA reverse transcription product was amplified with specific primers by PCR.

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: Ten days post-differentiation, EBs in the supernatant were harvested by centrifugation (BeckmanAllegra-6R, 1000 rpm, 5 min) and RNA was isolated using the RNeasy Micro Kit (Qiagen). .. Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 and used as template in subsequent PCR with Taq DNA Polymerase.

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines
    Article Snippet: RNA extraction and Quantitative real time PCR (qRT-PCR) RNeasy Micro Kit (Qiagen) was used to extract total RNA from 1 × 106 PLum-AD and PLum-AI cells. .. Platinum Taq Polymerase (Invitrogen) was used to perform PCR.

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: .. RT-PCR and PCR Total RNA was harvested using RNeasy Micro Kit (Qiagen) and quantified by spectrophotometer. .. 500 ng of RNA was used for cDNA synthesis using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 (Invitrogen).

    Western Blot:

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Total RNA was isolated using the RNeasy Micro Kit (Qiagen), and isoform‐specific expression analysis was performed as explained above. .. Total protein extracts from control and LC snap‐frozen tissue samples were analyzed by Western blotting following standard protocols (Singh et al , ) and using antibodies specific for CD63 (ab8219, Abcam), TSG101 (sc‐7964, Santa Cruz), and ACTB (ab6276, Abcam).

    Quantitative RT-PCR:

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines
    Article Snippet: .. RNA extraction and Quantitative real time PCR (qRT-PCR) RNeasy Micro Kit (Qiagen) was used to extract total RNA from 1 × 106 PLum-AD and PLum-AI cells. .. Super Script III First Strand Synthesis System for RT-PCR (Invitrogen) was used to generate cDNA from the extracted total RNA.

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate
    Article Snippet: Paragraph title: Gene expression analysis by qRT–PCR ... Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen).

    Spectrophotometry:

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: .. RT-PCR and PCR Total RNA was harvested using RNeasy Micro Kit (Qiagen) and quantified by spectrophotometer. .. 500 ng of RNA was used for cDNA synthesis using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 (Invitrogen).

    RNA Extraction:

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines
    Article Snippet: .. RNA extraction and Quantitative real time PCR (qRT-PCR) RNeasy Micro Kit (Qiagen) was used to extract total RNA from 1 × 106 PLum-AD and PLum-AI cells. .. Super Script III First Strand Synthesis System for RT-PCR (Invitrogen) was used to generate cDNA from the extracted total RNA.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Ionic currents in intimal cultured synoviocytes from the rabbit
    Article Snippet: Paragraph title: Total RNA isolation and RT-PCR for HAS2 and Kv1 expression. ... Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen).

    Article Title: Primary versus castration-resistant prostate cancer: modeling through novel murine prostate cancer cell lines
    Article Snippet: RNA extraction and Quantitative real time PCR (qRT-PCR) RNeasy Micro Kit (Qiagen) was used to extract total RNA from 1 × 106 PLum-AD and PLum-AI cells. .. Super Script III First Strand Synthesis System for RT-PCR (Invitrogen) was used to generate cDNA from the extracted total RNA.

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells
    Article Snippet: .. RT-PCR and PCR Total RNA was harvested using RNeasy Micro Kit (Qiagen) and quantified by spectrophotometer. .. 500 ng of RNA was used for cDNA synthesis using Superscript III Reverse Transcriptase primed with oligo(dT)12–18 (Invitrogen).

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    Qiagen rneasy micro kit
    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using <t>RNeasy</t> Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent <t>PCR</t> with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.
    Rneasy Micro Kit, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 2460 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Journal: PLoS ONE

    Article Title: Antibody-Directed Lentiviral Gene Transduction for Live-Cell Monitoring and Selection of Human iPS and hES Cells

    doi: 10.1371/journal.pone.0034778

    Figure Lengend Snippet: Characterization of endogenous pluripotent makers in selected iPS cell lines. Panel A. Total RNA was isolated using RNeasy Micro Kit from selected iPS cell lines (G1–G3, G5, G6), hES H9 cells (H9), and human primary fibroblasts (F). Total RNA (500 ng) was reverse-transcribed using Superscript III Reverse Transcriptase primed with oligo(dT) 12–18 and used as template in subsequent PCR with Taq DNA Polymerase. PCR analysis examined the expression of endogenous Oct4, Nanog, Sox2, as well as ABCG2, Rex1, DNMT3B and hTERT. GAPDH was used as an internal control. N, no template control (N). PCR products were analyzed on a 10% polyacrylamide TBE Precast Gel. Panel B. TRAP assay for telomerase activity. Selected iPS cells (G1–G3, G6), hES H9 cells (H9), and human primary fibroblasts (F) were analyzed for telomerase activity using the TRAPEZE RT Telomerase Detection Kit as described in M M. PCR products were separated on 10% polyacrylamide TBE Precast Gel. Individual samples are as indicated.

    Article Snippet: RT-PCR and PCR Total RNA was harvested using RNeasy Micro Kit (Qiagen) and quantified by spectrophotometer.

    Techniques: Isolation, Polymerase Chain Reaction, Expressing, TRAP Assay, Activity Assay

    Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.

    Journal: PLoS ONE

    Article Title: Global Array-Based Transcriptomics from Minimal Input RNA Utilising an Optimal RNA Isolation Process Combined with SPIA cDNA Probes

    doi: 10.1371/journal.pone.0017625

    Figure Lengend Snippet: Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input RNA. HUVEC total RNA was titrated to cover a range of input RNA from 50 ng–10 pg. 50 ng (n = 1), 500 pg (n = 2) and 250 pg (n = 2) of total RNA was used as input for the WT-Ovation FFPE system V2 while 500 pg (n = 2), 250 pg (n = 2), 100 pg (n = 2), 50 pg (n = 2) and 10 pg (n = 2) were used as input for the WT-Ovation One-Direct system (NuGen Technologies, Inc). All cDNA reactions were purified via Zymo Research Clean and Concentrator™-25 or Qiagen RNeasy MinElute Cleanup kits (WT-Ovation FFPE V2 and WT-Ovation One-Direct systems respectively) as recommended. All purified cDNA probes were assessed for quantity and quality using the Agilent 2100 Bioanalyzer and the Nanodrop-8000 RNA Nano chips. FL-Ovation™ cDNA Biotin Module V2 (NuGEN) was used for fragmentation and biotin labelling of 5 µg of cDNA and used for subsequent hybridisation to Affymetrix HGU133 Plus 2.0 microarrays.

    Article Snippet: Total RNA from minimal cell numbers was also extracted using the RNeasy Micro RNA isolation kit with the following modifications (process B) ( ).

    Techniques: Formalin-fixed Paraffin-Embedded, Purification, Hybridization

    Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Journal: EMBO Molecular Medicine

    Article Title: Non‐invasive lung cancer diagnosis by detection of GATA6 and NKX2‐1 isoforms in exhaled breath condensate

    doi: 10.15252/emmm.201606382

    Figure Lengend Snippet: Optimization of EBC ‐based expression analysis for LC diagnosis RTube is suitable for RNA isolation. Two main EBC collection devices, RTube and TurboDECCS, were compared for the total RNA yield ( y ‐axis, ng) obtained using the QIAGEN RNeasy Micro kit and 500 μl EBC as starting material. Triangles and rhombuses are used to denote RNA yield of each individual sample using EBCs collected from the different EBC collection devices. Data are represented as mean ± s.e.m.; n = 6. P ‐values after one‐way ANOVA. 500 μl of EBC is optimal for RNA isolation. Total RNA isolation with the RNeasy Micro kit was performed using 200, 350, 500, or 1,000 μl EBC as starting material. Data are represented as mean ± s.e.m.; n = 4. The High Capacity cDNA Reverse Transcriptase kit is more efficient than EpiScript Reverse Transcriptase. Two RT kits were tested for qRT–PCR‐based analysis of GATA6 Ad. CT values were plotted. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Serial dilution of cDNA template to determine the linear range of detection of GATA6 Ad. cDNA from control EBC was serially diluted and used as template for qRT–PCR‐based expression analysis of GATA6 Ad. Each dot represents the CT value for a technical triplicate. Data are represented as mean ± s.e.m.; n = 3. Delayed snap‐freezing of EBC after collection compromises mRNA integrity. Top, schematic representation of the precursor mRNAs from GAPDH (E) and HPRT1 (F) showing exons (boxes), introns (lines), and location of primer pairs (arrowheads) used for qRT–PCR‐based expression analysis. Bottom, EBCs were collected and incubated on ice for 0, 5, and 15 min prior to snap‐freezing in liquid nitrogen. Expression of GAPDH and HPRT1 was determined in the EBCs using the indicated primers, and the expression ratios (5′/3′) of each gene were calculated as indicators of mRNA integrity. RNA purified from EBCs with expression ratios of GAPDH and HPRT1 between 0.75 and 1.5 (dashed lines) was considered as acceptable for further analysis. Data are represented as mean ± s.e.m.; n = 3. Each colored triangle represents one individual. EBCs should be thawed on ice, for a maximum of 15 min, before further processing. EBCs, that were stored at −80°C, were thawed on ice (green line) or 25°C (red dashed line) for 15, 30, and 60 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity. Each triangle refers to the 5′/3′ expression ratio for GAPDH of one sample. Each sample was measured in triplicates and the mean was used for the calculation of the ratio. Long‐term storage at −80°C or transportation on dry ice did not compromise mRNA integrity. EBCs were collected either in Germany (GER, green line) or in Mexico (MEX, red dashed line) and subsequently transported to Germany on dry ice. EBCs were stored at −80°C for 7, 30, or 365 days before they were thawed on ice and further processed in less than 15 min. GAPDH 5′/3′ expression ratios were determined as in (E) as indicators of mRNA integrity.

    Article Snippet: Total RNA isolation from EBC was performed using 500 μl of sample and the RNeasy Micro Kit (Qiagen).

    Techniques: Expressing, Isolation, Quantitative RT-PCR, Serial Dilution, Incubation, Purification