total rna  (Qiagen)

 
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    Name:
    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 27926 article reviews
    Price from $9.99 to $1999.99
    total rna - by Bioz Stars, 2020-08
    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 "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:

    9) 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

    10) 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

    11) Product Images from "The lineage-specific transcription factor CDX2 navigates dynamic chromatin to control distinct stages of intestine development"

    Article Title: The lineage-specific transcription factor CDX2 navigates dynamic chromatin to control distinct stages of intestine development

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.172189

    Temporal-specific shifts in CDX2 binding is conserved between mice and humans, and corresponds to temporal shifts in intestinal gene expression. ; for E13.5, 12 embryos were pooled per replicate; for E16.5, three embryos pooled per replicate; for E17.5, two embryos pooled per replicate). The heatmap depicts CDX2 ChIP-seq intensity at each of the regions defined as embryo- or adult-enriched via k ). (B) The average ChIP-seq signals at the binding regions shown in A is plotted. (C) Embryo-enriched and adult-enriched binding sites, as identified in A, correspond to different sets of nearby target genes, which show stage-specific gene expression. RNA-seq was conducted on E12.5 or adult intestinal epithelium and genes linked to embryo-enriched or adult-enriched CDX2 binding (within 5 kb) were analyzed for their distribution along the E12.5-to-adult expression continuum using GSEA analysis. The GSEA plots indicate that embryo-enriched sites are nearby genes that are expressed at higher levels in the embryo than in the adult (leftward shift), whereas the opposite trend is observed for expression of genes nearby CDX2 sites that are stronger in the adult. CDX2-bound genes are also dependent upon CDX2 for expression, as revealed by RNA-seq analysis in Cdx2 .
    Figure Legend Snippet: Temporal-specific shifts in CDX2 binding is conserved between mice and humans, and corresponds to temporal shifts in intestinal gene expression. ; for E13.5, 12 embryos were pooled per replicate; for E16.5, three embryos pooled per replicate; for E17.5, two embryos pooled per replicate). The heatmap depicts CDX2 ChIP-seq intensity at each of the regions defined as embryo- or adult-enriched via k ). (B) The average ChIP-seq signals at the binding regions shown in A is plotted. (C) Embryo-enriched and adult-enriched binding sites, as identified in A, correspond to different sets of nearby target genes, which show stage-specific gene expression. RNA-seq was conducted on E12.5 or adult intestinal epithelium and genes linked to embryo-enriched or adult-enriched CDX2 binding (within 5 kb) were analyzed for their distribution along the E12.5-to-adult expression continuum using GSEA analysis. The GSEA plots indicate that embryo-enriched sites are nearby genes that are expressed at higher levels in the embryo than in the adult (leftward shift), whereas the opposite trend is observed for expression of genes nearby CDX2 sites that are stronger in the adult. CDX2-bound genes are also dependent upon CDX2 for expression, as revealed by RNA-seq analysis in Cdx2 .

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

    12) 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

    13) Product Images from "Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α"

    Article Title: Mouse and human microglial phenotypes in Alzheimer’s disease are controlled by amyloid plaque phagocytosis through Hif1α

    Journal: bioRxiv

    doi: 10.1101/639054

    Methoxy-XO4 purifies a molecularly distinct plaque-phagocytic population in 5xFAD mice. a , Schematic of the methodology employed in this study . b , Representative immunofluorescence image of the hippocampus of WT and 5xFAD mice injected with Methoxy-XO4 and stained with Iba1 (AlexaFluor 488, n =6 animals per genotype), scale bar=250 μm, inset 50 μm c , Representative FACS plot (from n =12-19 animals per genotype and age group) showing that XO4 + microglia are present in 6m 5xFAD plaque-affected regions (top panels). d , left, the percentage of XO4 + microglia isolated from plaque-affected regions in 1, 4, and 6m old WT and 5xFAD mice, ( n = 12-19 per genotype and age group; male and female mice pooled) and right, the percentage of XO4 + microglia isolated from plaque-affected and non-affected regions in 6m old male and female WT and 5xFAD mice ( n = 6-8 per genotype), expressed as mean ± SEM. e , PCA of bulk RNA-seq. Cx, Cortex; Cb, Cerebellum f, g Gene cytometry plots showing genes that are differentially expressed between XO4 + and XO4 − microglia and/or genes that are differentially expressed between old (4, 6 month) and young (1 month) microglia. Gene scores are calculated as the product of the log fold change and –log 10 (FDR). Example genes in each quadrant are labelled in red (upregulated over time or phagocytosis) or blue (downregulated). h i , Venn diagram showing the overlap between genes whose expression levels could be explained by the age, region and XO4 covariate as well as GO and KEGG terms associated with XO4 covariate genes. h ii , table showing the 21 core microglial neurodegeneration signature genes and their direction of differential expression in DAM 28 , CD11c + 29 , MGnD 30 and XO4 + microglia. i , heat map of targeted LC-SWATH-MS analysis of detected peptides within DEGs in n =3-5 biological replicates of WT (blue), XO4 − 5xFAD (orange) and XO4 + 5xFAD (green) microglia. j , comparison of RNA and protein expression for selected genes, and quantitation of a tryptic peptide in Aβ in microglia. Data are expressed as mean ± SEM log fold change compared to WT microglia, normalized relative to peptides in Supplementary table 2. p values in d and j were calculated by one-way ANOVA using Tukey’s multiple comparison test.
    Figure Legend Snippet: Methoxy-XO4 purifies a molecularly distinct plaque-phagocytic population in 5xFAD mice. a , Schematic of the methodology employed in this study . b , Representative immunofluorescence image of the hippocampus of WT and 5xFAD mice injected with Methoxy-XO4 and stained with Iba1 (AlexaFluor 488, n =6 animals per genotype), scale bar=250 μm, inset 50 μm c , Representative FACS plot (from n =12-19 animals per genotype and age group) showing that XO4 + microglia are present in 6m 5xFAD plaque-affected regions (top panels). d , left, the percentage of XO4 + microglia isolated from plaque-affected regions in 1, 4, and 6m old WT and 5xFAD mice, ( n = 12-19 per genotype and age group; male and female mice pooled) and right, the percentage of XO4 + microglia isolated from plaque-affected and non-affected regions in 6m old male and female WT and 5xFAD mice ( n = 6-8 per genotype), expressed as mean ± SEM. e , PCA of bulk RNA-seq. Cx, Cortex; Cb, Cerebellum f, g Gene cytometry plots showing genes that are differentially expressed between XO4 + and XO4 − microglia and/or genes that are differentially expressed between old (4, 6 month) and young (1 month) microglia. Gene scores are calculated as the product of the log fold change and –log 10 (FDR). Example genes in each quadrant are labelled in red (upregulated over time or phagocytosis) or blue (downregulated). h i , Venn diagram showing the overlap between genes whose expression levels could be explained by the age, region and XO4 covariate as well as GO and KEGG terms associated with XO4 covariate genes. h ii , table showing the 21 core microglial neurodegeneration signature genes and their direction of differential expression in DAM 28 , CD11c + 29 , MGnD 30 and XO4 + microglia. i , heat map of targeted LC-SWATH-MS analysis of detected peptides within DEGs in n =3-5 biological replicates of WT (blue), XO4 − 5xFAD (orange) and XO4 + 5xFAD (green) microglia. j , comparison of RNA and protein expression for selected genes, and quantitation of a tryptic peptide in Aβ in microglia. Data are expressed as mean ± SEM log fold change compared to WT microglia, normalized relative to peptides in Supplementary table 2. p values in d and j were calculated by one-way ANOVA using Tukey’s multiple comparison test.

    Techniques Used: Mouse Assay, Immunofluorescence, Injection, Staining, FACS, Isolation, RNA Sequencing Assay, Cytometry, Expressing, Quantitation Assay

    Microglial XO4 + signature can be manipulated through the Hif1a regulon. a , Top ten activators of the Hif1a regulon predicted by IPA. The activation z-score is a statistical measure of the match between the expected relationship direction of regulation and the observed gene expression; positive z-scores are indicative of predicted activation. p -value of overlap refers to the significance of the overlap between the Hif1a regulon gene set and the regulated target genes predicted by IPA. b , Cartoon diagram of hypothesis generated by IPA. c , Stimulation of iMGLs with MyD88-dependent TLR agonist Pam3csk (alone or with BMP9) induces XO4 + signature genes within the Hif1a regulon as identified by qPCR and d , cytometric bead array. MyD88-independent TLR stimulation (Poly:IC) does not shift iMGLs towards an XO4 + signature. Data are fold changes normalized to non-treated cells. e , MyD88-dependent XO4 + signature gene expression is modulated by rapamycin. Data are fold changes induced by rapamycin normalized to each respective treatment in the absence of rapamycin. p values throughout are calculated by one way ANOVA and Sidak post-test. f , Venn diagram showing the overlap between a XO4 + -like state induced in iMGLs using Pam3csk and reversed by rapamycin (RNA-seq) as predicted by ingenuity pathway analysis (IPA) with the mouse XO4 + signature, as measured by RNA-seq. Representative gene expression heatmap of the overlap, showing expression levels in mice (WT, 5xFAD XO4 − , 5xFAD XO4 + ) and human iMGLs (non-treated, Pam3csk and Pam3csk+rapamycin). g , Fluorescently labeled synaptosome internalization by iMGLs treated with amyloid fibrils, alone, or in combination with rapamycin for 48 h, as measured by FACS. The data are presented as mean ± SEM, and analysed by unpaired t- test. h , proposed model of generation and regulation of microglia diversity in AD.
    Figure Legend Snippet: Microglial XO4 + signature can be manipulated through the Hif1a regulon. a , Top ten activators of the Hif1a regulon predicted by IPA. The activation z-score is a statistical measure of the match between the expected relationship direction of regulation and the observed gene expression; positive z-scores are indicative of predicted activation. p -value of overlap refers to the significance of the overlap between the Hif1a regulon gene set and the regulated target genes predicted by IPA. b , Cartoon diagram of hypothesis generated by IPA. c , Stimulation of iMGLs with MyD88-dependent TLR agonist Pam3csk (alone or with BMP9) induces XO4 + signature genes within the Hif1a regulon as identified by qPCR and d , cytometric bead array. MyD88-independent TLR stimulation (Poly:IC) does not shift iMGLs towards an XO4 + signature. Data are fold changes normalized to non-treated cells. e , MyD88-dependent XO4 + signature gene expression is modulated by rapamycin. Data are fold changes induced by rapamycin normalized to each respective treatment in the absence of rapamycin. p values throughout are calculated by one way ANOVA and Sidak post-test. f , Venn diagram showing the overlap between a XO4 + -like state induced in iMGLs using Pam3csk and reversed by rapamycin (RNA-seq) as predicted by ingenuity pathway analysis (IPA) with the mouse XO4 + signature, as measured by RNA-seq. Representative gene expression heatmap of the overlap, showing expression levels in mice (WT, 5xFAD XO4 − , 5xFAD XO4 + ) and human iMGLs (non-treated, Pam3csk and Pam3csk+rapamycin). g , Fluorescently labeled synaptosome internalization by iMGLs treated with amyloid fibrils, alone, or in combination with rapamycin for 48 h, as measured by FACS. The data are presented as mean ± SEM, and analysed by unpaired t- test. h , proposed model of generation and regulation of microglia diversity in AD.

    Techniques Used: Indirect Immunoperoxidase Assay, Activation Assay, Expressing, Generated, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Mouse Assay, Labeling, FACS

    14) 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

    15) 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

    16) 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

    17) Product Images from "A CX3CR1 Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture"

    Article Title: A CX3CR1 Reporter hESC Line Facilitates Integrative Analysis of In-Vitro-Derived Microglia and Improved Microglia Identity upon Neuron-Glia Co-culture

    Journal: Stem Cell Reports

    doi: 10.1016/j.stemcr.2020.04.007

    H9.CX3CR1-tdTomato iMGLs Secrete Cytokines, Internalize Native Synaptic Material, and Can Be Readily Tracked in Co-cultures (A) Cytometric bead array for the cytokines and chemokines shown in H9.CX3CR1-tdTomato iMGLs basally (open circles), or stimulated for 24 h with LPS (closed circles, 100 ng/mL). Points represent the average of three individual wells from independent differentiations and are expressed as mean ± SEM. ∗p
    Figure Legend Snippet: H9.CX3CR1-tdTomato iMGLs Secrete Cytokines, Internalize Native Synaptic Material, and Can Be Readily Tracked in Co-cultures (A) Cytometric bead array for the cytokines and chemokines shown in H9.CX3CR1-tdTomato iMGLs basally (open circles), or stimulated for 24 h with LPS (closed circles, 100 ng/mL). Points represent the average of three individual wells from independent differentiations and are expressed as mean ± SEM. ∗p

    Techniques Used:

    Generation of a Dual Microglia Reporter ESC Line and Kinetics of Differentiation to iMGLs (A) Schematic illustrating the CRISPR vector used for insertion of the tdTomato and nanoluciferase gene into the genome. Long homology arm (LHA) and short homology arm (SHA) for CX3CR1 were designed, using an IRES linker for tdTomato expression. cDNAs encoding tdTomato and nanoluciferase were linked with a T2A fragment, allowing translation of both proteins. A neomycin/kanamycin resistance cassette under the control of a PGK promoter was included for positive selection of correctly targeted clones. A DTA coding cassette was also included for negative selection of cells that do not correctly integrate the donor vector. (B) Detection of luciferase secretion at various differentiation stages in H9 CX3CR1-tdTomato or H9 iMGLs. Error bars are SEM. (C) Expression of CX3CR1 on iMGLs corresponds to tdTomato expression, as demonstrated by FACS. H9 CX3CR1-tdTomato iMGLs express CD11b, CD45, and TREM2, but not CCR2 as determined by FACS. (D) iMGLs, but not H9 CX3CR1-tdTomato ESCs or H9 CX3CR1-tdTomato HPCs, express P2RY12, TREM2, as well as tdTomato. Scale bars, 50 μm. (E and F) River plot showing the kinetics of cell identity transitions during differentiation of iPSCs to iHPCs (E) and iHPCs to iMGLs (F), as measured by expression of the markers TRA-1-60, CD43, CD11b, CD45, CX3CR1-tdTomato, and TREM2. Population proportions are presented as gated on the markers shown. Ungated FACS plots of the live cell populations are in Figures S2 A and S2B. See also Figures S1 and S2 .
    Figure Legend Snippet: Generation of a Dual Microglia Reporter ESC Line and Kinetics of Differentiation to iMGLs (A) Schematic illustrating the CRISPR vector used for insertion of the tdTomato and nanoluciferase gene into the genome. Long homology arm (LHA) and short homology arm (SHA) for CX3CR1 were designed, using an IRES linker for tdTomato expression. cDNAs encoding tdTomato and nanoluciferase were linked with a T2A fragment, allowing translation of both proteins. A neomycin/kanamycin resistance cassette under the control of a PGK promoter was included for positive selection of correctly targeted clones. A DTA coding cassette was also included for negative selection of cells that do not correctly integrate the donor vector. (B) Detection of luciferase secretion at various differentiation stages in H9 CX3CR1-tdTomato or H9 iMGLs. Error bars are SEM. (C) Expression of CX3CR1 on iMGLs corresponds to tdTomato expression, as demonstrated by FACS. H9 CX3CR1-tdTomato iMGLs express CD11b, CD45, and TREM2, but not CCR2 as determined by FACS. (D) iMGLs, but not H9 CX3CR1-tdTomato ESCs or H9 CX3CR1-tdTomato HPCs, express P2RY12, TREM2, as well as tdTomato. Scale bars, 50 μm. (E and F) River plot showing the kinetics of cell identity transitions during differentiation of iPSCs to iHPCs (E) and iHPCs to iMGLs (F), as measured by expression of the markers TRA-1-60, CD43, CD11b, CD45, CX3CR1-tdTomato, and TREM2. Population proportions are presented as gated on the markers shown. Ungated FACS plots of the live cell populations are in Figures S2 A and S2B. See also Figures S1 and S2 .

    Techniques Used: CRISPR, Plasmid Preparation, Expressing, Selection, Clone Assay, Luciferase, FACS

    18) 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

    19) 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

    20) 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

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    27) 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

    28) 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:

    29) 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

    30) 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

    31) 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

    32) 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

    33) 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

    34) 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

    35) Product Images from "Discovery of genes required for body axis and limb formation by global identification of retinoic acid–regulated epigenetic marks"

    Article Title: Discovery of genes required for body axis and limb formation by global identification of retinoic acid–regulated epigenetic marks

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3000719

    ChIP-seq findings identify RAREs near genes required for NMP function. (A) Two RA-regulated ChIP-seq peaks for H3K27ac (red bars) near Sox2 are shown for trunk tissue from E8.5 WT versus Aldh1a2 -/- (KO). A RARE (green) was found in the 3′-noncoding peak (but not the 5′-noncoding peak), suggesting it may function as a RARE enhancer as the H3K27ac peak is decreased when RA is lost. (B) Shown are RA-regulated ChIP-seq peaks for H3K27me3 and H3K27ac near Fgf8 . In the 5′-noncoding region of Fgf8 , we found 2 RAREs on either end of the peak for H3K27me3 (repressive mark) that is decreased in KO, indicating they are candidate RARE silencers; the RARE furthest upstream in the 5′-noncoding region at −4.1 kb was shown by knockout studies to function as an RA-dependent RARE silencer required for caudal Fgf8 repression and somitogenesis [ 7 ]. We also found another RARE in the 3′-noncoding region of Fgf8 that is another candidate for a RARE silencer, as it is contained within an RA-regulated peak for H3K27ac (activating mark) that is increased when RA is lost. (C) Cdx2 has a peak for H3K27ac that is increased and an overlapping peak for H3K27me3 that is decreased, along with 3 RAREs included within both peaks, indicating that all these RAREs are candidates for RARE silencers. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO,; NMP, neuromesodermal progenitor; RA, retinoic acid; RARE, RA response element; WT, wild-type.
    Figure Legend Snippet: ChIP-seq findings identify RAREs near genes required for NMP function. (A) Two RA-regulated ChIP-seq peaks for H3K27ac (red bars) near Sox2 are shown for trunk tissue from E8.5 WT versus Aldh1a2 -/- (KO). A RARE (green) was found in the 3′-noncoding peak (but not the 5′-noncoding peak), suggesting it may function as a RARE enhancer as the H3K27ac peak is decreased when RA is lost. (B) Shown are RA-regulated ChIP-seq peaks for H3K27me3 and H3K27ac near Fgf8 . In the 5′-noncoding region of Fgf8 , we found 2 RAREs on either end of the peak for H3K27me3 (repressive mark) that is decreased in KO, indicating they are candidate RARE silencers; the RARE furthest upstream in the 5′-noncoding region at −4.1 kb was shown by knockout studies to function as an RA-dependent RARE silencer required for caudal Fgf8 repression and somitogenesis [ 7 ]. We also found another RARE in the 3′-noncoding region of Fgf8 that is another candidate for a RARE silencer, as it is contained within an RA-regulated peak for H3K27ac (activating mark) that is increased when RA is lost. (C) Cdx2 has a peak for H3K27ac that is increased and an overlapping peak for H3K27me3 that is decreased, along with 3 RAREs included within both peaks, indicating that all these RAREs are candidates for RARE silencers. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO,; NMP, neuromesodermal progenitor; RA, retinoic acid; RARE, RA response element; WT, wild-type.

    Techniques Used: Chromatin Immunoprecipitation, Knock-Out, ChIP-sequencing

    ChIP-seq findings for Pax6 and Spry4 that lack RARE enhancers or silencers. These genes are good candidates for being indirect transcriptional targets of RA as their RA-regulated ChIP-seq peaks do not contain RAREs. (A) Pax6 has 2 RA-regulated peaks (red bars) for H3K27ac (decreased) when RA is lost in E8.5 trunk tissue from Aldh1a2 -/- (KO) compared with WT; these RA-regulated peaks do not contain RAREs, suggesting that transcription of Pax6 is indirectly activated by RA. (B) Spry4 has an RA-regulated peak for H3K27me3 (decreased) when RA is lost with no associated RARE, suggesting that transcription of Spry4 is indirectly repressed by RA. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; RA, retinoic acid; RARE, RA response element; WT, wild-type.
    Figure Legend Snippet: ChIP-seq findings for Pax6 and Spry4 that lack RARE enhancers or silencers. These genes are good candidates for being indirect transcriptional targets of RA as their RA-regulated ChIP-seq peaks do not contain RAREs. (A) Pax6 has 2 RA-regulated peaks (red bars) for H3K27ac (decreased) when RA is lost in E8.5 trunk tissue from Aldh1a2 -/- (KO) compared with WT; these RA-regulated peaks do not contain RAREs, suggesting that transcription of Pax6 is indirectly activated by RA. (B) Spry4 has an RA-regulated peak for H3K27me3 (decreased) when RA is lost with no associated RARE, suggesting that transcription of Spry4 is indirectly repressed by RA. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; RA, retinoic acid; RARE, RA response element; WT, wild-type.

    Techniques Used: Chromatin Immunoprecipitation, ChIP-sequencing, Knock-Out

    ChIP-seq findings for Rarb , Crabp2 , Hoxa1 , and Cdx1 showing that RA-regulated peaks for H3K27ac and H3K7me3 are located near known RARE enhancers. (A) Shown for Rarb are RA-regulated ChIP-seq peaks for H3K27ac and H3K27me3 (red bars) when RA is lost in E8.5 trunk comparing WT versus Aldh1a2 -/- (KO) as well as RAREs (green). A RARE in the 5′-untranslated region is known to function as an RA-dependent enhancer in mouse transgene studies [ 23 ]; here, H3K27ac is decreased and H3K27me3 increased near the native RARE when RA is lost in trunk tissue, supporting its function as a RARE enhancer in vivo. We also found a RARE in the 5′-noncoding region of Rarb within an H3K27me3 ChIP-seq peak that is increased when RA is lost. (B) RA-regulated peaks for H3K27ac and RAREs are shown for Crabp2 . The 2 RAREs in the 5′-noncoding region were previously shown to function as RA-dependent enhancers in cell line studies [ 24 ]. Our epigenetic studies also identified another RARE enhancer in the 3′-noncoding region. (C) RA-regulated peaks for H3K27ac and/or H3K27me3 and RAREs are shown for Hoxa1 . KO studies in mouse embryos have shown that the RARE in the 3′-noncoding region is essential for hindbrain Hoxa1 expression and development [ 10 ]. (D) RA-regulated peaks for H3K27ac and H3K27me3 and RAREs are shown for Cdx1 . KO studies in mouse embryos have shown that the RARE in the 5′-noncoding region is essential for Cdx1 expression and body axis development [ 11 ]. RA-regulated peaks in the genome browser view shown here and elsewhere are for 1 replicate, with the other replicate showing a similar result. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; RA, retinoic acid; RARE, RA response element; WT, wild-type.
    Figure Legend Snippet: ChIP-seq findings for Rarb , Crabp2 , Hoxa1 , and Cdx1 showing that RA-regulated peaks for H3K27ac and H3K7me3 are located near known RARE enhancers. (A) Shown for Rarb are RA-regulated ChIP-seq peaks for H3K27ac and H3K27me3 (red bars) when RA is lost in E8.5 trunk comparing WT versus Aldh1a2 -/- (KO) as well as RAREs (green). A RARE in the 5′-untranslated region is known to function as an RA-dependent enhancer in mouse transgene studies [ 23 ]; here, H3K27ac is decreased and H3K27me3 increased near the native RARE when RA is lost in trunk tissue, supporting its function as a RARE enhancer in vivo. We also found a RARE in the 5′-noncoding region of Rarb within an H3K27me3 ChIP-seq peak that is increased when RA is lost. (B) RA-regulated peaks for H3K27ac and RAREs are shown for Crabp2 . The 2 RAREs in the 5′-noncoding region were previously shown to function as RA-dependent enhancers in cell line studies [ 24 ]. Our epigenetic studies also identified another RARE enhancer in the 3′-noncoding region. (C) RA-regulated peaks for H3K27ac and/or H3K27me3 and RAREs are shown for Hoxa1 . KO studies in mouse embryos have shown that the RARE in the 3′-noncoding region is essential for hindbrain Hoxa1 expression and development [ 10 ]. (D) RA-regulated peaks for H3K27ac and H3K27me3 and RAREs are shown for Cdx1 . KO studies in mouse embryos have shown that the RARE in the 5′-noncoding region is essential for Cdx1 expression and body axis development [ 11 ]. RA-regulated peaks in the genome browser view shown here and elsewhere are for 1 replicate, with the other replicate showing a similar result. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; RA, retinoic acid; RARE, RA response element; WT, wild-type.

    Techniques Used: Chromatin Immunoprecipitation, In Vivo, Expressing, ChIP-sequencing, Knock-Out

    ChIP-seq findings for Nr2f1 , Nr2f2 , Meis1 , and Meis2 identify RARE enhancers in gene families. (A-B) Nr2f1 and Nr2f2 have RA-regulated peaks (red bars) for both H3K27ac (decreased) and H3K27me3 (increased) when RA is lost in E8.5 trunk from Aldh1a2 -/- (KO) compared with WT. Each family member has one RARE (green) contained within these RA-regulated peaks that are candidates for RARE enhancers. (C-D) Meis1 and Meis2 have RA-regulated peaks for both H3K27ac (all decreased) and H3K27me3 (all increased) when RA is lost, along with associated RAREs for each peak that are candidates for RARE enhancers. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; Meis1 , Meis homeobox 1; Meis2 , Meis homeobox 2; Nr2f1 , nuclear receptor 2f1; Nr2f2 , nuclear receptor 2f2; RA, retinoic acid; RARE, RA response element; WT, wild-type.
    Figure Legend Snippet: ChIP-seq findings for Nr2f1 , Nr2f2 , Meis1 , and Meis2 identify RARE enhancers in gene families. (A-B) Nr2f1 and Nr2f2 have RA-regulated peaks (red bars) for both H3K27ac (decreased) and H3K27me3 (increased) when RA is lost in E8.5 trunk from Aldh1a2 -/- (KO) compared with WT. Each family member has one RARE (green) contained within these RA-regulated peaks that are candidates for RARE enhancers. (C-D) Meis1 and Meis2 have RA-regulated peaks for both H3K27ac (all decreased) and H3K27me3 (all increased) when RA is lost, along with associated RAREs for each peak that are candidates for RARE enhancers. ChIP-seq, chromatin immunoprecipitation sequencing; E, embryonic day; H3K27ac, histone H3 K27 acetylation; H3K27me3, histone H3 K27 trimethylation; KO, knockout; Meis1 , Meis homeobox 1; Meis2 , Meis homeobox 2; Nr2f1 , nuclear receptor 2f1; Nr2f2 , nuclear receptor 2f2; RA, retinoic acid; RARE, RA response element; WT, wild-type.

    Techniques Used: Chromatin Immunoprecipitation, ChIP-sequencing, Knock-Out

    36) Product Images from "Pre-TCR signaling intensity shapes the TCRβ repertoire"

    Article Title: Pre-TCR signaling intensity shapes the TCRβ repertoire

    Journal: bioRxiv

    doi: 10.1101/2020.05.04.074922

    T cell differentiation in L19A mice is impaired at the DN3a to DN3b transition. (a) Representative flow cytometry plots of double negative (DN) populations in WT, L19A and Cd3e -deficient (Cd3e -/- ) thymuses according to CD44 and CD25 markers (upper panels; CD25 +/lo CD44 - DN3 and CD25 - CD44 - DN4 gates indicated) and identification of DN3a and DN3b subpopulations within the DN3 gate according to forward scatter (FSC) and CD3ζ-GFP expression (bottom panels). On the right, quantification based on pooled data from six independent experiments showing the percentage of the DN3a, DN3b and DN4 populations. (b) Representative histograms (left) and quantification (right) of the intracellular TCRβ expression levels in the DN3a (CD44 - CD25 + ), DN3b (CD44 - CD25 lo ) and DN4 (CD44 - CD25 - ) subpopulations. Thymocytes from Rag1 -deficient mice were used as a control for intracellular staining and quantification is based on one out of four experiments. (c) Representative histograms (left) and quantification (right) of the extracellular TCRβ (top panels). Data in graph represent one out of four experiments. (d) pTα expression level for the indicated DN subsets, defined by level of CD25 expression. Data in graphs are based on one out of 3 experiments. Graphs present mean ± SEM. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p
    Figure Legend Snippet: T cell differentiation in L19A mice is impaired at the DN3a to DN3b transition. (a) Representative flow cytometry plots of double negative (DN) populations in WT, L19A and Cd3e -deficient (Cd3e -/- ) thymuses according to CD44 and CD25 markers (upper panels; CD25 +/lo CD44 - DN3 and CD25 - CD44 - DN4 gates indicated) and identification of DN3a and DN3b subpopulations within the DN3 gate according to forward scatter (FSC) and CD3ζ-GFP expression (bottom panels). On the right, quantification based on pooled data from six independent experiments showing the percentage of the DN3a, DN3b and DN4 populations. (b) Representative histograms (left) and quantification (right) of the intracellular TCRβ expression levels in the DN3a (CD44 - CD25 + ), DN3b (CD44 - CD25 lo ) and DN4 (CD44 - CD25 - ) subpopulations. Thymocytes from Rag1 -deficient mice were used as a control for intracellular staining and quantification is based on one out of four experiments. (c) Representative histograms (left) and quantification (right) of the extracellular TCRβ (top panels). Data in graph represent one out of four experiments. (d) pTα expression level for the indicated DN subsets, defined by level of CD25 expression. Data in graphs are based on one out of 3 experiments. Graphs present mean ± SEM. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p

    Techniques Used: Cell Differentiation, Mouse Assay, Flow Cytometry, Expressing, Staining, Two Tailed Test

    TIFRM analysis of the pre-TCR on CD44 - primary thymocytes. (a) Purification strategy of DN CD44 - thymocytes through two purification steps, depleting Lin + thymocytes from pools of three thymi of each transgenic mouse line, followed by sorting CD44 - LIN - cells with a cut-off of GFP fluorescence intensity of 10 3 relative units. (b) Median of the short time-lag diffusion coefficient (D 1-4 ) of the mobile particles detected in the analysis. (c) Quantification of the percentage (mean ± SEM) of mobile particles for confined, free and directed diffusion tracks (d) Quantification of the mean fluorescent intensity of each spot during the first 20 frames in which the particles are visible. Each dot in the graph presents single CD3ζ-GFP spot (e) Percentage (mean ± SEM) of mobile and immobile particles in the WT and L19A samples. Pooled data were obtained in 4 independent experiments encompassing on average 19.1 and 13.1 tracked particles per cell for 131 WT cells (range 20 - 57 cells/experiment) and 110 L19A cells (range 13 - 40 cells /experiment), respectively. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (***p
    Figure Legend Snippet: TIFRM analysis of the pre-TCR on CD44 - primary thymocytes. (a) Purification strategy of DN CD44 - thymocytes through two purification steps, depleting Lin + thymocytes from pools of three thymi of each transgenic mouse line, followed by sorting CD44 - LIN - cells with a cut-off of GFP fluorescence intensity of 10 3 relative units. (b) Median of the short time-lag diffusion coefficient (D 1-4 ) of the mobile particles detected in the analysis. (c) Quantification of the percentage (mean ± SEM) of mobile particles for confined, free and directed diffusion tracks (d) Quantification of the mean fluorescent intensity of each spot during the first 20 frames in which the particles are visible. Each dot in the graph presents single CD3ζ-GFP spot (e) Percentage (mean ± SEM) of mobile and immobile particles in the WT and L19A samples. Pooled data were obtained in 4 independent experiments encompassing on average 19.1 and 13.1 tracked particles per cell for 131 WT cells (range 20 - 57 cells/experiment) and 110 L19A cells (range 13 - 40 cells /experiment), respectively. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (***p

    Techniques Used: Purification, Transgenic Assay, Fluorescence, Diffusion-based Assay, Two Tailed Test

    Pre-TCR signaling by L19A thymocytes is reduced compared with WT counterparts. (a) Scheme of pre-TCR signaling pathways. (b) Representative histograms and normalized quantification of intracellular APA1/1 staining. Graphs show the mean ± SEM of pooled data from two independent experiments. (c) Representative histograms and normalized quantification of intracellular pZAP70 (P-Y319) labeling. Graphs show the mean ± SEM of three independent experiments. (c) Representative histograms and normalized quantification of pERK (P-Thr202/P-Tyr204) staining. Data show the mean ± SEM of two independent experiments. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p
    Figure Legend Snippet: Pre-TCR signaling by L19A thymocytes is reduced compared with WT counterparts. (a) Scheme of pre-TCR signaling pathways. (b) Representative histograms and normalized quantification of intracellular APA1/1 staining. Graphs show the mean ± SEM of pooled data from two independent experiments. (c) Representative histograms and normalized quantification of intracellular pZAP70 (P-Y319) labeling. Graphs show the mean ± SEM of three independent experiments. (c) Representative histograms and normalized quantification of pERK (P-Thr202/P-Tyr204) staining. Data show the mean ± SEM of two independent experiments. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p

    Techniques Used: Staining, Labeling, Two Tailed Test

    Reduction in size of thymic populations in L19A mice. (a) Thymus size and total number of thymocytes. Representative images of WT and L19A thymuses (left). Scale bar is equivalent to 0.25cm. Quantification of total number of thymocytes (right). Each dot shows the total number of thymocytes in an individual mouse. (b) Quantification of numbers (left) and percentage (right) of the major populations of thymocytes defined according to expression of CD4 and CD8. Results and statistical analysis shown are based on data from 10 independent experiments. (c) Representative plots of the DN (CD4-CD8-) subpopulations defined by the expression of CD44 and CD25 and (d) quantification in percentage (left) and cell number (right) of these populations. The DN population was pre-gated on the lineage negative (LIN-) population (CD4-, CD8-, CD19-; B220- and CD11c-). Graphs show the averages calculated on the basis of data obtained from 3 independent experiments. (e) Percentage and absolute numbers of DN3 and DN4 thymocytes in WT, L19A and Cd3z -deficient (Cd3z -/- ) mice. Data come from pooling data from 38 WT, 40 L19A and 13 Cd3z -/- mice. Graphs presents the mean ± SD. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p
    Figure Legend Snippet: Reduction in size of thymic populations in L19A mice. (a) Thymus size and total number of thymocytes. Representative images of WT and L19A thymuses (left). Scale bar is equivalent to 0.25cm. Quantification of total number of thymocytes (right). Each dot shows the total number of thymocytes in an individual mouse. (b) Quantification of numbers (left) and percentage (right) of the major populations of thymocytes defined according to expression of CD4 and CD8. Results and statistical analysis shown are based on data from 10 independent experiments. (c) Representative plots of the DN (CD4-CD8-) subpopulations defined by the expression of CD44 and CD25 and (d) quantification in percentage (left) and cell number (right) of these populations. The DN population was pre-gated on the lineage negative (LIN-) population (CD4-, CD8-, CD19-; B220- and CD11c-). Graphs show the averages calculated on the basis of data obtained from 3 independent experiments. (e) Percentage and absolute numbers of DN3 and DN4 thymocytes in WT, L19A and Cd3z -deficient (Cd3z -/- ) mice. Data come from pooling data from 38 WT, 40 L19A and 13 Cd3z -/- mice. Graphs presents the mean ± SD. P-values were calculated using an unpaired two-tailed Student’s t test with 95% CI (* p

    Techniques Used: Mouse Assay, Expressing, Two Tailed Test

    TCRβ diversity of DP and DN thymocytes in WT and L19A mice. (a) Sorting strategy for isolation of early DP and DN3 thymocytes. (b) Number of clones per sample estimated with the richness diversity ( 0 D ) for DP thymocytes. Solid lines: Rarefaction (interpolation) curve; dashed lines: extrapolation curves; symbols: observed diversity. Note that the observed values are, in all cases, close to the asymptotic part of the curves, consistent with the large values of the coverage (fig. S3). (c) Number of clones per sample estimated for DN3 thymocytes. Meaning of lines and symbols as in (b).
    Figure Legend Snippet: TCRβ diversity of DP and DN thymocytes in WT and L19A mice. (a) Sorting strategy for isolation of early DP and DN3 thymocytes. (b) Number of clones per sample estimated with the richness diversity ( 0 D ) for DP thymocytes. Solid lines: Rarefaction (interpolation) curve; dashed lines: extrapolation curves; symbols: observed diversity. Note that the observed values are, in all cases, close to the asymptotic part of the curves, consistent with the large values of the coverage (fig. S3). (c) Number of clones per sample estimated for DN3 thymocytes. Meaning of lines and symbols as in (b).

    Techniques Used: Mouse Assay, Isolation, Clone Assay

    37) Product Images from "Discovery of genes required for body axis and limb formation by global identification of retinoic acid regulated epigenetic marks"

    Article Title: Discovery of genes required for body axis and limb formation by global identification of retinoic acid regulated epigenetic marks

    Journal: bioRxiv

    doi: 10.1101/778191

    Analysis of differential gene expression of new RA target genes by qRT-PCR analysis of E8.5 wild-type vs Aldh1a2-l-trunk tis sue.
    Figure Legend Snippet: Analysis of differential gene expression of new RA target genes by qRT-PCR analysis of E8.5 wild-type vs Aldh1a2-l-trunk tis sue.

    Techniques Used: Expressing, Quantitative RT-PCR

    38) Product Images from "P38α Regulates Expression of DUX4 in Facioscapulohumeral Muscular Dystrophy"

    Article Title: P38α Regulates Expression of DUX4 in Facioscapulohumeral Muscular Dystrophy

    Journal: bioRxiv

    doi: 10.1101/700195

    Small molecule inhibitors of p38 alpha reduced expression of DUX4 in FSHD myotubes. ( A ) Diverse inhibitors of p38α/β reduce the expression of MBD3L2 in differentiating FSHD myotubes. Concentration-dependent responses were observed with all tested inhibitors. Four replicates per concentration were tested to measure reduction of MBD3L2 in immortalized C6 FSHD myotubes and bars indicate mean±SD. ( B ) P38α/β pathway inhibition in C6 FSHD myotubes. The ratio between phosphorylated HSP27 to total HSP27 was measured by an immunoassay (MSD) after 12h of treatment of C6 FSHD myotubes with the indicated inhibitors. Half maximal inhibitory concentrations (IC 50 ) observed for p-HSP27 were comparable to those obtained for reduction of MBD3L2 expression. Bars indicate mean±SD for four replicate wells. ( C ) Knockdown of p38α ( MAPK14 ) results in reduction of MBD3L2 expression. Immortalized C6 myoblasts were electroporated with siRNAs specific for MAPK14 (p38α) and MAPK12 (p38γ) plated and differentiated for 3 days. Expression of the indicated transcripts was measured using RT-qPCR and normalized against POLR2A . Reduction of MBD3L2 expression was observed when > 50% knockdown of MAPK14 was achieved. Bars indicate mean±SD. ( D ) P38α/β inhibition results in the reduction of DUX4 expression. After inhibition, correlated reduction of DUX4 mRNA, protein and downstream gene MBD3L2 was observed. To measure DUX4 protein a novel immunoassay was developed using previously described antibodies (see methods and Figure S4 ). Bars indicate mean±SD, t-test p value *
    Figure Legend Snippet: Small molecule inhibitors of p38 alpha reduced expression of DUX4 in FSHD myotubes. ( A ) Diverse inhibitors of p38α/β reduce the expression of MBD3L2 in differentiating FSHD myotubes. Concentration-dependent responses were observed with all tested inhibitors. Four replicates per concentration were tested to measure reduction of MBD3L2 in immortalized C6 FSHD myotubes and bars indicate mean±SD. ( B ) P38α/β pathway inhibition in C6 FSHD myotubes. The ratio between phosphorylated HSP27 to total HSP27 was measured by an immunoassay (MSD) after 12h of treatment of C6 FSHD myotubes with the indicated inhibitors. Half maximal inhibitory concentrations (IC 50 ) observed for p-HSP27 were comparable to those obtained for reduction of MBD3L2 expression. Bars indicate mean±SD for four replicate wells. ( C ) Knockdown of p38α ( MAPK14 ) results in reduction of MBD3L2 expression. Immortalized C6 myoblasts were electroporated with siRNAs specific for MAPK14 (p38α) and MAPK12 (p38γ) plated and differentiated for 3 days. Expression of the indicated transcripts was measured using RT-qPCR and normalized against POLR2A . Reduction of MBD3L2 expression was observed when > 50% knockdown of MAPK14 was achieved. Bars indicate mean±SD. ( D ) P38α/β inhibition results in the reduction of DUX4 expression. After inhibition, correlated reduction of DUX4 mRNA, protein and downstream gene MBD3L2 was observed. To measure DUX4 protein a novel immunoassay was developed using previously described antibodies (see methods and Figure S4 ). Bars indicate mean±SD, t-test p value *

    Techniques Used: Expressing, Concentration Assay, Inhibition, Quantitative RT-PCR

    Inhibition of the p38α/β pathway results in normalized gene expression in FSHD myotubes without affecting the differentiation process in vitro ( A ) Quantification of myotube differentiation after p38α/β inhibition. Two inhibitors were used to demonstrate the effects of p38α/β inhibition in a high-content imaging assay to quantify the number of nuclei that properly underwent differentiation by activation of expression of myofiber specific proteins (i.e. MHC). No changes were observed in the morphology of C6 myotubes treated for 5 days. Bars indicate mean±SD. ( B ) Heat map representing fold change of expression levels of differentially expressed genes after p38α/β inhibition in FSHD myotubes for 5 days. 86 genes showed significant changes in expression after treatment with two different inhibitors (abs(FC) > 4; FDR
    Figure Legend Snippet: Inhibition of the p38α/β pathway results in normalized gene expression in FSHD myotubes without affecting the differentiation process in vitro ( A ) Quantification of myotube differentiation after p38α/β inhibition. Two inhibitors were used to demonstrate the effects of p38α/β inhibition in a high-content imaging assay to quantify the number of nuclei that properly underwent differentiation by activation of expression of myofiber specific proteins (i.e. MHC). No changes were observed in the morphology of C6 myotubes treated for 5 days. Bars indicate mean±SD. ( B ) Heat map representing fold change of expression levels of differentially expressed genes after p38α/β inhibition in FSHD myotubes for 5 days. 86 genes showed significant changes in expression after treatment with two different inhibitors (abs(FC) > 4; FDR

    Techniques Used: Inhibition, Expressing, In Vitro, Imaging, Activation Assay

    Specific detection of DUX4 protein in mesoscale electro-chemiluminescent immunoassay (A) Recombinant GST-DUX4 calibrator curve. (B) C6 FSHD or wild type 5-day differentiated myotubes, DUX4 overexpressed 1-day differentiated myotubes infected with DUX4 bacmam, DUX4 overexpressed in 293 cells transfected with CMV-DUX4 plasmid. (C) C6 FSHD myotubes treated with scrambled or DUX4 anti-sense oligonucleotide or wild type control.
    Figure Legend Snippet: Specific detection of DUX4 protein in mesoscale electro-chemiluminescent immunoassay (A) Recombinant GST-DUX4 calibrator curve. (B) C6 FSHD or wild type 5-day differentiated myotubes, DUX4 overexpressed 1-day differentiated myotubes infected with DUX4 bacmam, DUX4 overexpressed in 293 cells transfected with CMV-DUX4 plasmid. (C) C6 FSHD myotubes treated with scrambled or DUX4 anti-sense oligonucleotide or wild type control.

    Techniques Used: Recombinant, Infection, Transfection, Plasmid Preparation

    Inhibition of the p38α/β pathway reduced the activation of programmed cell death in differentiating FSHD myotubes. ( A ) A high-content imaging assay was developed to measure cleaved caspase-3 in differentiating myotubes. C6 FSHD myotubes were differentiated and treated for 5 days as indicated above and stained to measure MHC, cleaved-caspase-3 and nuclei. Representative images show that cleaved caspase-3 was only detected in FSHD myotubes, not in wild-type controls or after inhibition of the p38 pathway. Six replicates were imaged and cleaved caspase-3 signal under MHC staining was quantified. ( B ) Stochastic expression of DUX4 target gene, SLC34A2 , in C6 FSHD myotubes. Expression of SLC34A2 was measured by immunostaining in similar conditions as image above. No expression was detected in wild-type control or p38 inhibitor-treated myotubes. Signal of SLC34A2 under MHC staining was quantified in two replicates ( C ) Concentration-dependent inhibition of the expression of DUX4 target genes is highly correlated to the inhibition of programmed cell death in C6 myotubes. Bars indicate mean±SD.
    Figure Legend Snippet: Inhibition of the p38α/β pathway reduced the activation of programmed cell death in differentiating FSHD myotubes. ( A ) A high-content imaging assay was developed to measure cleaved caspase-3 in differentiating myotubes. C6 FSHD myotubes were differentiated and treated for 5 days as indicated above and stained to measure MHC, cleaved-caspase-3 and nuclei. Representative images show that cleaved caspase-3 was only detected in FSHD myotubes, not in wild-type controls or after inhibition of the p38 pathway. Six replicates were imaged and cleaved caspase-3 signal under MHC staining was quantified. ( B ) Stochastic expression of DUX4 target gene, SLC34A2 , in C6 FSHD myotubes. Expression of SLC34A2 was measured by immunostaining in similar conditions as image above. No expression was detected in wild-type control or p38 inhibitor-treated myotubes. Signal of SLC34A2 under MHC staining was quantified in two replicates ( C ) Concentration-dependent inhibition of the expression of DUX4 target genes is highly correlated to the inhibition of programmed cell death in C6 myotubes. Bars indicate mean±SD.

    Techniques Used: Inhibition, Activation Assay, Imaging, Staining, Expressing, Immunostaining, Concentration Assay

    Description of an assay for the identification of inhibitors of DUX4 expression. ( A ) Schematic describing the cellular assay used to identify small molecules that result in the inhibition of DUX4 expression and activity. In short, immortalized FSHD myoblasts (C6, 6.5 D4Z4 RUs) were seeded in 96-well plates 2 days before differentiation was induced. After myoblasts reached confluence, media was replaced and compounds for treatment were added. At day 2, fusion was observed and at day 5, differentiated myotubes were harvested for gene expression analysis or fixed for immunostaining. Representative image of the alpha-actinin staining in differentiated myotubes. ( B ) DUX4 expression is rapidly induced after differentiation of immortalized FSHD myotubes in vitro . To measure DUX4 transcript, C6 FSHD myotubes were grown in 12-well plates similarly to A, cells were harvest on day 5 for RNA extraction. RT-qPCR was used to determine expression of DUX4 mRNA and its downstream gene MBD3L2 (normalized using HMBS as housekeeping). These transcripts were not detected in wild-type immortalized myotubes derived from healthy volunteers. ( C ) Canonical DUX4 target genes are specifically detected in FSHD myotubes and are downregulated when DUX4 is knocked down using a specific antisense oligonucleotide (ASO). RT-qPCR analysis was used to detect expression in immortalized myoblasts/myotubes. ASO knockdown in FSHD myotubes (mt) was carried out during the 5 days of differentiation. Bars indicate mean±SD. ( D ) A 96-well plate cell-based assay was optimized to screen for inhibitors of DUX4 expression. An assay measuring MBD3L2 by RT-qPCR was selected because of robust separation and specificity reporting DUX4 activity. MBD3L2 signal was normalized using POLR2A as a housekeeping gene. Bars indicate mean±SD. ( E) Hits identified in small molecule screen potently reduced the activity of DUX4. X and Y axis show the normalized MBD3L2 signal obtained from the two replicate wells analyzed.
    Figure Legend Snippet: Description of an assay for the identification of inhibitors of DUX4 expression. ( A ) Schematic describing the cellular assay used to identify small molecules that result in the inhibition of DUX4 expression and activity. In short, immortalized FSHD myoblasts (C6, 6.5 D4Z4 RUs) were seeded in 96-well plates 2 days before differentiation was induced. After myoblasts reached confluence, media was replaced and compounds for treatment were added. At day 2, fusion was observed and at day 5, differentiated myotubes were harvested for gene expression analysis or fixed for immunostaining. Representative image of the alpha-actinin staining in differentiated myotubes. ( B ) DUX4 expression is rapidly induced after differentiation of immortalized FSHD myotubes in vitro . To measure DUX4 transcript, C6 FSHD myotubes were grown in 12-well plates similarly to A, cells were harvest on day 5 for RNA extraction. RT-qPCR was used to determine expression of DUX4 mRNA and its downstream gene MBD3L2 (normalized using HMBS as housekeeping). These transcripts were not detected in wild-type immortalized myotubes derived from healthy volunteers. ( C ) Canonical DUX4 target genes are specifically detected in FSHD myotubes and are downregulated when DUX4 is knocked down using a specific antisense oligonucleotide (ASO). RT-qPCR analysis was used to detect expression in immortalized myoblasts/myotubes. ASO knockdown in FSHD myotubes (mt) was carried out during the 5 days of differentiation. Bars indicate mean±SD. ( D ) A 96-well plate cell-based assay was optimized to screen for inhibitors of DUX4 expression. An assay measuring MBD3L2 by RT-qPCR was selected because of robust separation and specificity reporting DUX4 activity. MBD3L2 signal was normalized using POLR2A as a housekeeping gene. Bars indicate mean±SD. ( E) Hits identified in small molecule screen potently reduced the activity of DUX4. X and Y axis show the normalized MBD3L2 signal obtained from the two replicate wells analyzed.

    Techniques Used: Expressing, Inhibition, Activity Assay, Immunostaining, Staining, In Vitro, RNA Extraction, Quantitative RT-PCR, Derivative Assay, Allele-specific Oligonucleotide, Cell Based Assay

    Differentiation of C6 FSHD myotubes was not affected by MAPK12 and MAPK14 partial knockdown that resulted in MBD3L2 level reduction.
    Figure Legend Snippet: Differentiation of C6 FSHD myotubes was not affected by MAPK12 and MAPK14 partial knockdown that resulted in MBD3L2 level reduction.

    Techniques Used:

    p38α/β inhibition results in the reduction of DUX4 activity and cell death across a variety of genotypes of FSHD1 and FSHD2 primary myotubes. ( A ) Levels of MBD3L2 expression across different primary and immortalized myotubes determined RT-qPCR. DUX4 activity is only detected in FSHD1/2 lines after 4 days of differentiation. Bars indicate mean±SD and repeat number is indicated in parenthesis in FSHD1 lines and SMCHD1 mutation for FSHD2 lines used. ( B ) Inhibition of the p38α/β pathway results in potent reduction of MBD3L2 expression activation across the entire set of FSHD primary cells tested. Three different inhibitors were used, and each circle indicates a different FSHD cell line tested. FSHD1 in blue and FSHD2 in green. Expression levels were measured by RT-qPCR in six replicates. ( C and D ) p38α/β pathway inhibition reduces activation of programmed cell death across primary FSHD cell lines with different genotypes. Stochastic activation of caspase-3 in a small number of FSHD myotubes was detected by immunostaining and quantified in all lines. Six replicates were used to quantify signal of cleaved caspase-3 under MHC stained myotubes. Wilconox test, P value **0.002, ***0.0002.
    Figure Legend Snippet: p38α/β inhibition results in the reduction of DUX4 activity and cell death across a variety of genotypes of FSHD1 and FSHD2 primary myotubes. ( A ) Levels of MBD3L2 expression across different primary and immortalized myotubes determined RT-qPCR. DUX4 activity is only detected in FSHD1/2 lines after 4 days of differentiation. Bars indicate mean±SD and repeat number is indicated in parenthesis in FSHD1 lines and SMCHD1 mutation for FSHD2 lines used. ( B ) Inhibition of the p38α/β pathway results in potent reduction of MBD3L2 expression activation across the entire set of FSHD primary cells tested. Three different inhibitors were used, and each circle indicates a different FSHD cell line tested. FSHD1 in blue and FSHD2 in green. Expression levels were measured by RT-qPCR in six replicates. ( C and D ) p38α/β pathway inhibition reduces activation of programmed cell death across primary FSHD cell lines with different genotypes. Stochastic activation of caspase-3 in a small number of FSHD myotubes was detected by immunostaining and quantified in all lines. Six replicates were used to quantify signal of cleaved caspase-3 under MHC stained myotubes. Wilconox test, P value **0.002, ***0.0002.

    Techniques Used: Inhibition, Activity Assay, Expressing, Quantitative RT-PCR, Mutagenesis, Activation Assay, Immunostaining, Staining

    Levels of phosphorylated-HSP27 increase during myogenic differentiation in C6 FSHD myotubes.
    Figure Legend Snippet: Levels of phosphorylated-HSP27 increase during myogenic differentiation in C6 FSHD myotubes.

    Techniques Used:

    39) 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

    40) 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

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    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.

    Isolation:

    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: 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.

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    Article Snippet: .. RNA isolation from vascular endothelial biopsies was also carried out using the RNeasy Micro RNA isolation kit (Qiagen) following process B ( ). ..

    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).

    Purification:

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    Article Snippet: .. One volume of freshly prepared RNase-free 70% ethanol was added to the lysate and total RNA purification was performed by following RNeasy® Micro user guide (QIAGEN, Cat #74004). .. RNase-free DNase set (QIAGEN, Cat#79254) was utilized to remove genomic DNA that can interfere with downstream applications.

    Reverse Transcription Polymerase Chain Reaction:

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    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).

    Expressing:

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    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).

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    Qiagen rneasy micro rna isolation kit
    Experimental workflow to assess efficiency of NuGen probe generation technologies using low amounts of input <t>RNA.</t> 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 <t>RNeasy</t> 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.
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    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

    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

    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.

    Journal: American Journal of Physiology - Cell Physiology

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

    doi: 10.1152/ajpcell.00028.2010

    Figure Lengend 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.

    Article Snippet: Total RNA was obtained from synoviocyte cell cultures using an RNeasy Micro Kit (Qiagen).

    Techniques: RNA Extraction, Amplification, Cell Culture